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Sample records for polymorphic conformational epitopes

  1. Characterization of Conformation-dependent Prion Protein Epitopes*

    PubMed Central

    Kang, Hae-Eun; Weng, Chu Chun; Saijo, Eri; Saylor, Vicki; Bian, Jifeng; Kim, Sehun; Ramos, Laylaa; Angers, Rachel; Langenfeld, Katie; Khaychuk, Vadim; Calvi, Carla; Bartz, Jason; Hunter, Nora; Telling, Glenn C.

    2012-01-01

    Whereas prion replication involves structural rearrangement of cellular prion protein (PrPC), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27–30), a protease-resistant counterpart of the pathogenic scrapie form (PrPSc) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126–218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27–30) both renature to a common structure that reconstitutes the globular domain. PMID:22948149

  2. Dissecting antibodies with regards to linear and conformational epitopes.

    PubMed

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

    2015-01-01

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

  3. Characterization of a cashew allergen, 11S globulin (Ana o 2), conformational epitope.

    PubMed

    Robotham, Jason M; Xia, Lixin; Willison, LeAnna N; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H

    2010-05-01

    Both linear and conformational epitopes likely contribute to the allergenicity of tree nut allergens, yet, due largely to technical issues, few conformational epitopes have been characterized. Using the well studied recombinant cashew allergen, Ana o 2, an 11S globulin or legumin, we identified a murine monoclonal antibody which recognizes a conformational epitope and competes with patient IgE Ana o 2-reactive antibodies. This epitope is expressed on the large subunit of Ana o 2, but only when associated with an 11S globulin small subunit. Both Ana o 2 and the homologous soybean Gly m 6 small subunits can foster epitope expression, even when the natural N-terminal to C-terminal subunit order is reversed in chimeric molecules. The epitope, which is also expressed on native Ana o 2, is readily susceptible to destruction by physical and chemical denaturants. PMID:20362336

  4. Rapid fine conformational epitope mapping using comprehensive mutagenesis and deep sequencing.

    PubMed

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

    2015-10-30

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

  5. Conformational epitope mapping of Pru du 6, a major allergen from almond nut.

    PubMed

    Willison, LeAnna N; Zhang, Qian; Su, Mengna; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H

    2013-10-01

    Tree nuts are a widely consumed food. Although enjoyed safely by most individuals, allergic reactions to tree nuts, including almond, are not uncommon. Almond prunin (Pru du 6), an 11S globulin (legumin), is an abundant nut seed protein and a major allergen. Conformational epitope mapping studies of prunin have been performed with a murine monoclonal antibody (mAb) 4C10. This mAb reacts with non-reduced but not reduced prunin in immunoblotting assays, indicating the recognition of a conformational epitope. 4C10 competes with patient IgE, as assessed by ELISA, indicating clinical significance of the epitope. To characterize the 4C10 epitope, hydrogen/deuterium exchange (HDX) monitored by 14.5 T Fourier transform ion cyclotron resonance mass spectrometry (MS) was performed on the native prunin-4C10 complex and on uncomplexed native prunin. Several epitope candidate peptides that differ in deuterium uptake between the complexed and uncomplexed forms were identified. The epitope was further mapped by analyzing chimeric molecules incorporating segments of the homologous soybean allergen, Gly m 6, in immunoassays. These data indicate that the 4C10 epitope overlaps with a subset of patient IgE binding epitopes on almond prunin and further supports HDX-MS as a valid technique for mapping conformational epitopes. PMID:23498967

  6. Bioinformatics Resources and Tools for Conformational B-Cell Epitope Prediction

    PubMed Central

    Sun, Pingping; Ju, Haixu; Liu, Zhenbang; Ning, Qiao; Zhang, Jian; Zhao, Xiaowei; Huang, Yanxin; Ma, Zhiqiang; Li, Yuxin

    2013-01-01

    Identification of epitopes which invoke strong humoral responses is an essential issue in the field of immunology. Localizing epitopes by experimental methods is expensive in terms of time, cost, and effort; therefore, computational methods feature for its low cost and high speed was employed to predict B-cell epitopes. In this paper, we review the recent advance of bioinformatics resources and tools in conformational B-cell epitope prediction, including databases, algorithms, web servers, and their applications in solving problems in related areas. To stimulate the development of better tools, some promising directions are also extensively discussed. PMID:23970944

  7. Effects of vector fusion peptides on the conformation and immune reactivity of epitope-shuffled, recombinant multi-epitope antigens.

    PubMed

    Wang, Jian; Lin, Yahui; Cai, Pengfei; Wang, Heng

    2011-01-01

    The use of multi-epitopes has been considered as a promising strategy to overcome the obstacle of antigenic variation in malarial vaccine development. Previously, we constructed a multi-epitope artificial antigen, Malaria Random Constructed Antigen-1(M.RCAg-1), to optimize expression of the antigen, and we subcloned the gene into three prokaryotic expression vectors that contain different fusion tags at the N-terminus. Three recombinant proteins expressed by these vectors, named M.RCAg-1/Exp.V-1, V-2, and V-3, were purified after the cleavage of the fusion tag. All three recombinant proteins were able to induce similar levels of antigenicity in BALB/c murine models. However, the antibody responses against the individual epitope peptides of the recombinant products were dramatically different. Additionally, the different epitopes elicited various CD4(+) T-cell responses, as shown by the resulting lymphocyte proliferation and varied IFN-γ and IL-4 levels determined by EILSPOT; however, each could be distinctly recognized by sera derived from malaria patients. Additionally, the rabbit antibody induced by these proteins showed diverse efficacy in malaria parasite growth inhibition assays in vitro. Furthermore, analysis via circular dichroism spectroscopy confirmed that the secondary structure was different among these recombinant proteins. These results suggest that the expressed multi-epitope artificial antigens originating from the different vector fusion peptides indeed affect the protein folding and, subsequently, the epitope exposure. Thus, these proteins are able to induce both distinct humoral and cellular immune responses in animal models, and they affect the efficacy of immune inhibition against the parasite. This work should lead to a further understanding of the impact of vector fusion peptides on the conformation and immune reactivity of recombinant proteins and could provide a useful reference for the development of artificial multi-epitope vaccines. PMID

  8. A novel conformational B-cell epitope prediction method based on mimotope and patch analysis.

    PubMed

    Sun, Pingping; Qi, Jialiang; Zhao, Yizhu; Huang, Yanxin; Yang, Guifu; Ma, Zhiqiang; Li, Yuxin

    2016-04-01

    A B-cell epitope is a group of residues on the surface of an antigen that stimulates humoral immune responses. Identifying B-cell epitopes is important for effective vaccine design. Predicting epitopes by experimental methods is expensive in terms of time, cost and effort; therefore, computational methods that have a low cost and high speed are widely used to predict B-cell epitopes. Recently, epitope prediction based on random peptide library screening has been viewed as a promising method. Some novel software and web-based servers have been proposed that have succeeded in some test cases. Herein, we propose a novel epitope prediction method based on amino acid pairs and patch analysis. The method first divides antigen surfaces into overlapping patches based on both radius (R) and number (N), then predict epitopes based on Amino Acid Pairs (AAPs) from mimotopes and the surface patch. The proposed method yields a mean sensitivity of 0.53, specificity of 0.77, ACC of 0.75 and F-measure of 0.45 for 39 test cases. Compared with mimotope-based methods, patch-based methods and two other prediction methods, the sensitivity of the new method offers a certain improvement. Our findings demonstrate that this proposed method was successful for patch and AAPs analysis and allowed for conformational B-cell epitope prediction. PMID:26804644

  9. Unravelling viral camouflage: approaches to the study and characterization of conformational epitopes.

    PubMed

    Augustin, T; Cehlar, O; Skrabana, R; Majerova, P; Hanes, J

    2015-06-01

    Antibodies are broadly used in clinical and basic research. Many of monoclonal antibodies are successfully adopted for therapeutic and diagnostic targeting of viral pathogens. Efficacy of antiviral neutralizing or protective antibodies depends on their ability to recognize epitopes interfering with viral infection. However, viruses are able to incessantly change their antigenic determinants to escape surveillance of humoral immune system and therefore the successful antiviral therapies require continuous development. Characterization of interactions of antibodies with prevalently conformational viral epitopes is important for understanding antibody mode of action and can help to identify conserved regions that may be exploited in designing new vaccines and virus neutralizing antibodies. In this article, we are reviewing techniques in use for characterization of conformational epitopes of monoclonal antibodies with focus on viruses. PMID:26104327

  10. Structure of Respiratory Syncytial Virus Fusion Glycoprotein in the Postfusion Conformation Reveals Preservation of Neutralizing Epitopes

    SciTech Connect

    McLellan, Jason S.; Yang, Yongping; Graham, Barney S.; Kwong, Peter D.

    2011-09-16

    Respiratory syncytial virus (RSV) invades host cells via a type I fusion (F) glycoprotein that undergoes dramatic structural rearrangements during the fusion process. Neutralizing monoclonal antibodies, such as 101F, palivizumab, and motavizumab, target two major antigenic sites on the RSV F glycoprotein. The structures of these sites as peptide complexes with motavizumab and 101F have been previously determined, but a structure for the trimeric RSV F glycoprotein ectodomain has remained elusive. To address this issue, we undertook structural and biophysical studies on stable ectodomain constructs. Here, we present the 2.8-{angstrom} crystal structure of the trimeric RSV F ectodomain in its postfusion conformation. The structure revealed that the 101F and motavizumab epitopes are present in the postfusion state and that their conformations are similar to those observed in the antibody-bound peptide structures. Both antibodies bound the postfusion F glycoprotein with high affinity in surface plasmon resonance experiments. Modeling of the antibodies bound to the F glycoprotein predicts that the 101F epitope is larger than the linear peptide and restricted to a single protomer in the trimer, whereas motavizumab likely contacts residues on two protomers, indicating a quaternary epitope. Mechanistically, these results suggest that 101F and motavizumab can bind to multiple conformations of the fusion glycoprotein and can neutralize late in the entry process. The structural preservation of neutralizing epitopes in the postfusion state suggests that this conformation can elicit neutralizing antibodies and serve as a useful vaccine antigen.

  11. Probing the Impact of Local Structural Dynamics of Conformational Epitopes on Antibody Recognition.

    PubMed

    Liang, Yu; Guttman, Miklos; Davenport, Thaddeus M; Hu, Shiu-Lok; Lee, Kelly K

    2016-04-19

    Antibody-antigen interactions are governed by recognition of specific residues and structural complementarity between the antigen epitope and antibody paratope. While X-ray crystallography has provided detailed insights into static conformations of antibody-antigen complexes, factors such as conformational flexibility and dynamics, which are not readily apparent in the structures, can also have an impact on the binding event. Here we investigate the contribution of dynamics in the HIV-1 gp120 glycoprotein to antibody recognition of conserved conformational epitopes, including the CD4- and coreceptor-binding sites, and an inner domain site that is targeted by ADCC-active antibodies. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) was used to measure local structural dynamics across a panel of variable loop truncation mutants of HIV-1 gp120, including full-length gp120, ΔV3, ΔV1/V2, and extended core, which includes ΔV1/V2 and V3 loop truncations. CD4-bound full-length gp120 was also examined as a reference state. HDX-MS revealed a clear trend toward an increased level of order of the conserved subunit core resulting from loop truncation. Combined with biolayer interferometry and enzyme-linked immunosorbent assay measurements of antibody-antigen binding, we demonstrate that an increased level of ordering of the subunit core was associated with better recognition by an array of antibodies targeting complex conformational epitopes. These results provide detailed insight into the influence of structural dynamics on antibody-antigen interactions and suggest the importance of characterizing the structural stability of vaccine candidates to improve antibody recognition of complex epitopes. PMID:27003615

  12. Mapping of a conformational epitope on the cashew allergen Ana o 2: a discontinuous large subunit epitope dependent upon homologous or heterologous small subunit association.

    PubMed

    Xia, Lixin; Willison, LeAnna N; Porter, Lauren; Robotham, Jason M; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H

    2010-05-01

    The 11S globulins are members of the cupin protein superfamily and represent an important class of tree nut allergens for which a number of linear epitopes have been mapped. However, specific conformational epitopes for these allergens have yet to be described. We have recently reported a cashew Ana o 2 conformational epitope defined by murine mAb 2B5 and competitively inhibited by a subset of patient IgE antibodies. The 2B5 epitope appears to reside on the large (acidic) subunit, is dependent upon small (basic) subunit association for expression, and is highly susceptible to denaturation. Here we fine map the epitope using a combination of recombinant chimeric cashew Ana o 2-soybean Gly m 6 chimeras, deletion and point mutations, molecular modeling, and electron microscopy of 2B5-Ana o 2 immune complexes. Key residues appear confined to a 24 amino acid segment near the N-terminus of the large subunit peptide, a portion of which makes direct contact with the small subunit. These data provide an explanation for both the small subunit dependence and the structurally labile nature of the epitope. PMID:20362338

  13. Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus

    PubMed Central

    Gustafsson, Erika; Haas, Pieter-Jan; Walse, Björn; Hijnen, Marcel; Furebring, Christina; Ohlin, Mats; van Strijp, Jos AG; van Kessel, Kok PM

    2009-01-01

    Background The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. Results In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31–121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. Conclusion Conformational epitopes recognized by human antibodies have been mapped on the

  14. Protein polymorphism of a human plasma apolipoprotein D antigenic epitope.

    PubMed

    Camato, R; Marcel, Y L; Milne, R W; Lussier-Cacan, S; Weech, P K

    1989-06-01

    Based on our previous observation that monoclonal antibody anti-apoD-4E11 reacted with several HDL proteins we studied them further with three questions in mind: i) is there common protein polymorphism in healthy individuals? ii) how many proteins are present and what are their characteristics? iii) are they all apolipoproteins and do they have the same lipoprotein distribution as apoD? Isolated, delipidated apoD was used as a standard for radioimmunometric assay of plasma with antibody 4E11. The antigen varied from 3 to 11 mumol-equivalents of apoD per liter of plasma (equivalent to 5-20 mg apoD/dl plasma) with means of 6.1 and 6.8 mumol/l in men and women, respectively. Two-dimensional electrophoresis of plasma found up to eight 4E11-antigenic-proteins of different Mr, each heterogeneous in pI. All plasmas tested contained apoD and an Mr 38,000 antigen, the latter being the most immunoreactive. Six proteins of Mr 70,000-94,000 were found, but the number varied between subjects. Eighty nine percent of the plasma antigen was associated with lipoproteins: 83% with HDL and VHDL, 5% with LDL and VLDL. Lipoproteins of all sizes, separated by polyacrylamide gradient gel electrophoresis, contained the antigen. ApoD was almost the only 4E11-antigen in LDL, and was in two states: the one free, the other an apoD-apoB mixed disulfide complex. The apparent proportions of higher Mr antigens increased with increasing lipoprotein density, and the proportion of apoD decreased reciprocally. None of these 4E11-antigenic-proteins cross-reacted with antiserum to retinol-binding protein. PMID:2477480

  15. New polymorphs of an old drug: conformational and synthon polymorphism of 5-nitrofurazone.

    PubMed

    Pogoda, Dorota; Janczak, Jan; Videnova-Adrabinska, Veneta

    2016-04-01

    Two new polymorphic forms of 5-nitrofurazone (5-nitro-2-furaldehyde semicarbazone) have been synthesized and structurally characterized by single-crystal and powder X-ray diffraction methods, vibrational spectroscopy (FT-IR and temperature Raman), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and Hirshfeld surface analysis. The compound crystallizes in three different polymorphic forms P21/a (polymorph α), P21 (polymorph β) and P21/c (polymorph γ), the crystal structures of two of which (polymorphs β and γ) represent new structure determinations. The solid-state molecular organization in the three crystal forms is analyzed and discussed in terms of molecular conformation, crystal packing and hydrogen-bonded networks. All three crystals are formed from trans geometrical isomers, but the molecular conformation of the α-polymorph is syn-anti-anti-anti, while that of β- and γ-polymorphs is syn-anti-syn-syn. As a consequence of this the hydrogen-bond donor and acceptor sites of the molecules are oriented differently, which in turn results in different hydrogen-bond connectivity and packing patterns. PMID:27048728

  16. Neutralizing human monoclonal antibodies to conformational epitopes of human T-cell lymphotropic virus type 1 and 2 gp46.

    PubMed Central

    Hadlock, K G; Rowe, J; Perkins, S; Bradshaw, P; Song, G Y; Cheng, C; Yang, J; Gascon, R; Halmos, J; Rehman, S M; McGrath, M S; Foung, S K

    1997-01-01

    Ten human monoclonal antibodies derived from peripheral B cells of a patient with human T-cell lymphotropic virus (HTLV)-associated myelopathy are described. One monoclonal antibody recognized a linear epitope within the carboxy-terminal 43 amino acids of HTLV gp21, and two monoclonal antibodies recognized linear epitopes within HTLV type 1 (HTLV-1) gp46. The remaining seven monoclonal antibodies recognized denaturation-sensitive epitopes within HTLV-1 gp46 that were expressed on the surfaces of infected cells. Two of these antibodies also bound to viable HTLV-2 infected cells and immunoprecipitated HTLV-2 gp46. Virus neutralization was determined by syncytium inhibition assays. Eight monoclonal antibodies, including all seven that recognized denaturation-sensitive epitopes within HTLV-1 gp46, possessed significant virus neutralization activity. By competitive inhibition analysis it was determined that these antibodies recognized at least four distinct conformational epitopes within HTLV-1 gp46. These findings indicate the importance of conformational epitopes within HTLV-1 gp46 in mediating a neutralizing antibody response to HTLV infection. PMID:9223472

  17. Conformational B-Cell Epitopes Prediction from Sequences Using Cost-Sensitive Ensemble Classifiers and Spatial Clustering

    PubMed Central

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

    2014-01-01

    B-cell epitopes are regions of the antigen surface which can be recognized by certain antibodies and elicit the immune response. Identification of epitopes for a given antigen chain finds vital applications in vaccine and drug research. Experimental prediction of B-cell epitopes is time-consuming and resource intensive, which may benefit from the computational approaches to identify B-cell epitopes. In this paper, a novel cost-sensitive ensemble algorithm is proposed for predicting the antigenic determinant residues and then a spatial clustering algorithm is adopted to identify the potential epitopes. Firstly, we explore various discriminative features from primary sequences. Secondly, cost-sensitive ensemble scheme is introduced to deal with imbalanced learning problem. Thirdly, we adopt spatial algorithm to tell which residues may potentially form the epitopes. Based on the strategies mentioned above, a new predictor, called CBEP (conformational B-cell epitopes prediction), is proposed in this study. CBEP achieves good prediction performance with the mean AUC scores (AUCs) of 0.721 and 0.703 on two benchmark datasets (bound and unbound) using the leave-one-out cross-validation (LOOCV). When compared with previous prediction tools, CBEP produces higher sensitivity and comparable specificity values. A web server named CBEP which implements the proposed method is available for academic use. PMID:25045691

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

    PubMed

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

    2014-01-01

    B-cell epitopes are regions of the antigen surface which can be recognized by certain antibodies and elicit the immune response. Identification of epitopes for a given antigen chain finds vital applications in vaccine and drug research. Experimental prediction of B-cell epitopes is time-consuming and resource intensive, which may benefit from the computational approaches to identify B-cell epitopes. In this paper, a novel cost-sensitive ensemble algorithm is proposed for predicting the antigenic determinant residues and then a spatial clustering algorithm is adopted to identify the potential epitopes. Firstly, we explore various discriminative features from primary sequences. Secondly, cost-sensitive ensemble scheme is introduced to deal with imbalanced learning problem. Thirdly, we adopt spatial algorithm to tell which residues may potentially form the epitopes. Based on the strategies mentioned above, a new predictor, called CBEP (conformational B-cell epitopes prediction), is proposed in this study. CBEP achieves good prediction performance with the mean AUC scores (AUCs) of 0.721 and 0.703 on two benchmark datasets (bound and unbound) using the leave-one-out cross-validation (LOOCV). When compared with previous prediction tools, CBEP produces higher sensitivity and comparable specificity values. A web server named CBEP which implements the proposed method is available for academic use. PMID:25045691

  19. Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis

    PubMed Central

    GORA, M; GARDAS, A; WIKTOROWICZ, W; HOBBY, P; WATSON, P F; WEETMAN, A P; SUTTON, B J; BANGA, J P

    2004-01-01

    Autoantibodies to thyroid peroxidase (TPO) recognize predominantly conformational epitopes, which are restricted to two distinct determinants, termed immunodominant domain region (IDR) A and B. These dominant determinants reside in the region with structural homology to myeloperoxidase (MPO)-like domain and may extend into the adjacent complement control protein (CCP) domain. We have explored the location of these determinants on the MPO-like domain of the structural model of TPO, by identifying exposed hydrophilic loops that are potential candidates for the autoantigenic sites, generating rabbit antipeptide antisera, and competing with well characterized murine monoclonal antibodies (mabs) specific for these two IDRs. We recently defined the location of IDR-B, and here report our findings on the location of IDR-A and its relationship to IDR-B, defined with a new panel of 15 antipeptide antisera. Moreover, in combination with single amino acid replacements by in vitro mutagenesis, we have defined the limits of the IDR-B region on the TPO model. The combination of antisera to peptides P12 (aa 549–563), P14 (aa 599–617) and P18 (aa 210–225) inhibited the binding of the mab specific for IDR-A (mab 2) by 75. The same combination inhibited the binding of autoantibodies to native TPO from 67 to 94% (mean 81·5%) at autoantibody levels of 5 IU. Fabs prepared from the antipeptide IgG and pooled in this combination were also effective in competition assays, thus defining the epitopes more precisely. IDR-A was found to lie immediately adjacent to IDR-B and thus the two immunodominant epitopes form an extended patch on the surface of TPO. Finally, by single amino acid mutagenesis, we show that IDR-B extends to residue N642, thus further localizing the boundary of this autoantigenic region on the structural model. PMID:15030525

  20. Monoclonal anti-idiotypes induce neutralizing antibodies to enterovirus 70 conformational epitopes.

    PubMed Central

    Wiley, J A; Hamel, J; Brodeur, B R

    1992-01-01

    Monoclonal antibodies (MAbs) directed against the prototype enterovirus 70 (EV-70) strain J670/71 were generated and characterized in order to produce anti-idiotypic MAbs (MAb2s) for use as surrogate immunogens. Western immunoblot and radioimmunoprecipitation assays suggested that all the MAbs recognize conformational epitopes on the virion surface. An EV-70-neutralizing antibody, MAb/ev-12 (MAb1), was selected for the production of MAb2s. Five MAb2s were selected for their capacities to inhibit the interaction of MAb/ev-12 with EV-70 in dot immunobinding inhibition and immunofluorescence assays. In addition, these five MAb2s inhibited virus neutralization mediated by MAb/ev-12, suggesting that they recognize paratope-associated idiotopes. In competition enzyme immunosorbent assays, none of the five MAb2s recognized other neutralizing and nonneutralizing EV-70-specific MAbs, demonstrating that the MAb2s were specific for private idiotopes. Immunization with each of the MAb2s was carried out for the production of anti-anti-idiotypic antibodies (Ab3). All five MAb2s induced an immune response. Moreover, results suggested that they share idiotopes, since MAb2-MAb/ev-12 binding could be inhibited by homologous as well as heterologous Ab3s. Ab3 sera were shown to possess antibodies capable of immunoprecipitating 35S-labeled viral proteins in the same manner as MAb/ev-12. Nine of 15 mice immunized with MAb2s demonstrated Ab3 neutralizing activity specific for the prototype EV-70 strain, J670/71. The potential application of MAb2s to serve as surrogate immunogens for conformational epitopes is substantiated by the results presented in this report. Images PMID:1382141

  1. Peptide Conformations for a Microarray Surface-Tethered Epitope of the Tumor Suppressor p53

    SciTech Connect

    Feng, Jun; Wong, Ka-Yiu; Lynch, Gillian C.; Gao, Xiaolian; Pettitt, Bernard M.

    2007-12-13

    The research described in this product was performed in part in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. Peptides or proteins near surfaces exhibit different structural properties from those present in a homogeneous solution, and these differences give rise to varied biological activity. Therefore, understanding the detailed molecular structure of these molecules tethered to a surface is important for interpreting the performance of the various microarrays based on the activities of the immobilized peptides or proteins. We performed molecular dynamics simulations of a pentapeptide, RHSVV, an epitope of the tumor suppressor protein p53, tethered via a spacer on a functionalized silica surface and free in solution, to study their structural and conformational differences. These calculations allowed analyses of the peptide-surface interactions, the sequence orientations, and the translational motions of the peptide on the surface to be performed. Conformational similarities are found among dominant structures of the tethered and free peptide. In the peptide microarray simulations, the peptide fluctuates between a parallel and tilted orientation driven in part by the hydrophobic interactions between the nonpolar peptide residues and the methyl-terminated silica surface. The perpendicular movement of the peptide relative to the surface is also restricted due to the hydrophobic nature of the microarray surface. With regard to structures available for recognition and binding, we find that similar conformations to those found in solution are available to the peptide tethered to the surface, but with a shifted equilibrium constant. Comparisons with experimental results show important implications of this for peptide microarray design and assays.

  2. Flexible vs Rigid Epitope Conformations for Diagnostic- and Vaccine-Oriented Applications: Novel Insights from the Burkholderia pseudomallei BPSL2765 Pal3 Epitope.

    PubMed

    Gori, Alessandro; Peri, Claudio; Quilici, Giacomo; Nithichanon, Arnone; Gaudesi, Davide; Longhi, Renato; Gourlay, Louise; Bolognesi, Martino; Lertmemongkolchai, Ganjana; Musco, Giovanna; Colombo, Giorgio

    2016-03-11

    Peptides seldom retain stable conformations if separated from their native protein structure. In an immunological context, this potentially affects the development of selective peptide-based bioprobes and, from a vaccine perspective, poses inherent limits in the elicitation of cross-reactive antibodies by candidate epitopes. Here, a 1,4-disubstituted-1,2,3-triazole-mediated stapling strategy was used to stabilize the native α-helical fold of the Pal3 peptidic epitope from the protein antigen PalBp (BPSL2765) from Burkholderia pseudomallei, the etiological agent of melioidosis. Whereas Pal3 shows no propensity to fold outside its native protein context, the engineered peptide (Pal3H) forms a stable α-helix, as assessed by MD, NMR, and CD structural analyses. Importantly, Pal3H shows an enhanced ability to discriminate between melioidosis patient subclasses in immune sera reactivity tests, demonstrating the potential of the stapled peptide for diagnostic purposes. With regard to antibody elicitation and related bactericidal activities, the linear peptide is shown to elicit a higher response. On these bases, we critically discuss the implications of epitope structure engineering for diagnostic- and vaccine-oriented applications. PMID:27623032

  3. Reversible conformational change of tau2 epitope on exposure to detergent in glial cytoplasmic inclusions of multiple system atrophy.

    PubMed

    Shibuya, Katsuhiko; Uchihara, Toshiki; Nakamura, Ayako; Ishiyama, Miyako; Yamaoka, Keiko; Yagishita, Saburo; Iwabuchi, Kiyoshi; Kosaka, Kenji

    2003-05-01

    Tau-like immunoreactivity (IR) on glial cytoplasmic inclusions (GCIs) of multiple system atrophy (MSA) was investigated with a panel of anti-tau antibodies and we found that tau2, one of the phosphorylation-independent antibodies, preferentially immunolabeled GCIs. Co-presence (0.03%) of polyethyleneglycol- p-isooctylphenyl ether (Triton X-100, TX) with tau2, however, abolished this IR on GCIs, but did not abolish tau2 IR on neurofibrillary tangles (NFTs). Tau2-immunoreactive bands on immunoblot of brain homogenates from MSA brains were retrieved mainly in a TRIS-saline-soluble fraction, as reported in normal brains. This was in contrast to SDS-soluble fractions from brain with Down's syndrome, which contained tau2-immunoreactive bands of higher molecular weight. It indicates that the appearance of tau2 IR on GCIs is not related to hyperphosphorylation of tau. These tau2-immunoreactive bands, except those from bovine brain, were similarly abolished in the presence of TX (0.06%), and repeated washing after exposure to TX restored the tau2 IR on immunohistochemistry and on immunoblot. These findings can be explained if the modified tau2 epitope undergoes a reversible conformational change on exposure to TX, which is reversible after washing. Because the conformation centered at Ser101 of bovine tau is crucial for its affinity to tau2, the Ser-like conformation mimicked by its human counterpart Pro may represent pathological modification of tau shared by GCIs and NFTs. The relative resistance of tau2 epitope on NFTs on exposure to TX suggests that tau woven into NFTs confers additional stability to the pathological conformation of tau2 epitope. The conformation of the tau2 epitope in GCIs is not as stable as in NFTs, suggesting that tau proteins are not the principal constituents of the fibrillary structures of GCIs, even though they were immunodecorated with tau2. The difference in the susceptibility of the tau2 epitope to TX may distinguish its conformational states

  4. Virus-epitope vaccine design: informatic matching the HLA-I polymorphism to the virus genome.

    PubMed

    Vider-Shalit, Tal; Raffaeli, Shai; Louzoun, Yoram

    2007-02-01

    Attempts to develop peptide vaccines, based on a limited number of peptides face two problems: HLA polymorphism and the high mutation rate of viral epitopes. We have developed a new genomic method that ensures maximal coverage and thus maximal applicability of the peptide vaccine. The same method also promises a large number of epitopes per HLA to prevent escape via mutations. Our design can be applied swiftly in order to face rapidly emerging viral diseases. We use a genomic scan of all candidate peptides and join them optimally. For a given virus, we use algorithms computing: peptide cleavage probability, transfer through TAP and MHC binding for a large number of HLA alleles. The resulting peptide libraries are pruned for peptides that are not conserved or are too similar to self peptides. We then use a genetic algorithm to produce an optimal protein composed of peptides from this list properly ordered for cleavage. The selected peptides represent an optimal combination to cover all HLA alleles and all viral proteins. We have applied this method to HCV and found that some HCV proteins (mainly envelope proteins) represent much less peptide than expected. A more detailed analysis of the peptide variability shows a balance between the attempts of the immune system to detect less mutating peptides, and the attempts of viruses to mutate peptides and avoid detection by the immune system. In order to show the applicability of our method, we have further used it on HIV-I, Influenza H3N2 and the Avian Flu Viruses. PMID:16930710

  5. Force-dependent polymorphism in type IV pili reveals hidden epitopes.

    PubMed

    Biais, Nicolas; Higashi, Dustin L; Brujic, Jasna; So, Magdalene; Sheetz, Michael P

    2010-06-22

    Through evolution, nature has produced exquisite nanometric structures, with features unrealized in the most advanced man-made devices. Type IV pili (Tfp) represent such a structure: 6-nm-wide retractable filamentous appendages found in many bacteria, including human pathogens. Whereas the structure of Neisseria gonorrhoeae Tfp has been defined by conventional structural techniques, it remains difficult to explain the wide spectrum of functions associated with Tfp. Here we uncover a previously undescribed force-induced quaternary structure of the N. gonorrhoeae Tfp. By using a combination of optical and magnetic tweezers, atomic force microscopy, and molecular combing to apply forces on purified Tfp, we demonstrate that Tfp subjected to approximately 100 pN of force will transition into a new conformation. The new structure is roughly 3 times longer and 40% narrower than the original structure. Upon release of the force, the Tfp fiber regains its original form, indicating a reversible transition. Equally important, we show that the force-induced conformation exposes hidden epitopes previously buried in the Tfp fiber. We postulate that this transition provides a means for N. gonorrhoeae to maintain attachment to its host while withstanding intermittent forces encountered in the environment. Our findings demonstrate the need to reassess our understanding of Tfp dynamics and functions. They could also explain the structural diversity of other helical polymers while presenting a unique mechanism for polymer elongation and exemplifying the extreme structural plasticity of biological polymers. PMID:20534431

  6. Molecular docking study of conformational polymorph: building block of crystal chemistry.

    PubMed

    Dubey, Rashmi; Tewari, Ashish Kumar; Singh, Ved Prakash; Singh, Praveen; Dangi, Jawahar Singh; Puerta, Carmen; Valerga, Pedro; Kant, Rajni

    2013-01-01

    Two conformational polymorphs of novel 2-[2-(3-cyano-4,6-dimethyl-2-oxo-2H-pyridin-1-yl)-ethoxy]-4,6-dimethyl nicotinonitrile have been developed. The crystal structure of both polymorphs (1a and 1b) seems to be stabilized by weak interactions. A difference was observed in the packing of both polymorphs. Polymorph 1b has a better binding affinity with the cyclooxygenase (COX-2) receptor than the standard (Nimesulide). PMID:24250264

  7. The Transport Signal on Sec22 for Packaging into COPII-Coated Vesicles is a Conformational Epitope

    SciTech Connect

    Mancias,J.; Goldberg, J.

    2007-01-01

    The mechanism of cargo concentration into ER-derived vesicles involves interactions between the COPII vesicular coat complex and cargo transport signals-peptide sequences of 10-15 residues. The SNARE protein Sec22 contains a signal that binds the COPII subcomplex Sec23/24 and specifies its endoplasmic reticulum (ER) exit as an unassembled SNARE. The 200 kDa crystal structure of Sec22 bound to Sec23/24 reveals that the transport signal is a folded epitope rather than a conventional short peptide sequence. The NIE segment of the SNARE motif folds against the N-terminal longin domain, and this closed form of Sec22 binds at the Sec23/24 interface. Thus, COPII recognizes unassembled Sec22 via a folded epitope, whereas Sec22 assembly into SNARE complexes would mask the NIE segment. The concept of a conformational epitope as a transport signal suggests packaging mechanisms in which a coat is sensitive to the folded state of a cargo protein or the assembled state of a multiprotein complex.

  8. A comprehensive analysis of the naturally occurring polymorphisms in HIV-1 Vpr: Potential impact on CTL epitopes

    PubMed Central

    Srinivasan, Alagarsamy; Ayyavoo, Velpandi; Mahalingam, Sundarasamy; Kannan, Aarthi; Boyd, Anne; Datta, Debduti; Kalyanaraman, Vaniambadi S; Cristillo, Anthony; Collman, Ronald G; Morellet, Nelly; Sawaya, Bassel E; Murali, Ramachandran

    2008-01-01

    The enormous genetic variability reported in HIV-1 has posed problems in the treatment of infected individuals. This is evident in the form of HIV-1 resistant to antiviral agents, neutralizing antibodies and cytotoxic T lymphocytes (CTLs) involving multiple viral gene products. Based on this, it has been suggested that a comprehensive analysis of the polymorphisms in HIV proteins is of value for understanding the virus transmission and pathogenesis as well as for the efforts towards developing anti-viral therapeutics and vaccines. This study, for the first time, describes an in-depth analysis of genetic variation in Vpr using information from global HIV-1 isolates involving a total of 976 Vpr sequences. The polymorphisms at the individual amino acid level were analyzed. The residues 9, 33, 39, and 47 showed a single variant amino acid compared to other residues. There are several amino acids which are highly polymorphic. The residues that show ten or more variant amino acids are 15, 16, 28, 36, 37, 48, 55, 58, 59, 77, 84, 86, 89, and 93. Further, the variant amino acids noted at residues 60, 61, 34, 71 and 72 are identical. Interestingly, the frequency of the variant amino acids was found to be low for most residues. Vpr is known to contain multiple CTL epitopes like protease, reverse transcriptase, Env, and Gag proteins of HIV-1. Based on this, we have also extended our analysis of the amino acid polymorphisms to the experimentally defined and predicted CTL epitopes. The results suggest that amino acid polymorphisms may contribute to the immune escape of the virus. The available data on naturally occurring polymorphisms will be useful to assess their potential effect on the structural and functional constraints of Vpr and also on the fitness of HIV-1 for replication. PMID:18721481

  9. Identification of neutralization and diagnostic epitopes on PIM, the polymorphic immunodominant molecule of Theileria parva.

    PubMed Central

    Toye, P; Nyanjui, J; Goddeeris, B; Musoke, A J

    1996-01-01

    The polymorphic immunodominant molecule (PIM) of Theileria parva is expressed by the schizont and sporozoite stages of the parasite. We have recently cloned the cDNA encoding the PIM antigen from two stocks of the parasite: the cattle-derived T. parva (Muguga) stock and a buffalo-derived stock. The cDNAs were used in transient-transfection assays to assess the reactivity of the antigen with monoclonal antibodies (MAb) previously raised against schizont-infected cells and used for parasite strain identification. We demonstrate that 19 of the 25 MAb are specific for PIM. Antibody reactivities with deletion mutants of a fusion protein containing PIM and Pepscan analysis of the Muguga version of the molecule with 13 of the MAb indicate that there are at least 10 different epitopes throughout the molecule. None of the MAb react with a tetrapeptide repeat present in the central region of the molecule, probably because of an inability of BALB/c mice to produce antibodies to this repeat. In contrast, sera from infected cattle react strongly with the repeat region, suggesting that this region alone may be useful as a diagnostic reagent. Previous studies showed that MAb to PIM inhibit sporozoite infectivity of bovine lymphocytes in vitro, which suggests that the antigen may be useful in immunizing cattle against T. parva infection. Pepscan analysis revealed that sera from infected cattle reacted with peptides recognized by the neutralizing MAb, as did sera from cattle inoculated with a PIM-containing recombinant protein. The latter sera did not, however, neutralize sporozoite infectivity in vitro. These results will be useful in exploiting the strain identification, diagnostic, and immunizing potentials of this family of antigens. PMID:8613398

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

    PubMed Central

    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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

  14. Comprehensive Analysis of Contributions from Protein Conformational Stability and Major Histocompatibility Complex Class II-Peptide Binding Affinity to CD4+ Epitope Immunogenicity in HIV-1 Envelope Glycoprotein

    PubMed Central

    Li, Tingfeng; Steede, N. Kalaya; Nguyen, Hong-Nam P.; Freytag, Lucy C.; McLachlan, James B.; Mettu, Ramgopal R.; Robinson, James E.

    2014-01-01

    ABSTRACT Helper T-cell epitope dominance in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is not adequately explained by peptide binding to major histocompatibility complex (MHC) proteins. Antigen processing potentially influences epitope dominance, but few, if any, studies have attempted to reconcile the influences of antigen processing and MHC protein binding for all helper T-cell epitopes of an antigen. Epitopes of gp120 identified in both humans and mice occur on the C-terminal flanks of flexible segments that are likely to be proteolytic cleavage sites. In this study, the influence of gp120 conformation on the dominance pattern in gp120 from HIV strain 89.6 was examined in CBA mice, whose MHC class II protein has one of the most well defined peptide-binding preferences. Only one of six dominant epitopes contained the most conserved element of the I-Ak binding motif, an aspartic acid. Destabilization of the gp120 conformation by deletion of single disulfide bonds preferentially enhanced responses to the cryptic I-Ak motif-containing sequences, as reported by T-cell proliferation or cytokine secretion. Conversely, inclusion of CpG in the adjuvant with gp120 enhanced responses to the dominant CD4+ T-cell epitopes. The gp120 destabilization affected secretion of some cytokines more than others, suggesting that antigen conformation could modulate T-cell functions through mechanisms of antigen processing. IMPORTANCE CD4+ helper T cells play an essential role in protection against HIV and other pathogens. Thus, the sites of helper T-cell recognition, the dominant epitopes, are targets for vaccine design; and the corresponding T cells may provide markers for monitoring infection and immunity. However, T-cell epitopes are difficult to identify and predict. It is also unclear whether CD4+ T cells specific for one epitope are more protective than T cells specific for other epitopes. This work shows that the three-dimensional (3D) structure of an

  15. Detecting high-resolution polymorphisms in human coding loci by combining PCR and single-strand conformation polymorphism (SSCP) analysis.

    PubMed

    Poduslo, S E; Dean, M; Kolch, U; O'Brien, S J

    1991-07-01

    A strategy is described that allows the development of polymorphic genetic markers to be characterized in individual genes. Segments of the 3' untranslated regions are amplified, and polymorphisms are detected by digestion with frequently cutting enzymes and with the detection of single-stranded conformation polymorphisms. This allows these genes, or DNA segments, to be placed on the linkage maps of human chromosomes. Polymorphisms in two genes have been identified using this approach. A HaeIII polymorphism was detected in the KIT proto-oncogene, physically assigned to chromosome 4q11-12. This polymorphism is linked to other chromosome 4p markers and is in linkage disequilibrium with a HindIII polymorphism previously described at this locus. We have also identified in the insulin-like growth factor1 receptor gene (IGF1R) a 2-bp deletion that is present at a frequency of .25 in the Caucasian population. Pedigree analysis with this insertion/deletion polymorphism placed the IGF1R gene at the end of the current linkage map of chromosome 15q, consistent with the physical assignment of 15q2526. Thus, polymorphisms in specific genes can be used to related the physical, genetic, and comparative maps of mammalian genomes and to simplify the testing of candidate genes for human diseases. PMID:1676559

  16. Using small molecule reagents to selectively modify epitopes based on their conformation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PrPSc is an infectious protein. The only experimentally verified difference between PrPSc and their normal cellular isoform (PrPC) is conformational. This work describes an approach to determining the presence of surface exposed or sequestered amino acids present in the PrPSc isoform. The N-hydroxys...

  17. Alpha S1-casein polymorphisms in camel (Camelus dromedarius) and descriptions of biological active peptides and allergenic epitopes.

    PubMed

    Erhardt, Georg; Shuiep, El Tahir Salih; Lisson, Maria; Weimann, Christina; Wang, Zhaoxin; El Zubeir, Ibtisam El Yas Mohamed; Pauciullo, Alfredo

    2016-06-01

    Milk samples of 193 camels (Camelus dromedarius) from different regions of Sudan were screened for casein variability by isoelectric focusing. Kappa-casein and beta-casein were monomorphic, whereas three protein patterns named αs1-casein A, C, and D were identified. The major allele A revealed frequencies of 0.79 (Lahaoi), 0.75 (Shanbali), 0.90 (Arabi Khali), and 0.88 (Arabi Gharbawi) in the different ecotypes. CSN1S1*C shows a single G > T nucleotide substitution in the exon 5, leading to a non-synonymous amino acid exchange (p.Glu30 > Asp30) in comparison to CSN1S1*A and D. At cDNA level, no further single nucleotide polymorphisms could be identified in CSN1S1* A, C, and D, whereas the variants CSN1S1*A and CSN1S1*C are characterized by missing of exon 18 compared to the already described CSN1S1*B, as consequence of DNA insertion of 11 bp at intron 17 which alter the pre-mRNA spliceosome machinery. A polymerase chain-restriction fragment length polymorphism method (PCR-RFLP) was established to type for G > T nucleotide substitution at genomic DNA level. The occurrence and differences of IgE-binding epitopes and bioactive peptides between αs1-casein A, C, and D after digestion were analyzed in silico. The amino acid substitutions and deletion affected the arising peptide pattern and thus modifications between IgE-binding epitopes and bioactive peptides of the variants were found. The allergenic potential of these different peptides will be investigated by microarray immunoassay using sera from milk-sensitized individuals, as it was already demonstrated for bovine αs1-casein variants. PMID:26922739

  18. Human anti-Aβ IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

    PubMed

    Welzel, Alfred T; Williams, Angela D; McWilliams-Koeppen, Helen P; Acero, Luis; Weber, Alfred; Blinder, Veronika; Mably, Alex; Bunk, Sebastian; Hermann, Corinna; Farrell, Michael A; Ehrlich, Hartmut J; Schwarz, Hans P; Walsh, Dominic M; Solomon, Alan; O'Nuallain, Brian

    2012-01-01

    Soluble non-fibrillar assemblies of amyloid-beta (Aβ) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer's disease (AD). Anti-Aβ immunotherapy is a promising and advanced therapeutic strategy, but the precise Aβ species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aβ conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aβ dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aβ extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aβ's conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody's nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aβ monomers and peptide fragments. The antibody's lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aβ NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aβ NAbs are warranted. PMID:23209707

  19. Human Anti-Aβ IgGs Target Conformational Epitopes on Synthetic Dimer Assemblies and the AD Brain-Derived Peptide

    PubMed Central

    Welzel, Alfred T.; Williams, Angela D.; McWilliams-Koeppen, Helen P.; Acero, Luis; Weber, Alfred; Blinder, Veronika; Mably, Alex; Bunk, Sebastian; Hermann, Corinna; Farrell, Michael A.; Ehrlich, Hartmut J.; Schwarz, Hans P.; Walsh, Dominic M.; Solomon, Alan; O’Nuallain, Brian

    2012-01-01

    Soluble non-fibrillar assemblies of amyloid-beta (Aβ) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer’s disease (AD). Anti-Aβ immunotherapy is a promising and advanced therapeutic strategy, but the precise Aβ species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aβ conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aβ dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aβ extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aβ’s conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody’s nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aβ monomers and peptide fragments. The antibody’s lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aβ NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aβ NAbs are warranted. PMID:23209707

  20. Correlation between nanomechanics and polymorphic conformations in amyloid fibrils.

    PubMed

    Usov, Ivan; Mezzenga, Raffaele

    2014-11-25

    Amyloid fibrils occur in diverse morphologies, but how polymorphism affects the resulting mechanical properties is still not fully appreciated. Using formalisms from the theory of elasticity, we propose an original way of averaging the second area moment of inertia for non-axisymmetric fibrils, which constitutes the great majority of amyloid fibrils. By following this approach, we derive theoretical expressions for the bending properties of the most common polymorphic forms of amyloid fibrils (twisted ribbons, helical ribbons, and nanotubes), and we benchmark the predictions to experimental cases. These results not only allow an accurate estimation of the amyloid fibrils' elastic moduli but also bring insight into the structure-property relationships in the nanomechanics of amyloid systems, such as in the closure of helical ribbons into nanotubes. PMID:25275956

  1. Amyloid-β-Anti-Amyloid-β Complex Structure Reveals an Extended Conformation in the Immunodominant B-Cell Epitope

    SciTech Connect

    Miles, Luke A; Wun, Kwok S; Crespi, Gabriela A.N.; Fodero-Tavoletti, Michelle T; Galatis, Denise; Bagley, Christopher J; Beyreuther, Konrad; Masters, Colin L; Cappai, Roberto; McKinstry, William J; Barnham, Kevin J; Parker, Michael W

    2008-04-29

    Alzheimer's disease (AD) is the most common form of dementia. Amyloid-β (Aβ) peptide, generated by proteolytic cleavage of the amyloid precursor protein, is central to AD pathogenesis. Most pharmaceutical activity in AD research has focused on Aβ, its generation and clearance from the brain. In particular, there is much interest in immunotherapy approaches with a number of anti-Aβ antibodies in clinical trials. We have developed a monoclonal antibody, called WO2, which recognises the Aβ peptide. To this end, we have determined the three-dimensional structure, to near atomic resolution, of both the antibody and the complex with its antigen, the Aβ peptide. The structures reveal the molecular basis for WO2 recognition and binding of Aβ. The Aβ peptide adopts an extended, coil-like conformation across its major immunodominant B-cell epitope between residues 2 and 8. We have also studied the antibody-bound Aβ peptide in the presence of metals known to affect its aggregation state and show that WO2 inhibits these interactions. Thus, antibodies that target the N-terminal region of Aβ, such as WO2, hold promise for therapeutic development.

  2. Amyloid-β-Anti-Amyloid-β Complex Structure Reveals an Extended Conformation in the Immunodominant B-Cell Epitope

    SciTech Connect

    Miles, Luke A; Wun, Kwok S; Crespi, Gabriela A.N.; Fodero-Tavoletti, Michelle T; Galatis, Denise; Bagley, Christopher J; Beyreuther, Konrad; Masters, Colin L; Cappai, Roberto; McKinstry, William J; Barnham, Kevin J; Parker, Michael W

    2012-04-17

    Alzheimer's disease (AD) is the most common form of dementia. Amyloid-β (Aβ) peptide, generated by proteolytic cleavage of the amyloid precursor protein, is central to AD pathogenesis. Most pharmaceutical activity in AD research has focused on Aβ, its generation and clearance from the brain. In particular, there is much interest in immunotherapy approaches with a number of anti-Aβ antibodies in clinical trials. We have developed a monoclonal antibody, called WO2, which recognises the Aβ peptide. To this end, we have determined the three-dimensional structure, to near atomic resolution, of both the antibody and the complex with its antigen, the Aβ peptide. The structures reveal the molecular basis for WO2 recognition and binding of Aβ. The Aβ peptide adopts an extended, coil-like conformation across its major immunodominant B-cell epitope between residues 2 and 8. We have also studied the antibody-bound Aβ peptide in the presence of metals known to affect its aggregation state and show that WO2 inhibits these interactions. Thus, antibodies that target the N-terminal region of Aβ, such as WO2, hold promise for therapeutic development.

  3. Association of PTPN22 1858C→T polymorphism, HLA-DRB1 shared epitope and autoantibodies with rheumatoid arthritis.

    PubMed

    Raslan, Hala M; Attia, Hanaa R; Salama, Iman; Ibrahim, Mona Hamed; Hassan, Eman Mahmoud; El Hussieny, Mohamed S; El Menyawi, Manal M; Amr, Khalda S

    2016-08-01

    To assess impact of PTPN22 1858C→T polymorphism, HLA shared epitope and autoantibodies on susceptibility and severity of rheumatoid arthritis (RA). A total of 150 RA patients and 150 controls were included in the study. Anti-cyclic citrullinated peptide (anti-CCP) and rheumatoid factor isotypes (IgG, IgM and IgA) were assayed by ELISA. PTPN22 1858C→T polymorphism was performed by RFLP analysis and HLA-DRB1 genotyping by PCR-SSP analysis. Single-view, anteroposterior radiographs of the hands and feet were obtained on all RA patients. The results showed association of PTPN22 1858 T allele with RA (OR = 2.3, 95 % CI 1.5-3.5) and bone erosion (OR = 2.9, 95 % CI 1.1-7.6). The associations increased with the combination of positive autoantibodies, HLA-DRB1 SE with PTPN22 1858 T allele carriage. The highest association was with the combination with anti-CCP antibodies (OR = 47.3, 95 % CI 10.9-204.4 for RA and OR = 69.4, 95 % CI 15.8-305.5 for erosion p < 0.001). Combination of PTPN22 1858 T allele carriage with negative RF isotypes or with absence HLA-DRB1 SE showed no significant association with RA. The presence of PTPN22 1858C→T polymorphism with HLA SE and autoantibodies increases risk of RA development and erosive disease. PMID:27324632

  4. Genetic mapping of the human tryptophan hydroxylase gene on chromosome 11, using an intronic conformational polymorphism

    SciTech Connect

    Nielsen, D.A.; Goldman, D. ); Dean, M. )

    1992-12-01

    The identification of polymorphic alleles at loci coding for functional genes is crucial for genetic association and linkage studies. Since the tryptophan hydroxylase (TPH) gene codes for the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, it would be advantageous to identify a polymorphism in this gene. By examining introns of the human TPH gene by PCR amplification and analysis by the single-strand conformation polymorphism (SSCP) technique, an SSCP was revealed with two alleles that occur with frequencies of .40 and .60 in unrelated Caucasians. DNAs from 24 informative CEPH families were typed for the TPH intron polymorphism and analyzed with respect to 10 linked markers on chromosome 11, between p13 and p15, with the result that TPH was placed between D11S151 and D11S134. This region contains loci for several important genes, including those for Beckwith-Wiedemann syndrome and tyrosine hydroxylase. 37 refs., 2 figs., 1 tab.

  5. Mapping the conformational epitope of a neutralizing antibody (AcV1) directed against the AcMNPV GP64 protein

    SciTech Connect

    Zhou Jian; Blissard, Gary W. . E-mail: gwb1@cornell.edu

    2006-09-01

    The envelope glycoprotein GP64 of Autographa californica nucleopolyhedrovirus (AcMNPV) is necessary and sufficient for the acid-induced membrane fusion activity that is required for fusion of the budded virus (BV) envelope and the endosome membrane during virus entry. Infectivity of the budded virus (BV) is neutralized by AcV1, a monoclonal antibody (MAb) directed against GP64. Prior studies indicated that AcV1 recognizes a conformational epitope and does not inhibit virus attachment to the cell, but instead inhibits entry at a step following virus attachment. We found that AcV1 recognition of GP64 was lost upon exposure of GP64 to low pH (pH 4.5) and restored by returning GP64 to pH 6.2. In addition, the AcV1 epitope was lost upon denaturation of GP64 in SDS, but the AcV1 epitope was restored by refolding the protein in the absence of SDS. Using truncated GP64 proteins expressed in insect cells, we mapped the AcV1 epitope to a 24 amino acid region in the central variable domain of GP64. When sequences within the mapped AcV1 epitope were substituted with a c-Myc epitope and the resulting construct was used to replace wt GP64 in recombinant AcMNPV viruses, the modified GP64 protein appeared to function normally. However, an anti-c-Myc monoclonal antibody did not neutralize infectivity of those viruses. Because binding of the c-Myc MAb to the same site in the GP64 sequence did not result in neutralization, these studies suggest that AcV1 neutralization may result from a specific structural constraint caused by AcV1 binding and not simply by steric hindrance caused by antibody binding at this position in GP64.

  6. Definition of a possible genetic basis for susceptibility to acute myelogenous leukemia associated with the presence of a polymorphic Ia epitope.

    PubMed Central

    Seremetis, S; Cuttner, J; Winchester, R

    1985-01-01

    The polymorphic Ia epitope recognized by monoclonal antibody 109d6 is detectable on the leukemic cells of a significantly increased number of individuals with acute myelogenous leukemia, compared with its frequency in normal healthy control individuals. In control individuals, the presence of the 109d6 epitope is closely correlated with but not identical to the DRw53 allo-specificity. However, the frequency of particular conventional Ia allodeterminants, including DRw53, is not significantly elevated in the leukemia group. Considerable evidence supports the conclusion that the high frequency of the 109d6 epitope reflects an inherited basis for susceptibility to the development of acute myelogenous leukemia and not a differentiation event occurring in the leukemic lineage. The 109d6 determinant is expressed by leukemic myeloblasts as well as by homologous normal B cells and monocytes obtained from the same individuals during remission of the leukemia. Furthermore, in healthy family members the 109d6 epitope is encoded by Ia haplotypes that are shared with the patient. Of special interest, certain of these haplotypes have combinations of the 109d6 epitope and Ia specificities not commonly seen in normal individuals; here, also, healthy family members share these haplotypes. PMID:2414320

  7. A new Leishmania-specific hypothetical protein and its non-described specific B cell conformational epitope applied in the serodiagnosis of canine visceral leishmaniasis.

    PubMed

    Lage, Daniela P; Martins, Vívian T; Duarte, Mariana C; Costa, Lourena E; Garde, Esther; Dimer, Laura M; Kursancew, Amanda C S; Chávez-Fumagalli, Miguel A; de Magalhães-Soares, Danielle F; Menezes-Souza, Daniel; Roatt, Bruno M; Machado-de-Ávila, Ricardo A; Soto, Manuel; Tavares, Carlos A P; Coelho, Eduardo A F

    2016-04-01

    The serodiagnosis of canine visceral leishmaniasis (CVL) presents problems related to its sensitivity and/or specificity. In the present study, a new Leishmania-specific hypothetical protein, LiHyD, was produced as a recombinant protein (rLiHyD) and evaluated in ELISA experiments for the CVL serodiagnosis. LiHyD was characterized as antigenic in a recent immunoproteomic search performed with Leishmania infantum proteins and the sera of dogs developing visceral leishmaniasis (VL). Aiming to compare the efficacy between whole proteins and synthetic peptides, two linear and one conformational B cell epitopes of LiHyD were synthesized and also evaluated as diagnostic markers. The four antigens were recognized by the sera of dogs suffering VL. On the contrary, low reactivity was observed when they were assayed with sera from non-infected healthy dogs living in endemic or non-endemic areas of leishmaniasis. In addition, no reactivity was found against them using sera from dogs experimentally infected by Trypanosoma cruzi, Babesia canis, or Ehrlichia canis, or sera from animals vaccinated with the Leish-Tec® vaccine, a prophylactic preparation commercially available for CVL prevention in Brazil. As comparative diagnostic tools, a recombinant version of the amastigote-specific A2 protein and a soluble crude Leishmania extract were studied. Both antigens presented lower sensitivity and/or specificity values than the LiHyD-based products. The rLiHyD presented better results for the CVL serodiagnosis than its linear epitopes, although the peptide recreating the conformational epitope resulted also appropriate as a diagnostic marker of CVL. To the best of our knowledge, this is the first study showing the use of a conformational epitope derived from a Leishmania protein for serodiagnosis of CVL. PMID:26782811

  8. A Conformational Switch in Human Immunodeficiency Virus gp41 Revealed by the Structures of Overlapping Epitopes Recognized by Neutralizing Antibodies ▿

    PubMed Central

    Pejchal, Robert; Gach, Johannes S.; Brunel, Florence M.; Cardoso, Rosa M.; Stanfield, Robyn L.; Dawson, Philip E.; Burton, Dennis R.; Zwick, Michael B.; Wilson, Ian A.

    2009-01-01

    The membrane-proximal external region (MPER) of the human immunodeficiency virus (HIV) envelope glycoprotein (gp41) is critical for viral fusion and infectivity and is the target of three of the five known broadly neutralizing HIV type 1 (HIV-1) antibodies, 2F5, Z13, and 4E10. Here, we report the crystal structure of the Fab fragment of Z13e1, an affinity-enhanced variant of monoclonal antibody Z13, in complex with a 12-residue peptide corresponding to the core epitope (W670NWFDITN677) at 1.8-Å resolution. The bound peptide adopts an S-shaped conformation composed of two tandem, perpendicular helical turns. This conformation differs strikingly from the α-helical structure adopted by an overlapping MPER peptide bound to 4E10. Z13e1 binds to an elbow in the MPER at the membrane interface, making relatively few interactions with conserved aromatics (Trp672 and Phe673) that are critical for 4E10 recognition. The comparison of the Z13e1 and 4E10 epitope structures reveals a conformational switch such that neutralization can occur by the recognition of the different conformations and faces of the largely amphipathic MPER. The Z13e1 structure provides significant new insights into the dynamic nature of the MPER, which likely is critical for membrane fusion, and it has significant implications for mechanisms of HIV-1 neutralization by MPER antibodies and for the design of HIV-1 immunogens. PMID:19515770

  9. Genotyping of Enteric Adenoviruses by Using Single-Stranded Conformation Polymorphism Analysis and Heteroduplex Mobility Assay

    PubMed Central

    Soares, Caroline C.; Volotão, Eduardo M.; Albuquerque, Maria Carolina M.; Nozawa, Carlos M.; Linhares, Rosa Elisa C.; Volokhov, Dmitriy; Chizhikov, Vladimir; Lu, Xiaoyan; Erdman, Dean; Santos, Norma

    2004-01-01

    Single-stranded conformation polymorphism (SSCP) analysis and heteroduplex mobility assays (HMAs) were used to identify and genotype enteric adenoviruses (EAd). The results were compared to those of restriction endonuclease assays, species-specific PCRs, and direct nucleotide sequence analyses. Of the 31 stool samples tested, 15 isolates were identified as EAd and 7 were identified as nonenteric Ad by all methods. An agreement of 100% was found between the SSCP and HMA results. PMID:15071032

  10. Generation of Neutralizing Monoclonal Antibodies against a Conformational Epitope of Human Adenovirus Type 7 (HAdv-7) Incorporated in Capsid Encoded in a HAdv-3-Based Vector

    PubMed Central

    Li, Xiao; Zhou, Zhichao; Li, Chenyang; Zhou, Rong

    2014-01-01

    The generation of monoclonal antibodies (MAbs) by epitope-based immunization is difficult because the immunogenicity of simple peptides is poor and T cells must be potently stimulated and immunological memory elicited. A strategy in which antigen is incorporated into the adenoviral capsid protein has been used previously to develop antibody responses against several vaccine targets and may offer a solution to this problem. In this study, we used a similar strategy to develop HAdv-7-neutralizing MAbs using rAdMHE3 virions into which hexon hypervariable region 5 (HVR5) of adenovirus type 7 (HAdv-7) was incorporated. The epitope mutant rAdMHE3 was generated by replacing HVR5 of Ad3EGFP, a recombinant HAdv-3-based vector expressing enhanced green fluorescence protein, with HVR5 of HAdv-7. We immunized BALB/c mice with rAdMHE3 virions and produced 22 different MAbs against them, four of which showed neutralizing activity against HAdv-7 in vitro. Using an indirect enzyme-linked immunosorbent assay (ELISA) analysis and an antibody-binding-competition ELISA with Ad3EGFP, HAdv-7, and a series of chimeric adenoviral particles containing epitope mutants, we demonstrated that the four MAbs recognize the neutralization site within HVR5 of the HAdv-7 virion. Using an immunoblotting analysis and ELISA with HAdv-7, recombinant peptides, and a synthetic peptide, we also showed that the neutralizing epitope within HVR5 of the HAdv-7 virion is a conformational epitope. These findings suggest that it is feasible to use a strategy in which antigen is incorporated into the adenoviral capsid protein to generate neutralizing MAbs. This strategy may also be useful for developing therapeutic neutralizing MAbs and designing recombinant vector vaccines against HAdv-7, and in structural analysis of adenoviruses. PMID:25054273

  11. Identification of a hexapeptide that mimics a conformation-dependent binding site of acetylcholine receptor by use of a phage-epitope library.

    PubMed Central

    Balass, M; Heldman, Y; Cabilly, S; Givol, D; Katchalski-Katzir, E; Fuchs, S

    1993-01-01

    Monoclonal antibody (mAb) 5.5 is directed against the ligand-binding site of the nicotinic acetylcholine receptor. The epitope for this antibody is conformation-dependent, and the antibody does not react with synthetic peptides derived from the receptor sequence. We have identified a ligand peptide that mimics this conformation-dependent epitope from a phage-epitope library composed of filamentous phage displaying random hexapeptides. Among 38 positive phage clones, individually selected from the library, 34 positive clones carried the sequence Asp-Leu-Val-Trp-Leu-Leu (DLVWLL), 1 positive clone had the sequence Asp-Ile-Val-Trp-Leu-Leu (DIVWLL), and 3 positive clones expressed the sequence Leu-Ile-Glu-Trp-Leu-Leu (LIEWLL), none of which are significantly homologous with the nicotinic acetylcholine receptor alpha subunit sequence. All of these phages bind specifically to mAb 5.5. The synthetic peptide DLVWLL inhibits binding of mAb 5.5 to the related peptide-presenting phage and to the nicotinic acetylcholine receptor in a concentration-dependent manner; the IC50 value is of the order of 10(-4) M. Bioactivity of the peptide "mimotope" DLVWLL was demonstrated in vivo in hatched chickens by inhibition of the mAb 5.5 effect by the peptide. The neuromuscular block and myasthenia gravis-like symptoms that are induced in chicken by passive transfer of mAb 5.5 were specifically abolished by DLVWLL. This study shows the potential of a random peptide phage-epitope library for selecting a mimotope for an antibody that recognizes a folded form of the protein, where peptides from the linear amino acid sequence of the protein are not applicable. Images Fig. 5 PMID:7504273

  12. Molecular typing of isolates of the fish pathogen, Flavobacterium columnare, by single-strand conformation polymorphism analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavobacterium columnare intraspecies diversity was revealed by analyzing the 16S rRNA gene and the 16S-23S internal spacer region (ISR). Standard restriction fragment length polymorphism (RFLP) of these sequences was compared with single strand conformation polymorphism (SSCP). Diversity indexes sh...

  13. Polymorphism versus thermochromism: interrelation of color and conformation in overcrowded bistricyclic aromatic enes.

    PubMed

    Biedermann, P Ulrich; Stezowski, John J; Agranat, Israel

    2006-04-12

    The nature of the thermochromic form of overcrowded bistricyclic aromatic enes (BAEs) has been controversial for a century. We report the single-crystal X-ray structure analysis of the deep-purple and yellow polymorphs of 9-(2,7-dimethyl-9H-fluoren-9-ylidene)-9H-xanthene (11), which revealed the molecules in a twisted and a folded conformation, respectively. Therefore, the deeply colored thermochromic form B of BAEs is identified as having a twisted conformation and the ambient-temperature form A as having a folded conformation. This relationship between the color and the conformation is further supported by the X-ray structures of the deep-purple crystals of the twisted 9-(9H-fluoren-9-ylidene)-9H-xanthene (10), and of the yellow crystals of the folded 9-(11H-benzo[b]fluoren-11-ylidene)-9H-xanthene (12). Based on this conclusive crystallographic evidence, eleven previously proposed rationales of thermochromism in BAEs are refuted. In the twisted structures, the tricyclic moieties are nearly planar and the central double bond is elongated to 1.40 A and twisted by 42 degrees . In the folded structures, the xanthylidene moieties are folded by 45 degrees and the fluorenylidene moieties by 18-20 degrees . Factors stabilizing the twisted and folded conformations are discussed, including twisting of formal single or double bonds, intramolecular overcrowding, and the significance of a dipolar aromatic "xanthenylium-fluorenide" push-pull structure. PMID:16440387

  14. Identification of one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of porcine circovirus type 2

    PubMed Central

    2011-01-01

    Background Porcine circovirus type 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome (PMWS) in pigs. Currently, there is considerable interest in the immunology of PCV2; in particular, the immunological properties of the capsid protein. This protein is involved in PCV2 immunogenicity and is a potential target for vaccine development. In this study, we identified one critical amino acid that determines a conformational neutralizing epitope in the capsid protein of PCV2. Results One monoclonal antibody (mAb; 8E4), against the capsid protein of PCV2, was generated and characterized in this study. 8E4 reacted with the genotype PCV2a (CL, LG and JF2) strains but not PCV2b (YJ, SH and JF) strains by an immunoperoxidase monolayer assay (IPMA) and a capture ELISA. Furthermore, the mAb had the capacity to neutralize PCV2a (CL, LG and JF2) strains but not PCV2b (YJ, SH and JF) strains. One critical amino acid that determined a conformational neutralizing epitope was identified using mAb 8E4 and PCV2 infectious clone technique. Amino acid residues 47-72 in the capsid protein of PCV2a/CL were replaced with the corresponding region of PCV2b/YJ, and the reactivity of mAb 8E4 was lost. Further experiments demonstrated that one amino acid substitution, the alanine for arginine at position 59 (A59R) in the capsid protein of PCV2a (CL, LG and JF2) strains, inhibited completely the immunoreactivity of three PCV2a strains with mAb 8E4. Conclusions It is concluded that the alanine at position 59 in the capsid protein of PCV2a (CL, LG and JF2) strains is a critical amino acid, which determines one neutralizing epitope of PCV2a (CL, LG and JF2) strains. This study provides valuable information for further in-depth mapping of the conformational neutralizing epitope, understanding antigenic difference among PCV2 strains, and development of a useful vaccine for control of PCV2-associated disease. PMID:21859462

  15. Broadly neutralizing HIV antibodies define a glycan-dependent epitope on the pre-fusion conformation of the gp41 protein on cleaved Envelope trimers

    PubMed Central

    Falkowska, Emilia; Le, Khoa M.; Ramos, Alejandra; Doores, Katie J.; Lee, Jeong Hyun; Blattner, Claudia; Ramirez, Alejandro; Derking, Ronald; van Gils, Marit J.; Liang, Chi-Hui; Mcbride, Ryan; von Bredow, Benjamin; Shivatare, Sachin S.; Wu, Chung-Yi; Chan-Hui, Po-Ying; Liu, Yan; Feizi, Ten; Zwick, Michael B.; Koff, Wayne C.; Seaman, Michael S.; Swiderek, Kristine; Moore, John P.; Evans, David; Paulson, James C.; Wong, Chi-Huey; Ward, Andrew B.; Wilson, Ian A.; Sanders, Rogier W.; Poignard, Pascal; Burton, Dennis R.

    2014-01-01

    SUMMARY Broadly neutralizing antibodies to HIV are much sought-after (a) to guide vaccine design, both as templates and to inform on the authenticity of vaccine candidates, (b) to assist in structural studies and (c) as potential therapeutics. However, the number of targets on the viral envelope spike for such antibodies is limited. Here, we describe a set of human monoclonal antibodies that define a previously undefined target on HIV Env. The antibodies recognize a glycan-dependent epitope on the prefusion conformation of gp41 and unambiguously distinguish cleaved from uncleaved Env trimers, an important property given increasing evidence that cleavage is required for vaccine candidates that seek to mimic the functional HIV envelope spike. The availability of this set of antibodies expands the number of vaccine targets on HIV and provides reagents to characterize the native envelope spike. PMID:24768347

  16. Conformational polymorphism in a Schiff-base macrocyclic organic ligand: an experimental and theoretical study.

    PubMed

    Lo Presti, Leonardo; Soave, Raffaella; Longhi, Mariangela; Ortoleva, Emanuele

    2010-10-01

    Polymorphism in the highly flexible organic Schiff-base macrocycle ligand 3,6,9,17,20,23-hexa-azapentacyclo(23.3.1.1(11,15).0(2,6).0(16,20))triaconta-1(29),9,11,13,15(30),23,25,27-octaene (DIEN, C(24)H(30)N(6)) has been studied by single-crystal X-ray diffraction and both solid-state and gas-phase density functional theory (DFT) calculations. In the literature, only solvated structures of the title compound are known. Two new polymorphs and a new solvated form of DIEN, all obtained from the same solvent with different crystallization conditions, are presented for the first time. They all have P\\bar 1 symmetry, with the macrocycle positioned on inversion centres. The two unsolvated polymorphic forms differ in the number of molecules in the asymmetric unit Z', density and cohesive energy. Theoretical results confirm that the most stable form is (II°), with Z' = 1.5. Two distinct molecular conformations have been found, named `endo' or `exo' according to the orientation of the imine N atoms, which can be directed towards the interior or the exterior of the macrocycle. The endo arrangement is ubiquitous in the solid state and is shared by two independent molecules which constitute an invariant supramolecular synthon in all the known crystal forms of DIEN. It is also the most stable arrangement in the gas phase. The exo form, on the other hand, appears only in phase (II°), which contains both the conformers. Similarities and differences among the occurring packing motifs, as well as solvent effects, are discussed with the aid of Hirshfeld surface fingerprint plots and correlated to the results of the energy analysis. A possible interconversion path in the gas phase between the endo and the exo conformers has been found by DFT calculations; it consists of a two-step mechanism with activation energies of the order of 30-40 kJ mol(-1). These findings have been related to the empirical evidence that the most stable phase (II°) is also the last appearing one, in

  17. Self-assembled monolayers of shape-persistent macrocycles on graphite: interior design and conformational polymorphism

    PubMed Central

    Vollmeyer, Joscha; Eberhagen, Friederike; Höger, Sigurd

    2014-01-01

    Summary Three shape-persistent naphthylene–phenylene–acetylene macrocycles of identical backbone structures and extraannular substitution patterns but different (empty, apolar, polar) nanopore fillings are self-assembled at the solid/liquid interface of highly oriented pyrolytic graphite and 1,2,4-trichlorobenzene. Submolecularly resolved images of the resulting two-dimensional (2D) crystalline monolayer patterns are obtained by in situ scanning tunneling microscopy. A concentration-dependent conformational polymorphism is found, and open and more dense packing motifs are observed. For all three compounds alike lattice parameters are found, therefore the intermolecular macrocycle distances are mainly determined by their size and symmetry. This is an excellent example that the graphite acts as a template for the macrocycle organization independent from their specific interior. PMID:25550743

  18. Capillary Electrophoresis Single-Strand Conformational Polymorphisms as a Method to Differentiate Algal Species.

    PubMed

    Jernigan, Alice; Hestekin, Christa

    2015-01-01

    Capillary electrophoresis single-strand conformational polymorphism (CE-SSCP) was explored as a fast and inexpensive method to differentiate both prokaryotic (blue-green) and eukaryotic (green and brown) algae. A selection of two blue-green algae (Nostoc muscorum and Anabaena inaequalis), five green algae (Chlorella vulgaris, Oedogonium foveolatum, Mougeotia sp., Scenedesmus quadricauda, and Ulothrix fimbriata), and one brown algae (Ectocarpus sp.) were examined and CE-SSCP electropherogram "fingerprints" were compared to each other for two variable regions of either the 16S or 18S rDNA gene. The electropherogram patterns were remarkably stable and consistent for each particular species. The patterns were unique to each species, although some common features were observed between the different types of algae. CE-SSCP could be a useful method for monitoring changes in an algae species over time as potential shifts in species occurred. PMID:26101693

  19. Capillary Electrophoresis Single-Strand Conformational Polymorphisms as a Method to Differentiate Algal Species

    PubMed Central

    Jernigan, Alice; Hestekin, Christa

    2015-01-01

    Capillary electrophoresis single-strand conformational polymorphism (CE-SSCP) was explored as a fast and inexpensive method to differentiate both prokaryotic (blue-green) and eukaryotic (green and brown) algae. A selection of two blue-green algae (Nostoc muscorum and Anabaena inaequalis), five green algae (Chlorella vulgaris, Oedogonium foveolatum, Mougeotia sp., Scenedesmus quadricauda, and Ulothrix fimbriata), and one brown algae (Ectocarpus sp.) were examined and CE-SSCP electropherogram “fingerprints” were compared to each other for two variable regions of either the 16S or 18S rDNA gene. The electropherogram patterns were remarkably stable and consistent for each particular species. The patterns were unique to each species, although some common features were observed between the different types of algae. CE-SSCP could be a useful method for monitoring changes in an algae species over time as potential shifts in species occurred. PMID:26101693

  20. Aeromonas detection and characterization using genus-specific PCR and single-strand conformation polymorphism (SSCP).

    PubMed

    Delamare, Ana Paula Longaray; Lucena, Roberto Francisco; Thomazi, Guilherme; Ferrarini, Shana; Zacaria, Jucimar; Echeverrigaray, Sergio

    2012-10-01

    Based on sequence alignment, oligonucleotide primers targeting the Aeromonas extracellular lipase gene were developed for PCR detection of member of the genus. A pair of primers designed for conserved regions of the gene amplified a 276 bp sequence in all Aeromonas species and tested strains, but did not have a positive result with other Gram-positive and Gram-negative bacteria, showing high specificity and sensitivity. Selective enrichment in alkaline peptone water, followed by centrifugation, and direct usage of cells suspension as template, detected initial populations of 10 c.f.u. ml⁻¹. Single-strand conformation polymorphism analysis of the PCR products allowed the characterization of Aeromonas strains with a high discriminatory power (Simpson's index = 0.988). The method presented here provides a useful tool for the rapid detection of Aeromonas and the characterization of Aeromonas isolates. PMID:22806741

  1. Molecular identification of Amazonian stingless bees using polymerase chain reaction single-strand conformation polymorphism.

    PubMed

    Souza, M T; Carvalho-Zilse, G A

    2014-01-01

    In countries containing a mega diversity of wildlife, such as Brazil, identifying and characterizing biological diversity is a continuous process for the scientific community, even in face of technological and scientific advances. This activity demands initiatives for the taxonomic identification of highly diverse groups, such as stingless bees, including molecular analysis strategies. This type of bee is distributed in all of the Brazilian states, with the highest species diversity being found in the State of Amazônia. However, the estimated number of species diverges among taxonomists. These bees are considered the main pollinators in the Amazon rainforest, in which they obtain food and shelter; however, their persistence is constantly threatened by deforestation pressure. Hence, it is important to classify the number and abundance of bee specie, to measure their decline and implement meaningful, priority conservation strategies. This study aims to maximize the implementation of more direct, economic and successful techniques for the taxonomic identification of stingless bees. Specifically, the genes 16S rRNA and COI from mitochondrial DNA were used as molecular markers to differentiate 9 species of Amazonian stingless bees based on DNA polymorphism, using the polymerase chain reaction-single-strand conformation polymorphism technique. We registered different, exclusive SSCP haplotypes for both genes in all species analyzed. These results demonstrate that SSCP is a simple and cost-effective technique that is applicable to the molecular identification of stingless bee species. PMID:25117306

  2. Identification and differentiation of Cryptosporidium species by capillary electrophoresis single-strand conformation polymorphism.

    PubMed

    Power, Michelle L; Holley, Marita; Ryan, Una M; Worden, Paul; Gillings, Michael R

    2011-01-01

    Cryptosporidium species generally lack distinguishing morphological traits, and consequently, molecular methods are commonly used for parasite identification. Various methods for Cryptosporidium identification have been proposed, each with their advantages and disadvantages. In this study, we show that capillary electrophoresis coupled with single-strand conformation polymorphism (CE-SSCP) is a rapid, simple and cost-effective method for the identification of Cryptosporidium species and genotypes. Species could be readily differentiated based on the SSCP mobility of amplified 18S rRNA gene molecules. Clones that differed by single-nucleotide polymorphisms could be distinguished on CE-SSCP mobility. Profiles of species known to have heterogenic copies of 18S rRNA gene contained multiple peaks. Cloning and sequencing of Cryptosporidium parvum, Cryptosporidium hominis, Cryptosporidium fayeri and Cryptosporidium possum genotype 18S rRNA gene amplicons confirmed that these multiple peaks represented type A and type B 18S rRNA gene copies. CE-SSCP provides a reliable and sensitive analysis for epidemiological studies, environmental detection and diversity screening. PMID:21087296

  3. Rapid and sensitive genotyping of hepatitis C virus by single-strand conformation polymorphism.

    PubMed

    Lareu, R R; Swanson, N R; Fox, S A

    1997-02-01

    There is an increasing demand for genotyping hepatitis C virus (HCV) isolates due to the rapidly expanding list of distinct HCV genotypes and the mounting evidence of genotype-specific clinical consequences. We describe an SSCP-based assay for determining genotypes in HCV infections. HCV RNA extracted from serum was amplified by a sensitive nested-PCR assay producing a 287 bp fragment of the conserved 5' non-coding region (NCR) and analysed by non-denaturing polyacrylamide gel electrophoresis. Following empirical optimisation of the SSCP assay we identified distinct conformation polymorphisms (characteristic band patterns) corresponding to types 1a, 1b, 2a, 2b, 2c, 3 and 4 found in the Western Australian population. Seventy-three HCV RNA-positive samples were used to evaluate the SSCP genotyping assay for accuracy and efficiency by comparison with the previously established genotyping methods of manual direct sequencing and dideoxy fingerprinting. SSCP genotyping was in concord with control methods while performing more rapidly and at a fraction of the cost. Moreover, SSCP detected two co-infected samples that were not shown by the control methods. The PCR-SSCP assay provides an accurate and rapid method for genotyping of HCV RNA-positive samples at the 5' NCR by type-specific sequence polymorphisms which is applicable to large-scale screening. PMID:9029525

  4. Characterization of Citrus Tristeza Virus Isolates by Single-strand Conformation Polymorphism Analysis of the Coat Protein Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A method is needed to rapidly assess Citrus tristeza virus (CTV) strains and to identify mixed populations in tristeza-infected trees. Single-strand conformation polymorphism (SSCP) can detect point mutations in DNA fragments and determine the structure of viral populations. Previous reports utili...

  5. Conformational and Linear B-Cell Epitopes of Asp f 2, a Major Allergen of Aspergillus fumigatus, Bind Differently to Immunoglobulin E Antibody in the Sera of Allergic Bronchopulmonary Aspergillosis Patients

    PubMed Central

    Banerjee, Banani; Greenberger, Paul A.; Fink, Jordan N.; Kurup, Viswanath P.

    1999-01-01

    Asp f 2 is a major Aspergillus fumigatus allergen involved in allergic bronchopulmonary aspergillosis. Knowledge of the B-cell epitopes may contribute to the understanding of immunoregulation and immunodiagnosis. To elucidate the immunoglobulin E (IgE) binding epitopes in the linear sequence of Asp f 2, we synthesized decamer peptides spanning the whole molecule of Asp f 2 on derivatized cellulose membranes and evaluated IgE binding in ABPA patient and control sera. Peptides three to five amino acids long were synthesized based on amino acid sequences within the IgE binding regions and evaluated for the specificity of epitope antibody interactions. Nine IgE binding regions were recognized in this protein of 268 amino acid residues. Of the nine epitopes, seven (ATQRRQI, RKYFG, HWR, YTTRR, DHFAD, ALEAYA, and THEGGQ) are present in the hydrophilic regions of Asp f 2. Immunologic evaluation of the three recombinant fragments, Asp f 2A encompassing the N-terminal epitope region, Asp f 2B without N- and C-terminal regions of the protein, and Asp f 2C representing C-terminal epitopes, revealed that either the N- or C-terminal region of the protein is essential for the correct folding and conformation for IgE antibody binding. PMID:10225885

  6. Conformational Switching and Nanoscale Assembly of Human Prion Protein into Polymorphic Amyloids via Structurally Labile Oligomers.

    PubMed

    Dalal, Vijit; Arya, Shruti; Bhattacharya, Mily; Mukhopadhyay, Samrat

    2015-12-29

    Conformational switching of the prion protein (PrP) from an α-helical normal cellular form (PrP(C)) to an aggregation-prone and self-propagating β-rich scrapie form (PrP(Sc)) underlies the molecular basis of pathogenesis in prion diseases. Anionic lipids play a critical role in the misfolding and conformational conversion of the membrane-anchored PrP into the amyloidogenic pathological form. In this work, we have used a diverse array of techniques to interrogate the early intermediates during amyloid formation from recombinant human PrP in the presence of a membrane mimetic anionic detergent such as sodium dodecyl sulfate. We have been able to detect and characterize two distinct types of interconvertible oligomers. Our results demonstrate that highly ordered large β-oligomers represent benign off-pathway intermediates that lack the ability to mature into amyloid fibrils. On the contrary, structurally labile small oligomers are capable of switching to an ordered amyloid-state that exhibits profound toxicity to mammalian cells. Our fluorescence resonance energy transfer measurements revealed that the partially disordered PrP serves as precursors to small amyloid-competent oligomers. These on-pathway oligomers are eventually sequestered into higher order supramolecular assemblies that conformationally mature into polymorphic amyloids possessing varied nanoscale morphology as evident by the atomic force microscopy imaging. The nanoscale diversity of fibril architecture is attributed to the heterogeneous ensemble of early obligatory oligomers and offers a plausible explanation for the existence of multiple prion strains in vivo. PMID:26645611

  7. Characterization of desmoglein-3 epitope region peptides as synthetic antigens: analysis of their in vitro T cell stimulating efficacy, cytotoxicity, stability, and their conformational features.

    PubMed

    Szabados, Hajnalka; Uray, Katalin; Majer, Zsuzsa; Silló, Pálma; Kárpáti, Sarolta; Hudecz, Ferenc; Bősze, Szilvia

    2015-09-01

    Desmoglein-3 (Dsg3) adhesion protein is the main target of autoantibodies and autoreactive T cells in Pemphigus vulgaris (PV) autoimmune skin disorder. Several mapping studies of Dsg3 T cell epitope regions were performed, and based on those data, we designed and synthesized four peptide series corresponding to Dsg3 T cell epitope regions. Each peptide series consists of a 17mer full-length peptide (Dsg3/189-205, Dsg3/206-222, Dsg3/342-358, and Dsg3/761-777) and its N-terminally truncated derivatives, resulting in 15 peptides altogether. The peptides were prepared on solid phase and were chemically characterized. In order to establish a structure-activity relationship, the solution conformation of the synthetic peptides has been investigated using electronic circular dichroism spectroscopy. The in vitro T cell stimulating efficacy of the peptides has been determined on peripheral blood mononuclear cells isolated from whole blood of PV patients and also from healthy donors. After 20 h of stimulation, the interferon (IFN)-γ content of the supernatants was measured by enzyme-linked immunosorbent assay. In the in vitro conditions, peptides were stable and non-cytotoxic. The in vitro IFN-γ production profile of healthy donors and PV patients, induced by peptides as synthetic antigens, was markedly different. The most unambiguous differences were observed after stimulation with 17mer peptide Dsg3/342-358, and three truncated derivatives from two other peptide series, namely, peptides Dsg3/192-205, Dsg3/763-777, and Dsg3/764-777. Comparative analysis of in vitro activity and the capability of oligopeptides to form ordered or unordered secondary structure showed that peptides bearing high solvent sensibility and backbone flexibility were the most capable to distinguish between healthy and PV donors. PMID:26250896

  8. A Cryo-Electron Microscopy Study Identifies the Complete H16.V5 Epitope and Reveals Global Conformational Changes Initiated by Binding of the Neutralizing Antibody Fragment

    PubMed Central

    Lee, Hyunwook; Brendle, Sarah A.; Bywaters, Stephanie M.; Guan, Jian; Ashley, Robert E.; Yoder, Joshua D.; Makhov, Alexander M.; Conway, James F.; Christensen, Neil D.

    2014-01-01

    ABSTRACT Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16, we pursued a structural study to elucidate HPV capsids and antibody interactions. The cryo-electron microscopy (cryo-EM) structures of a mature HPV16 particle and an altered capsid particle were solved individually and as complexes with fragment of antibody (Fab) from the neutralizing antibody H16.V5. Fitted crystal structures provided a pseudoatomic model of the virus-Fab complex, which identified a precise footprint of H16.V5, including previously unrecognized residues. The altered-capsid–Fab complex map showed that binding of the Fab induced significant conformational changes that were not seen in the altered-capsid structure alone. These changes included more ordered surface loops, consolidated so-called “invading-arm” structures, and tighter intercapsomeric connections at the capsid floor. The H16.V5 Fab preferentially bound hexavalent capsomers likely with a stabilizing effect that directly correlated with the number of bound Fabs. Additional cryo-EM reconstructions of the virus-Fab complex for different incubation times and structural analysis provide a model for a hyperstabilization of the capsomer by H16.V5 Fab and showed that the Fab distinguishes subtle differences between antigenic sites. IMPORTANCE Our analysis of the cryo-EM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5 conformational epitope and demonstrated a detailed neutralization mechanism of this clinically important monoclonal antibody against HPV16. The Fab bound and ordered the apical loops of HPV16. This conformational change was transmitted to the lower region of the capsomer, resulting in enhanced intercapsomeric interactions evidenced by the more ordered capsid floor and “invading-arm” structures. This study advances the understanding of the neutralization mechanism used

  9. The alloantigenic sites of alpha3alpha4alpha5(IV) collagen: pathogenic X-linked alport alloantibodies target two accessible conformational epitopes in the alpha5NC1 domain.

    PubMed

    Kang, Jeong Suk; Kashtan, Clifford E; Turner, A Neil; Heidet, Laurence; Hudson, Billy G; Borza, Dorin-Bogdan

    2007-04-01

    Anti-glomerular basement membrane (GBM) antibody nephritis is caused by an autoimmune or alloimmune reaction to the NC1 domains of alpha3alpha4alpha5(IV) collagen. Some patients with X-linked Alport syndrome (XLAS) develop post-transplant nephritis mediated by pathogenic anti-GBM alloantibodies to collagen IV chains present in the renal allograft but absent from the tissues of the patient. In this work, the epitopes targeted by alloantibodies from these patients were identified and characterized. All XLAS alloantibodies recognized conformational epitopes in the NC1 domain of alpha5(IV) collagen, which were mapped using chimeric alpha1/alpha5 NC1 domains expressed in mammalian cells. Allograft-eluted alloantibodies mainly targeted two conformational alloepitopes mapping to alpha5NC1 residues 1-45 and 114-168. These regions also encompassed the major epitopes of circulating XLAS alloantibodies, which in some patients additionally targeted alpha5NC1 residues 169-229. Both kidney-eluted and circulating alloantibodies to alpha5NC1 distinctively targeted epitopes accessible in the alpha3alpha4alpha5NC1 hexamers of human GBM, unlike anti-GBM autoantibodies, which targeted sequestered alpha3NC1 epitopes. The results identify two immunodominant alpha5NC1 epitopes as major alloantigenic sites of alpha3alpha4alpha5(IV) collagen specifically implicated in the pathogenesis of post-transplant nephritis in XLAS patients. The contrast between the accessibility of these alloepitopes and the crypticity of autoepitopes indicates that distinct molecular forms of antigen may initiate the immunopathogenic processes in the two forms of anti-GBM disease. PMID:17293596

  10. Genetic polymorphism of toll-like receptors 4 gene by polymerase chain reaction-restriction fragment length polymorphisms, polymerase chain reaction-single-strand conformational polymorphism to correlate with mastitic cows

    PubMed Central

    Gupta, Pooja H.; Patel, Nirmal A.; Rank, D. N.; Joshi, C. G.

    2015-01-01

    Aim: An attempt has been made to study the toll-like receptors 4 (TLR4) gene polymorphism from cattle DNA to correlate with mastitis cows. Materials and Methods: In present investigation, two fragments of TLR4 gene named T4CRBR1 and T4CRBR2 of a 316 bp and 382 bp were amplified by polymerase chain reaction (PCR), respectively from Kankrej (22) and Triple cross (24) cattle. The genetic polymorphisms in the two populations were detected by a single-strand conformational polymorphism in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results: Results showed that both alleles (A and B) of two loci were found in all the two populations and the value of polymorphism information content indicated that these were highly polymorphic. Statistical results of χ2 test indicated that two polymorphism sites in the two populations fit with Hardy–Weinberg equilibrium (p<0.05). Meanwhile, the effect of polymorphism of TLR4 gene on the somatic cell score (SCS) indicated the cattle with allele a in T4CRBR1 showed lower SCS than that of allele B (p<0.05). Thus, the allele A might play an important role in mastitis resistance in cows. Conclusion: The relationship between the bovine mastitis trait and the polymorphism of TLR4 gene indicated that the bovine TLR4 gene may play an important role in mastitis resistance. PMID:27047144

  11. Radiation-induced radicals in different polymorphic modifications of D-mannitol: Structure, conformations and dosimetric implications

    NASA Astrophysics Data System (ADS)

    Sosulin, Ilya S.; Shiryaeva, Ekaterina S.; Feldman, Vladimir I.

    2015-12-01

    The structure and conformation of radicals produced by X-ray irradiation of three polymorphic forms of D-mannitol were investigated using EPR spectroscopy. In all the cases, primary species were identified as radicals resulting from hydrogen abstraction from position 3 or 4 of the mannitol molecule. It was found that molecular packing in crystals of different polymorphic modifications had noticeable effect on the conformation of radicals observed after irradiation at room temperature and the dehydration of the primary radicals occurring at 400 K. The radicals trapped in stable modifications (β- and δ-forms) were found to be very stable at room temperature. Relatively high radical yields and remarkable stability of radicals suggest that D-mannitol can be used as an EPR dosimeter or irradiation marker.

  12. Color polymorphs of aldose reductase inhibitor epalrestat: configurational, conformational and synthon differences.

    PubMed

    Swapna, Battini; Suresh, Kuthuru; Nangia, Ashwini

    2016-03-01

    We report five crystalline polymorphs and an amorphous phase of epalrestat together with configurational isomerism and color behavior: form I (deep red), form II (deep orange), form III (bright yellow), form IV (yellow), and form V (orange) are in the E,Z configuration of the drug, and a Z,Z isomer (bright yellow). Two pathways are identified for polymorph conversion: direct transformation of the E,Z isomer and another pathway via the Z,Z isomer to the E,Z polymorphs. From a pharmaceutical perspective, the stability of polymorphs was established under grinding, solvent slurry and thermal conditions: form I (thermodynamic) > form II > form V > form III > form IV (least stable). PMID:26889760

  13. Conformational polymorphism of solid tetramesityldisilene Mes2Si=SiMes2 (Raman, UV-vis, IR and fluorescence study).

    PubMed

    Leites, L A; Bukalov, S S; Mangette, J E; Schmedake, T A; West, R

    2003-07-01

    Conformational polymorphism of solid tetramesityldisilene (1) has been studied by the methods of optical spectroscopy. The three known modifications of 1: orange unsolvated 1a and two 1:1 solvates with toluene (1b) and THF (1c) have been found to transform under specific conditions to a new, most thermodynamically stable polymorph, yellow unsolvated powder 1d. The latter has been characterized by the Raman, IR, UV-vis and fluorescence data. All forms of 1 exhibit Raman spectra differing in details, which reflect their different crystal and molecular structures. Unsolvated 1a and 1d differ significantly in electronic absorption and fluorescence emission. The yellow form 1d can be converted to the orange form 1a upon illumination with laser light in the region 514-457 nm. Similarity of the Raman and UV-vis spectra of 1d to those of the solutions of 1 provides some evidence for a quasi-trans conformation of 1d. PMID:12788451

  14. DNA polymorphism in crystals: three stable conformations for the decadeoxynucleotide d(GCATGCATGC).

    PubMed

    Thirugnanasambandam, Arunachalam; Karthik, Selvam; Artheswari, Gunanithi; Gautham, Namasivayam

    2016-06-01

    High-resolution structures of DNA fragments determined using X-ray crystallography or NMR have provided descriptions of a veritable alphabet of conformations. They have also shown that DNA is a flexible molecule, with some sequences capable of adopting two different structures. Here, the first example is presented of a DNA fragment that can assume three different and distinct conformations in crystals. The decanucleotide d(GCATGCATGC) was previously reported to assume a single-stranded double-fold structure. In one of the two crystal structures described here the decamer assumes both the double-fold conformation and, simultaneously, the more conventional B-type double-helical structure. In the other crystal the sequence assumes the A-type double-helical conformation. These results, taken together with CD spectra, which were recorded as the decamer was titrated against four metal ions and spermine, indicate that the molecule may exist as a mixed population of structures in solution. Small differences in the environmental conditions, such as the concentration of metal ion, may decide which of these crystallizes out. The results also support the idea that it may be possible for DNA to change its structure to suit the binding requirements of proteins or drugs. PMID:27303798

  15. Comparison of rotation models for describing DNA conformations: application to static and polymorphic forms.

    PubMed Central

    Mazur, J; Jernigan, R L

    1995-01-01

    A new method, based on a space-fixed rotation axis, or local helix axis, is proposed for the calculation of the relative orientation variables for a sequence of base pairs. With this method, orientation variables are determined through the rotation of a base pair about this axis. These variables uniquely determine a set of helical variables, similar to the roll, tilt, and twist, commonly used for a description of spatial orientations of internally rigid base pairs. The proposed identification of roll and tilt with the direction cosines of the space-fixed rotation axis agrees well with their customary definitions as the openings of the angles between adjoining base pairs toward the minor groove and toward the ascending (5' to 3') backbone strand, respectively. These new variables permit a more direct physical comprehension of DNA conformations and also the behavior of self-complementary sequences. These direction cosines, together with the rotation angle about the space-fixed axis, form a set of three independent orientation variables of the bases that afford some advantages over the variously defined twist, roll, and tilt angles, either for static or average forms. An example for the static form of these variables is shown through their use to interpret crystal coordinates. An example for the average of orientation variables is based on statistical calculations. In this example, the orientation variables, together with the translational variables that describe the relative displacements of a pair of adjacent base pairs, form a canonically distributed ensemble in phase space spanned by these variables. Two sets of conformational variables are generated by using two different methods for performing rotation operations on the sequences of base pairs. The first method is based on the new single rotation about a space-fixed axis of rotation. This space-fixed axis of rotation is, in fact, the local helical axis as constructed previously by others. The second method is

  16. Characterisation of CAH alleles with non-radioactive DNA single strand conformation polymorphism analysis of the CYP21 gene.

    PubMed

    Bobba, A; Iolascon, A; Giannattasio, S; Albrizio, M; Sinisi, A; Prisco, F; Schettini, F; Marra, E

    1997-03-01

    The major cause of congenital adrenal hyperplasia (CAH), a common recessive genetic disease, is the deficiency of steroid 21-hydroxylase (21OH), a microsomal enzyme encoded by the CYP21 gene. Although several CAH causing mutations have been identified in the CYP21 gene of patients with 21OH deficiency, genotyping of the 21OH locus is quite complex because of the high frequency of gene conversion and the presence of multiple mutations on single CAH alleles. In order to perform the complete characterisation of the CYP21 gene coding region more simply, we developed a highly sensitive, non-radioactive method allowing DNA single strand conformation polymorphism (DNA-SSCP) analysis. This method was applied to the characterisation of all the exons and intron-exon junctions of the CYP21 gene in five patients affected by the simple virilising form and one affected by the salt wasting form. In all samples showing SSCP signals, direct sequence analysis showed the presence of more than one single sequence variant. In particular, four mutations which are already known to cause the disease, 16 polymorphisms, and one newly identified C to T transition at position 849 were detected. A random sequence analysis, performed on 31 out of 81 exons showing a normal SSCP pattern, shows the method to be highly sensitive: no sequence variant was detected, thus confirming the validity of this non-radioactive DNA-SSCP analysis in characterising the CYP21 gene in patients with steroid 21OH deficiency. Notwithstanding the complete characterisation of all exons and exon/intron junctions of the CYP21 gene, no complete genotype/phenotype correlation was found in the panel of patients analysed, thus suggesting that characterisation of CAH alleles must be extended to outside the coding region of the CYP21 gene, most probably into the promoter region. PMID:9132494

  17. Characterisation of CAH alleles with non-radioactive DNA single strand conformation polymorphism analysis of the CYP21 gene.

    PubMed Central

    Bobba, A; Iolascon, A; Giannattasio, S; Albrizio, M; Sinisi, A; Prisco, F; Schettini, F; Marra, E

    1997-01-01

    The major cause of congenital adrenal hyperplasia (CAH), a common recessive genetic disease, is the deficiency of steroid 21-hydroxylase (21OH), a microsomal enzyme encoded by the CYP21 gene. Although several CAH causing mutations have been identified in the CYP21 gene of patients with 21OH deficiency, genotyping of the 21OH locus is quite complex because of the high frequency of gene conversion and the presence of multiple mutations on single CAH alleles. In order to perform the complete characterisation of the CYP21 gene coding region more simply, we developed a highly sensitive, non-radioactive method allowing DNA single strand conformation polymorphism (DNA-SSCP) analysis. This method was applied to the characterisation of all the exons and intron-exon junctions of the CYP21 gene in five patients affected by the simple virilising form and one affected by the salt wasting form. In all samples showing SSCP signals, direct sequence analysis showed the presence of more than one single sequence variant. In particular, four mutations which are already known to cause the disease, 16 polymorphisms, and one newly identified C to T transition at position 849 were detected. A random sequence analysis, performed on 31 out of 81 exons showing a normal SSCP pattern, shows the method to be highly sensitive: no sequence variant was detected, thus confirming the validity of this non-radioactive DNA-SSCP analysis in characterising the CYP21 gene in patients with steroid 21OH deficiency. Notwithstanding the complete characterisation of all exons and exon/intron junctions of the CYP21 gene, no complete genotype/phenotype correlation was found in the panel of patients analysed, thus suggesting that characterisation of CAH alleles must be extended to outside the coding region of the CYP21 gene, most probably into the promoter region. Images PMID:9132494

  18. A conformational epitope mapped in the bovine herpesvirus type 1 envelope glycoprotein B by phage display and the HSV-1 3D structure.

    PubMed

    Almeida, Greyciele R; Goulart, Luiz Ricardo; Cunha-Junior, Jair P; Bataus, Luiz A M; Japolla, Greice; Brito, Wilia M E D; Campos, Ivan T N; Ribeiro, Cristina; Souza, Guilherme R L

    2015-08-01

    The selected dodecapeptide (1)DRALYGPTVIDH(12) from a phage-displayed peptide library and the crystal structure of the envelope glycoprotein B (Env gB) from Herpes Simplex Virus type 1 (HSV-1) led us to the identification of a new discontinuous epitope on the Bovine herpesvirus type 1 (BoHV-1) Env gB. In silico analysis revealed a short BoHV-1 gB motif ((338)YKRD(341)) within a epitope region, with a high similarity to the motifs shared by the dodecapeptide N-terminal region ((5)YxARD(1)) and HSV-1 Env gB ((326)YARD(329)), in which the (328)Arg residue is described to be a neutralizing antibody target. Besides the characterization of an antibody-binding site of the BoHV-1 Env gB, we have demonstrated that the phage-fused peptide has the potential to be used as a reagent for virus diagnosis by phage-ELISA assay, which discriminated BoHV-1 infected serum samples from negative ones. PMID:26267086

  19. Solution structure of Atg8 reveals conformational polymorphism of the N-terminal domain

    SciTech Connect

    Schwarten, Melanie; Stoldt, Matthias; Mohrlueder, Jeannine; Willbold, Dieter

    2010-05-07

    During autophagy a crescent shaped like membrane is formed, which engulfs the material that is to be degraded. This membrane grows further until its edges fuse to form the double membrane covered autophagosome. Atg8 is a protein, which is required for this initial step of autophagy. Therefore, a multistage conjugation process of newly synthesized Atg8 to phosphatidylethanolamine is of critical importance. Here we present the high resolution structure of unprocessed Atg8 determined by nuclear magnetic resonance spectroscopy. Its C-terminal subdomain shows a well-defined ubiquitin-like fold with slightly elevated mobility in the pico- to nanosecond timescale as determined by heteronuclear NOE data. In comparison to unprocessed Atg8, cleaved Atg8{sup G116} shows a decreased mobility behaviour. The N-terminal domain adopts different conformations within the micro- to millisecond timescale. The possible biological relevance of the differences in dynamic behaviours between both subdomains as well as between the cleaved and uncleaved forms is discussed.

  20. Polymorphic Ab protofilaments exhibit distinct conformational dynamics as calculated by normal mode analysis

    NASA Astrophysics Data System (ADS)

    Armbruster, Matthew; Soto, Patricia

    2012-02-01

    This project proposes to test the hypothesis that the physicochemical milieu modulates the conformational dynamics of synthetic Alzheimer's Ab protofilament structures, the main component of Alzheimer's senile plaques. To this end, 3D solid-state NMR structures of Ab protofilaments were used as initial structures for molecular dynamics simulations in explicit water and a water/hexane environment. The initial structures of the simulations and representative structures from the simulation-generated trajectories were taken to perform computational normal mode analysis. We developed a code in python with a graphical user-friendly interface. The program incorporated the ProDy (0.7.1) package. With the application, we examined cross-correlation plots of Ca positions of the 2-fold Ab protofilaments along the most collective mode and the slowest mode. The protofilament structures were highly correlated in the water environment. We hypothesized the protofilament would move as one in water because of the viscosity. The square fluctuation of Ca positions was calculated for the slowest mode for the hexane model and the MD generated ensemble. The two plots match up until midway through the structure. At the midway point a phase shift emerged between the two structures most likely where the surrounding changes. The in-house developed code made it easy to perform analysis and will be used by other students in the research group.

  1. [Microbial community structure in different wastewater treatment processes characterized by single-strand-conformation polymorphism (SSCP) technique].

    PubMed

    Zhao, Yang-guo; Wang, Ai-jie; Ren, Nan-qi; Zhao, Yan

    2006-07-01

    In order to investigate microbial community structures in different wastewater treatment processes and understand the relationship between the structures and the status of processes, the microbial community diversity, variety and distribution in five wastewater treatment processes were studied by a culture-independent genetic fingerprinting technique single-strand-conformation polymorphism (SSCP). The five processes included a denitrification and phosphorus removal bioreactor (N), Chinese traditional medicine wastewater treatment bioreactor (P), beer wastewater treatment bioreactor (W), fermentative biohydrogen production bioreactor (H) and sulfate-reduction bioreactor (S). The results indicate that the microbial community profiles in the same wastewater bioreactors are very similar. The diversity of microbial populations is correlated with the complexity of organic contaminants in wastewater. Chinese traditional medicine wastewater contains more complex organic components, so the population diversity is higher than that of simple nutrient bioreactors fed with molasses wastewater. Compared with the strain bands in a simulated community, the relative proportion of some functional microbial populations in bioreactors was not dominant, namely, fermentative biohydrogen producer Ethanologenbacterium sp. in the better condition bioreactor had only 5% band density, and the Desulfovibrio sp. in the sulfate-reducing bioreactor had less than 1.5% band density. SSCP profiles can define the difference in microbial community structures in wastewater treatment processes and monitor some of the functional microbes in these processes, providing useful guidance for improving its efficiency. PMID:16881324

  2. The application of single strand conformation polymorphism (SSCP) analysis in determining Hepatitis E virus intra-host diversity.

    PubMed

    Černi, S; Prpić, J; Jemeršić, L; Škorić, D

    2015-09-01

    Genetic heterogeneity of RNA populations influences virus pathogenesis, epidemiology and evolution. Therefore, accurate information regarding virus genetic structure is highly important for both diagnostic and scientific purposes. For the Hepatitis E virus (HEV), the causal agent of hepatitis in humans, the intra-host population structure has been poorly investigated, mainly using the less sensitive RFLP-based approach. The objective of this study was to assess the suitability and the accuracy of single strand conformation polymorphism (SSCP) analysis, a well-established tool in genetic variation research, for the characterization of HEV quasispecies. The analysis was conducted on 50 clones of five swine isolates and 30 clones of three human HEV isolates. To identify and quantify the sequence variants present in each HEV isolate, 348bp long fragments of the amplified conserved ORF2 region were separated by cloning. Ten clones per isolate were subjected to SSCP and sequenced in a parallel experiment. The results show a high correlation of SSCP haplotype profiling with the sequencing results, confirming the sensitivity and reliability of this simple, rapid and low cost approach in the characterization of HEV quasispecies. PMID:25920567

  3. The Relationship between B-cell Epitope and Mimotope Sequences.

    PubMed

    Zhang, Chunhua; Li, Yunyun; Tang, Weina; Zhou, Zhiguo; Sun, Pingping; Ma, Zhiqiang

    2016-01-01

    B-cell epitope is a group of residues which is on the surface of an antigen. It invokes humoral responses. Locating B-cell epitope is important for effective vaccine design, and the development of diagnostic reagents. Mimotope-based B-cell epitope prediction method is a kind of conformational B-cell epitope prediction, and the core idea of the method is mapping the mimotope sequences which are obtained from a random phage display library. However, current mimotope-based B-cell epitope prediction methods cannot maintain a high degree of satisfaction in the circumstances of employing only mimotope sequences. In this study, we did a multi-perspective analysis on parameters for conformational B-cell epitopes and characteristics between epitope and mimotope on a benchmark datasets which contains 67 mimotope sets, corresponding to 40 unique complex structures. In these 67 cases, there are 25 antigen-antibody complexes and 42 protein-protein interactions. We analyzed the two parts separately. The results showed the mimotope sequences do have some epitope features, but there are also some epitope properties that mimotope sequences do not contain. In addition, the numbers of epitope segments with different lengths were obviously different between the antigen-antibody complexes and the protein-protein interactions. This study reflects how similar do mimotope sequence and genuine epitopes have; and evaluates existing mimotope-based B-cell epitope prediction methods from a novel viewpoint. PMID:26715528

  4. An examination of polymorphic stability and molecular conformational flexibility as a function of crystal size associated with the nucleation and growth of benzophenone.

    PubMed

    Hammond, Robert B; Pencheva, Klimentina; Roberts, Kevin J

    2007-01-01

    The polymorphic behaviour of the aromatic ketone, benzophenone, which is a conformationally flexible molecule and forms crystal structures dominated by van der Waals intermolecular interactions, is examined. Crystallization of this material from the undercooled molten state yields the two known polymorphic forms, i.e. the stable alpha-form and the metastable beta-form. The relative, energetic stabilities are examined using both crystal lattice and molecular conformational modelling techniques. Examination of nano-sized faceted molecular clusters of these forms, with cluster sizes ranging from 3 to 100 molecules, reveals that at very small cluster size (< 5 molecules) the relative energetic stability of clusters representative for the two forms become very similar, indicating that for high melting undercooling (i.e. small critical cluster size for nucleation) crystallization of the metastable beta-phase becomes more likely. Detailed analysis of the variation in molecular conformations within the simulated molecular clusters reveals more disordered three-dimensional structures at small compared to larger cluster sizes. The conformational disorder was found to be higher for the metastable beta-form. This observation, together with the lower stability of clusters for this form is indicative of the difficulty in achieving crystallization of the metastable beta-form from the melt, which requires a considerable undercooling. PMID:17955805

  5. Conformational polymorphs of isobutyl-6-amino-5-cyano-2-methyl-4-phenyl-4H-pyran-3-carboxylate: spectroscopic, structural and DFT approach.

    PubMed

    Prasad, A Aditya; Kumar, C Udhaya; Prakasam, B Arul; Meenakshisundaram, S P

    2016-06-01

    The crystal structure of a new crystalline phase, polymorph (II) of isobutyl-6-amino-5-cyano-2-methyl-4-phenyl-4H-pyran-3-carboxylate, was accurately determined by single-crystal X-ray diffraction analysis providing a clean identification of polymorphic forms. Comparison with a known phase, referred to as polymorph (I), reveals the type of supramolecular assembly. Inter- and intramolecular hydrogen-bonding interactions exhibit various supramolecular architectures in crystal packing and these variations confirm well the polymorphism in isobutyl-6-amino-5-cyano-2-methyl-4-phenyl-4H-pyran-3-carboxylate (IAPC) crystal structure. Crystal cohesion is achieved by N-H...N, N-H...O and C-H...H-C interactions, responsible for the formation and strengthening of the supramolecular assembly. The objective of this investigation is to study crystalline forms which can offer enhanced physicochemical properties, and also to recognize the molecular orientations between such forms. The conformational polymorphs of IAPC were compared spectroscopically by FT-IR and FT-Raman. The bulk phases were studied by X-ray powder diffraction patterns. External morphology was investigated using scanning electron microscopic images. The molecular interactions were quantified using Hirshfeld surface and fingerprint analysis. Density functional theory (DFT) computations were used to optimize the structure. The optimized structure is further subjected to an analysis of Mulliken population, natural population and electrostatic potential. PMID:27240761

  6. High-resolution, three-dimensional modeling of human leukocyte antigen class I structure and surface electrostatic potential reveals the molecular basis for alloantibody binding epitopes.

    PubMed

    Kosmoliaptsis, Vasilis; Dafforn, Timothy R; Chaudhry, Afzal N; Halsall, David J; Bradley, J Andrew; Taylor, Craig J

    2011-11-01

    The potential of human leukocyte antigens (HLA) to stimulate humoral alloimmunity depends on the orientation, accessibility and physiochemical properties of polymorphic amino acids. We have generated high-resolution structural and physiochemical models of all common HLA class I alleles and analyzed the impact of amino acid polymorphisms on surface electrostatic potential. Atomic resolution three-dimensional structural models of HLA class I molecules were generated using the MODELLER computer algorithm. The molecular surface electrostatic potential was calculated using the DelPhi program. To confirm that electrostatic surface topography reflects known HLA B cell epitopes, we examined Bw4 and Bw6 and ascertained the impact of amino acid polymorphisms on their tertiary and physiochemical composition. The HLA protein structures generated performed well when subjected to stereochemical and energy-based testing for structural integrity. The electrostatic pattern and conformation of Bw4 and Bw6 epitopes are maintained among HLA molecules even when expressed in a different structural context. Importantly, variation in epitope amino acid composition does not always translate into a different electrostatic motif, providing an explanation for serologic cross-reactivity. Mutations of critical amino acids that abrogate antibody binding also induce distinct changes in epitope electrostatic properties. In conclusion, high-resolution structural modeling provides a physiochemical explanation for serologic patterns of antibody binding and provides novel insights into HLA immunogenicity. PMID:21840357

  7. Analysis of the conformation and thermal stability of the high-affinity IgE Fc receptor β chain polymorphic proteins.

    PubMed

    Terada, Tomoyoshi; Takahashi, Teppei; Arikawa, Hajime; Era, Seiichi

    2016-07-01

    The high-affinity IgE Fc receptor (FcεRI) β chain acts as a signal amplifier through the immunoreceptor tyrosine-based activation motif in its C-terminal intracellular region. Polymorphisms in FcεRI β have been linked to atopy, asthma, and allergies. We investigated the secondary structure, conformation, and thermal stability of FcεRI β polymorphic (β-L172I, β-L174V, and β-E228G) proteins. Polymorphisms did not affect the secondary structure and conformation of FcεRI β. However, we calculated Gibbs free energy of unfolding (ΔGunf) and significant differences were observed in ΔGunf values between the wild-type FcεRI β (β-WT) and β-E228G. These results suggested that β-E228G affected the thermal stability of FcεRI β. The role of β-E228G in biological functions and its involvement in allergic reactions have not yet been elucidated in detail; therefore, differences in the thermal stability of β-E228G may affect the function of FcεRI β. PMID:26940508

  8. Allergen structures and epitopes.

    PubMed

    Meno, K H

    2011-07-01

    Human type 1 hypersensitivity diseases such as allergic rhinoconjunctivitis are characterized by allergen-specific IgE antibodies produced in allergic individuals after allergen exposure. IgE antibodies bound to receptors on the surface of effector cells trigger an allergic response by interacting with three-dimensional (conformational) epitopes on the allergen surface. Crystal structures are available for complexes of antibody specifically bound to five allergens, from birch pollen, bee venom, cockroach, cow's milk and timothy grass pollen. The details of the antibody-allergen interaction extending all the way to atomic resolution are available from such complexes. In vitro investigations using recombinant monoclonal antibodies and human basophils show that binding affinity is a key to triggering the allergic response. Continued molecular characterization of antibody-allergen interactions is paving the way for the use of recombinant allergens in allergen-specific diagnosis and immunotherapy. PMID:21668845

  9. Atomic-level mapping of antibody epitopes on a GPCR.

    PubMed

    Paes, Cheryl; Ingalls, Jada; Kampani, Karan; Sulli, Chidananda; Kakkar, Esha; Murray, Meredith; Kotelnikov, Valery; Greene, Tiffani A; Rucker, Joseph B; Doranz, Benjamin J

    2009-05-27

    Epitopes that define the immunodominant regions of conformationally complex integral membrane proteins have been difficult to reliably delineate. Here, a high-throughput approach termed shotgun mutagenesis was used to map the binding epitopes of five different monoclonal antibodies targeting the GPCR CCR5. The amino acids, and in some cases the atoms, that comprise the critical contact points of each epitope were identified, defining the immunodominant structures of this GPCR and their physicochemistry. PMID:19453194

  10. Extended haplotype analysis of ovine ADRB3 using polymerase chain reaction single strand conformational polymorphism on two regions of the gene.

    PubMed

    Yang, Guo; Hickford, Jon G H; Zhou, Huitong; Fang, Qian; Forrest, Rachel H

    2011-07-01

    The β3 adrenergic receptor (ADRB3) plays a critical role in the regulation of energy metabolism in mammals. In sheep, intronic polymorphism of the ADRB3 gene has been associated with lamb survival and various production traits. This study investigates variation in the ovine ADRB3 3' untranslated region (3'UTR), a region that may impact expression of the gene. Using PCR- single strand conformational polymorphism (SSCP), six unique patterns (named a-f) were observed in an approximately 304-bp amplicon. Sequencing revealed three single-nucleotide polymorphisms (c.*233A>C, c.*271G>C, c.*357A>T) and a single-nucleotide deletion (c.*257delG). Haplotype analyses showed that the previously described allele A defined by variation in the ovine ADRB3 intron can be divided into three haplotypes (Aa, Ab, and Ac). In total, 16 haplotypes through ovine ADRB3 were detected. This study suggests that ovine ADRB3 is highly polymorphic and that the extended haplotype analysis through the promoter, 5'UTR, coding sequence, intron, and 3'UTR needs to be performed to define the full extent of variation in this gene. PMID:21348572

  11. Proof of principle for epitope-focused vaccine design

    PubMed Central

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

    2014-01-01

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

  12. Proof of principle for epitope-focused vaccine design

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

  13. Conformational Stability of Mammalian Prion Protein Amyloid Fibrils Is Dictated by a Packing Polymorphism within the Core Region*

    PubMed Central

    Cobb, Nathan J.; Apostol, Marcin I.; Chen, Shugui; Smirnovas, Vytautas; Surewicz, Witold K.

    2014-01-01

    Mammalian prion strains are believed to arise from the propagation of distinct conformations of the misfolded prion protein PrPSc. One key operational parameter used to define differences between strains has been conformational stability of PrPSc as defined by resistance to thermal and/or chemical denaturation. However, the structural basis of these stability differences is unknown. To bridge this gap, we have generated two strains of recombinant human prion protein amyloid fibrils that show dramatic differences in conformational stability and have characterized them by a number of biophysical methods. Backbone amide hydrogen/deuterium exchange experiments revealed that, in sharp contrast to previously studied strains of infectious amyloid formed from the yeast prion protein Sup35, differences in β-sheet core size do not underlie differences in conformational stability between strains of mammalian prion protein amyloid. Instead, these stability differences appear to be dictated by distinct packing arrangements (i.e. steric zipper interfaces) within the amyloid core, as indicated by distinct x-ray fiber diffraction patterns and large strain-dependent differences in hydrogen/deuterium exchange kinetics for histidine side chains within the core region. Although this study was limited to synthetic prion protein amyloid fibrils, a similar structural basis for strain-dependent conformational stability may apply to brain-derived PrPSc, especially because large strain-specific differences in PrPSc stability are often observed despite a similar size of the PrPSc core region. PMID:24338015

  14. Mutations in the rpoB gene of Mycobacterium tuberculosis that interfere with PCR-single-strand conformation polymorphism analysis for rifampin susceptibility testing.

    PubMed Central

    Kim, B J; Kim, S Y; Park, B H; Lyu, M A; Park, I K; Bai, G H; Kim, S J; Cha, C Y; Kook, Y H

    1997-01-01

    Rifampin susceptibility of 32 rifampin-resistant and 26 rifampin-susceptible Mycobacterium tuberculosis strains was analyzed by PCR-single-strand conformation polymorphism (SSCP) and DNA sequencing within the 157-bp region of the rpoB gene (Ala500 to Val550). Two false-positive PCR-SSCP results were observed among the susceptible strains due to the silent mutation Gln513 (CAA-->CAG) and the deletion mutation Thr508 and Ser509. Another silent mutation [Leu511 (CTG-->CTA)], combined with the mutation Ser531-->Leu, was observed in a resistant strain. These results suggest that to rule out false-positive PCR-SSCP results, sequencing of the target DNA is required. PMID:9003625

  15. Single-strand conformation polymorphism for p53 mutation by a combination of neutral pH buffer and temperature gradient in capillary electrophoresis.

    PubMed

    Gelfi, Cecilia; Vigano, Agnese; De Palma, Sara; Righetti, Pier Giorgio; Righetti, Sabin Carla; Corna, Elisabetta; Zunino, Franco

    2002-05-01

    A large number of point mutations in the p53 gene have been detected by capillary zone electrophoresis via single-strand conformation polymorphism (SSCP) analysis. A much improved detection sensitivity was obtained via the following modifications in running conditions: use of low-viscosity 3% hydroxyethylcellulose (HEC), a neutral pH (pH 6.8) buffer, in which the standard Tris moiety was substituted with a 2-(N-morpholino)ethanesulfonic acid (MES)/Tris mixture, use of SYBR Green II for improved fluorescent signal at the lower pH adopted; and, finally, the use of a temperature gradient in the 15-25 degrees C interval, for favoring the conformational transitions in the mutated samples. The typical temperature gradient activated had a slope of 2 degrees C/min and were induced externally. A total of 24 samples from affected patients, both in the homo- and heterozygous state, were analyzed. All the mutations could be detected by this improved protocol, raising the sensitivity from the standard ca. 80% of conventional SSCP to essentially 100% with the present methodology. All the mutations were confirmed by sequence analysis of the affected samples. PMID:12116163

  16. Structure of allergens and structure based epitope predictions☆

    PubMed Central

    Dall’Antonia, Fabio; Pavkov-Keller, Tea; Zangger, Klaus; Keller, Walter

    2014-01-01

    The structure determination of major allergens is a prerequisite for analyzing surface exposed areas of the allergen and for mapping conformational epitopes. These may be determined by experimental methods including crystallographic and NMR-based approaches or predicted by computational methods. In this review we summarize the existing structural information on allergens and their classification in protein fold families. The currently available allergen-antibody complexes are described and the experimentally obtained epitopes compared. Furthermore we discuss established methods for linear and conformational epitope mapping, putting special emphasis on a recently developed approach, which uses the structural similarity of proteins in combination with the experimental cross-reactivity data for epitope prediction. PMID:23891546

  17. Monoclonal antibodies to HLA-E bind epitopes carried by unfolded β2 m-free heavy chains.

    PubMed

    Tremante, Elisa; Lo Monaco, Elisa; Ingegnere, Tiziano; Sampaoli, Camilla; Fraioli, Rocco; Giacomini, Patrizio

    2015-08-01

    Since HLA-E heavy chains accumulate free of their light β2 -microglobulin (β2 m) subunit, raising mAbs to folded HLA-E heterodimers has been difficult, and mAb characterization has been controversial. Herein, mAb W6/32 and 5 HLA-E-restricted mAbs (MEM-E/02, MEM-E/07, MEM-E/08, DT9, and 3D12) were tested on denatured, acid-treated, and natively folded (both β2 m-associated and β2 m-free) HLA-E molecules. Four distinct conformations were detected, including unusual, partially folded (and yet β2 m-free) heavy chains reactive with mAb DT9. In contrast with previous studies, epitope mapping and substitution scan on thousands of overlapping peptides printed on microchips revealed that mAbs MEM-E/02, MEM-E/07, and MEM-E/08 bind three distinct α1 and α2 domain epitopes. All three epitopes are linear since they span just 4-6 residues and are "hidden" in folded HLA-E heterodimers. They contain at least one HLA-E-specific residue that cannot be replaced by single substitutions with polymorphic HLA-A, HLA-B, HLA-C, HLA-F, and HLA-G residues. Finally, also the MEM-E/02 and 3D12 epitopes are spatially distinct. In summary, HLA-E-specific residues are dominantly immunogenic, but only when heavy chains are locally unfolded. Consequently, the available mAbs fail to selectively bind conformed HLA-E heterodimers, and HLA-E expression may have been inaccurately assessed in some previous oncology, reproductive immunology, virology, and transplantation studies. PMID:25982269

  18. Bioinformatics analysis of the epitope regions for norovirus capsid protein

    PubMed Central

    2013-01-01

    Background Norovirus is the major cause of nonbacterial epidemic gastroenteritis, being highly prevalent in both developing and developed countries. Despite of the available monoclonal antibodies (MAbs) for different sub-genogroups, a comprehensive epitope analysis based on various bioinformatics technology is highly desired for future potential antibody development in clinical diagonosis and treatment. Methods A total of 18 full-length human norovirus capsid protein sequences were downloaded from GenBank. Protein modeling was performed with program Modeller 9.9. The modeled 3D structures of capsid protein of norovirus were submitted to the protein antigen spatial epitope prediction webserver (SEPPA) for predicting the possible spatial epitopes with the default threshold. The results were processed using the Biosoftware. Results Compared with GI, we found that the GII genogroup had four deletions and two special insertions in the VP1 region. The predicted conformational epitope regions mainly concentrated on N-terminal (1~96), Middle Part (298~305, 355~375) and C-terminal (560~570). We find two common epitope regions on sequences for GI and GII genogroup, and also found an exclusive epitope region for GII genogroup. Conclusions The predicted conformational epitope regions of norovirus VP1 mainly concentrated on N-terminal, Middle Part and C-terminal. We find two common epitope regions on sequences for GI and GII genogroup, and also found an exclusive epitope region for GII genogroup. The overlapping with experimental epitopes indicates the important role of latest computational technologies. With the fast development of computational immunology tools, the bioinformatics pipeline will be more and more critical to vaccine design. PMID:23514273

  19. Applying bioinformatics for antibody epitope prediction using affinity-selected mimotopes - relevance for vaccine design.

    PubMed

    Denisova, Galina F; Denisov, Dimitri A; Bramson, Jonathan L

    2010-01-01

    To properly characterize protective polyclonal antibody responses, it is necessary to examine epitope specificity. Most antibody epitopes are conformational in nature and, thus, cannot be identified using synthetic linear peptides. Cyclic peptides can function as mimetics of conformational epitopes (termed mimotopes), thereby providing targets, which can be selected by immunoaffinity purification. However, the management of large collections of random cyclic peptides is cumbersome. Filamentous bacteriophage provides a useful scaffold for the expression of random peptides (termed phage display) facilitating both the production and manipulation of complex peptide libraries. Immunoaffinity selection of phage displaying random cyclic peptides is an effective strategy for isolating mimotopes with specificity for a given antiserum. Further epitope prediction based on mimotope sequence is not trivial since mimotopes generally display only small homologies with the target protein. Large numbers of unique mimotopes are required to provide sufficient sequence coverage to elucidate the target epitope. We have developed a method based on pattern recognition theory to deal with the complexity of large collections of conformational mimotopes. The analysis consists of two phases: 1) The learning phase where a large collection of epitope-specific mimotopes is analyzed to identify epitope specific "signs" and 2) The identification phase where immunoaffinity-selected mimotopes are interrogated for the presence of the epitope specific "signs" and assigned to specific epitopes. We are currently using computational methods to define epitope "signs" without the need for prior knowledge of specific mimotopes. This technology provides an important tool for characterizing the breadth of antibody specificities within polyclonal antisera. PMID:21067548

  20. Drug Development in Conformational Diseases: A Novel Family of Chemical Chaperones that Bind and Stabilise Several Polymorphic Amyloid Structures

    PubMed Central

    Bencomo, Alberto; Lara-Martínez, Reyna; Rivera-Marrero, Suchitil; Domínguez, Guadalupe; Pérez-Perera, Rafaela; Jiménez-García, Luis Felipe; Altamirano-Bustamante, Nelly F.; Diaz-Delgado, Massiel; Vedrenne, Fernand; Rivillas-Acevedo, Lina; Pasten-Hidalgo, Karina; Segura-Valdez, María de Lourdes; Islas-Andrade, Sergio; Garrido-Magaña, Eulalia; Perera-Pintado, Alejandro; Prats-Capote, Anaís; Rodríguez-Tanty, Chryslaine; Altamirano-Bustamante, Myriam M.

    2015-01-01

    The increasing prevalence of conformational diseases, including Alzheimer's disease, type 2 Diabetes Mellitus and Cancer, poses a global challenge at many different levels. It has devastating effects on the sufferers as well as a tremendous economic impact on families and the health system. In this work, we apply a cross-functional approach that combines ideas, concepts and technologies from several disciplines in order to study, in silico and in vitro, the role of a novel chemical chaperones family (NCHCHF) in processes of protein aggregation in conformational diseases. Given that Serum Albumin (SA) is the most abundant protein in the blood of mammals, and Bovine Serum Albumin (BSA) is an off-the-shelf protein available in most labs around the world, we compared the ligandability of BSA:NCHCHF with the interaction sites in the Human Islet Amyloid Polypeptide (hIAPP):NCHCHF, and in the amyloid pharmacophore fragments (Aβ17–42 and Aβ16–21):NCHCHF. We posit that the merging of this interaction sites is a meta-structure of pharmacophore which allows the development of chaperones that can prevent protein aggregation at various states from: stabilizing the native state to destabilizing oligomeric state and protofilament. Furthermore to stabilize fibrillar structures, thus decreasing the amount of toxic oligomers in solution, as is the case with the NCHCHF. The paper demonstrates how a set of NCHCHF can be used for studying and potentially treating the various physiopathological stages of a conformational disease. For instance, when dealing with an acute phase of cytotoxicity, what is needed is the recruitment of cytotoxic oligomers, thus chaperone F, which accelerates fiber formation, would be very useful; whereas in a chronic stage it is better to have chaperones A, B, C, and D, which stabilize the native and fibril structures halting self-catalysis and the creation of cytotoxic oligomers as a consequence of fiber formation. Furthermore, all the chaperones are

  1. A novel linear neutralizing epitope of hepatitis E virus.

    PubMed

    Tang, Zi-Min; Tang, Ming; Zhao, Min; Wen, Gui-Ping; Yang, Fan; Cai, Wei; Wang, Si-Ling; Zheng, Zi-Zheng; Xia, Ning-Shao

    2015-07-01

    Hepatitis E virus (HEV) is a serious public health problem that causes acute hepatitis in humans and is primarily transmitted through fecal and oral routes. The major anti-HEV antibody responses are against conformational epitopes located in a.a. 459-606 of HEV pORF2. All reported neutralization epitopes are present on the dimer domain constructed by this peptide. While looking for a neutralizing monoclonal antibody (MAb)-recognized linear epitope, we found a novel neutralizing linear epitope (L2) located in a.a. 423-437 of pORF2. Moreover, epitope L2 is proved non-immunodominant in the HEV-infection process. Using the hepatitis B virus core protein (HBc) as a carrier to display this novel linear epitope, we show herein that this epitope could induce a neutralizing antibody response against HEV in mice and could protect rhesus monkeys from HEV infection. Collectively, our results showed a novel non-immunodominant linear neutralizing epitope of hepatitis E virus, which provided additional insight of HEV vaccine. PMID:26051517

  2. Novel methods to enhance single strand conformation polymorphism (SSCP) senstivity and efficiency: Application to mutation detection in cystic fibrosis (CF)

    SciTech Connect

    Hagstrom, D.J.; Snow, K.; Yuan, Z.; Thibodeau, S.N.

    1994-09-01

    For single gene defects in which there are a variety of mutations with significant frequencies, it is a challenge to find an efficient and sensitive method for mutation detection. For example, although 70% to 75% of CF chromosomes in a North American Caucasian population have the mutation {delta}F508, more than 400 mutations (mostly single base pair substitutions) are represented on the remaining chromosomes. SSCP analysis is a relatively straightforward procedure and therefore suitable for routine use in a clinical laboratory. However, previous reports have demonstrated suboptimal sensitivity rates in screening for mutations. We have developed a novel set of conditions which greatly enhances sensitivity and efficiency of SSCP. Our protocol incorporates multiplex PCR, stepping of wattages during electrophoresis and increased salt concentration at the anode relative to the gel. To screen for mutations in the CFTR gene, three multiplex PCR reactions are performed using identical thermocycler parameters. Sizes of PCR products range from 441 bp to 196 bp: size differences of > 30 bp are necessary to ensure separation during electrophoresis. All PCR products are separated by electrophoresis at room temperature on a single gel containing 8% (37.5:1) polyacrylamide, 5% glycerol and 1x TBE. Using an anode buffer with increased salt (2x TBE) sharpens smaller sized bands, and stepping watts from 5W to 20W during electrophoresis enhances sensitivity. Positive controls were used to demonstrate that mutations could be detected. Other mutations or polymorphisms were verified by cycle sequencing of PCR products or by alternative PCR-based assays for the more common mutations. Thus, using 3 PCR reactions per patient and one gel condition, we are able to achieve a CF mutation detection rate of approximately 90% in a North American Caucasian population.

  3. Nonisotopic single-strand conformation polymorphism analysis of sequence variability in ribosomal DNA expansion segments within the genus Trichinella (Nematoda: Adenophorea).

    PubMed

    Gasser, Robin B; Hu, Min; Abs El-Osta, Youssef G; Zarlenga, Dante S; Pozio, Edoardo

    2004-10-01

    A nonisotopic single-strand conformation polymorphism (SSCP) approach was employed to 'fingerprint' sequence variability in the expansion segment 5 (ES5) of domain IV and the D3 domain of nuclear ribosomal DNA within and/or among isolates and individual muscle (first-stage) larvae representing all currently recognized species/genotypes of Trichinella. In addition, phylogenetic analyses of the D3 sequence data set, employing three different tree-building algorithms, examined the relationships among all of them. These analyses showed strong support that the encapsulated species T. spiralis and T. nelsoni formed a group to the exclusion of the other encapsulated species T. britovi and its related genotypes Trichinella T8 and T9 and T. murrelli, and T. nativa and Trichinella T6, and strong support that T. nativa and Trichinella T6 grouped together. Also, these eight encapsulated members grouped to the exclusion of the nonencapsulated species T. papuae and T. zimbabwensis and the three representatives of T. pseudospiralis investigated. The findings showed that nonencapsulated species constitute a complex group which is distinct from the encapsulated species and supported the current hypothesis that encapsulated Trichinella group external to the nonencapsulated forms, in accordance with independent biological and biochemical data sets. PMID:15490459

  4. Genotyping of Trichomonas vaginalis isolates in Iran by using single stranded conformational polymorphism-PCR technique and internal transcribed spacer regions.

    PubMed

    Matini, M; Rezaeian, M; Mohebali, M; Maghsood, A H; Rabiee, S; Rahimi-Foroushani, A; Fallah, M; Miahipour, A; Rezaie, S

    2012-12-01

    Infection with Trichomonas vaginalis, the causative agent of human urogenital infection, is the most prevalent nonviral sexually transmitted disease worldwide. In spite of the high prevalence and medical importance of trichomoniasis, there is little knowledge about genetic epidemiology and genetic characterisation of this parasite. For this purpose, a Single Stranded Conformation Polymorphism-PCR (SSCP-PCR) typing method was conducted for Iranian T. vaginalis isolates using 5.8s ribosomal gene (rRNA gene) and the flanking internal transcribed spacer (ITS) regions. Nine hundred and fifty vaginal swab samples were examined in which 50 (5.3%) samples were parasitologically positive and used for molecular identification based on SSCP-PCR and nucleotide sequence analyses. Results of the SSCP analysis showed two distinct reproducible banding patterns (I, II) which were confirmed by nucleotide sequence analysis in the ITS1 regions. Frequencies of the SSCP banding patterns I and II were 84% (42/50) and 16% (8/50), respectively. In conclusion, SSCP-PCR analysis provided a reliable and sensitive method for strain genotyping of T. vaginalis based on the ITS1/5.8s/ITS2 region. This finding may help us gain more information about correlation between genetic properties and biological features of this parasite. PMID:23202606

  5. Single strand conformation polymorphism analysis of androgen receptor gene mutations in patients with androgen insensitivity syndromes: Application for diagnosis, genetic counseling, and therapy

    SciTech Connect

    Hiort, O. Tufts-New England Medical Center, Boston, MA ); Huang, Q. ); Sinnecker, G.H.G.; Kruse, K. ); Sadeghi-Nejad, A.; Wolfe, H.J. ); Yandell, D.W. ) Harvard School of Public Health, Boston, MA )

    1993-07-01

    Recent studies indicate that mutations in the androgen receptor gene are associated with androgen insensitivity syndromes, a heterogeneous group of related disorders involving defective sexual differentiation in karyotypic males. In this report, the authors address the possibility of rapid mutational analysis of the androgen receptor gene for initial diagnosis, genetic counseling, and molecular subclassification of affected patients and their families. DNA from peripheral blood leukocytes of six patients from five families with various degrees of androgen insensitivity was studied. Exons 2 to 8 of the androgen receptor gene were analyzed using a combination of single strand conformation polymorphism analysis and direct DNA sequencing. Female family members were also studied to identify heterozygote carriers. Point mutations in the AR gene were identified in all six patients, and all mutations caused amino acid substitutions. One patient with incomplete androgen insensitivity was a mosaic for the mutation. Four of the five mothers, as well as a young sister of one patient, were carriers of the mutation present in the affected child. The data show that new mutations may occur in the androgen receptor gene leading to sporadic androgen insensitivity syndrome. Molecular genetic characterization of the variant allele can serve as a primary tool for diagnosis and subsequent therapy, and can provide a basis for distinguishing heterozygous carriers in familial androgen resistance. The identification of carriers is of substantial clinical importance for genetic counseling. 29 refs., 2 figs., 1 tab.

  6. Elicitation of structure-specific antibodies by epitope scaffolds

    PubMed Central

    Ofek, Gilad; Guenaga, F. Javier; Schief, William R.; Skinner, Jeff; Baker, David; Wyatt, Richard; Kwong, Peter D.

    2010-01-01

    Elicitation of antibodies against targets that are immunorecessive, cryptic, or transient in their native context has been a challenge for vaccine design. Here we demonstrate the elicitation of structure-specific antibodies against the HIV-1 gp41 epitope of the broadly neutralizing antibody 2F5. This conformationally flexible region of gp41 assumes mostly helical conformations but adopts a kinked, extended structure when bound by antibody 2F5. Computational techniques were employed to transplant the 2F5 epitope into select acceptor scaffolds. The resultant “2F5-epitope scaffolds” possessed nanomolar affinity for antibody 2F5 and a range of epitope flexibilities and antigenic specificities. Crystallographic characterization of the epitope scaffold with highest affinity and antigenic discrimination confirmed good to near perfect attainment of the target conformation for the gp41 molecular graft in free and 2F5-bound states, respectively. Animals immunized with 2F5-epitope scaffolds showed levels of graft-specific immune responses that correlated with graft flexibility (p < 0.04), while antibody responses against the graft—as dissected residue-by-residue with alanine substitutions—resembled more closely those of 2F5 than sera elicited with flexible or cyclized peptides, a resemblance heightened by heterologous prime-boost. Lastly, crystal structures of a gp41 peptide in complex with monoclonal antibodies elicited by the 2F5-epitope scaffolds revealed that the elicited antibodies induce gp41 to assume its 2F5-recognized shape. Epitope scaffolds thus provide a means to elicit antibodies that recognize a predetermined target shape and sequence, even if that shape is transient in nature, and a means by which to dissect factors influencing such elicitation. PMID:20876137

  7. Flanking p10 contribution and sequence bias in matrix based epitope prediction: revisiting the assumption of independent binding pockets

    PubMed Central

    Parry, Christian S

    2008-01-01

    Background Eluted natural peptides from major histocompatibility molecules show patterns of conserved residues. Crystallographic structures show that the bound peptide in class II major histocompatibility complex adopts a near uniform polyproline II-like conformation. This way allele-specific favoured residues are able to anchor into pockets in the binding groove leaving other peptide side chains exposed for recognition by T cells. The anchor residues form a motif. This sequence pattern can be used to screen large sequences for potential epitopes. Quantitative matrices extend the motif idea to include the contribution of non-anchor peptide residues. This report examines two new matrices that extend the binding register to incorporate the polymorphic p10 pocket of human leukocyte antigen DR1. Their performance is quantified against experimental binding measurements and against the canonical nine-residue register matrix. Results One new matrix shows significant improvement over the base matrix; the other does not. The new matrices differ in the sequence of the peptide library. Conclusion One of the extended quantitative matrices showed significant improvement in prediction over the original nine residue matrix and over the other extended matrix. Proline in the sequence of the peptide library of the better performing matrix presumably stabilizes the peptide conformation through neighbour interactions. Such interactions may influence epitope prediction in this test of quantitative matrices. This calls into question the assumption of the independent contribution of individual binding pockets. PMID:18925947

  8. Ampelomyces mycoparasites from apple powdery mildew identified as a distinct group based on single-stranded conformation polymorphism analysis of the rDNA ITS region.

    PubMed

    Szentiványi, Orsolya; Kiss, Levente; Russell, John C; Kovács, Gábor M; Varga, Krisztina; Jankovics, Tünde; Lesemann, Silke; Xu, Xiang-Ming; Jeffries, Peter

    2005-04-01

    Pycnidial fungi belonging to the genus Ampelomyces are the most common natural antagonists of powdery mildews worldwide. During a study of the interactions between apple powdery mildew (Podosphaera leucotricha) and Ampelomyces mycoparasites, 52 new Ampelomyces isolates were obtained from P. leucotricha and, in addition, 13 new isolates from other species of the Erysiphaceae in four European countries. Their genetic diversity was screened using single-stranded conformation polymorphism (SSCP) analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA). For comparison, 24 isolates obtained from genetic resource collections or other sources were included in this study. Based on the ITS-SSCP patterns, the isolates were placed in eight groups. The isolates belonged to two types based on their growth in culture. The faster-growing and the slower-growing isolates were included in different SSCP groups. A phylogenetic analysis of the ITS sequences of representatives of these groups confirmed the results obtained with the SSCP method, and showed that the faster-growing isolates do not belong to Ampelomyces as suggested by earlier studies. All the isolates from P. leucotricha fell into a distinct SSCP group of genetically homogeneous isolates. This suggests that Ampelomyces mycoparasites which occur in apple powdery mildew are slightly different from the other Ampelomyces groups which contain mycoparasites from various powdery mildew species. This may be because the main growth period of Ampelomyces mycoparasites in apple powdery mildew is isolated in time from that of Ampelomyces isolates that occur in other species of the Erysiphaceae. P. leucotricha starts its life-cycle early in the season, usually in March-April, while most powdery mildews are active in the same environments only late in the year. PMID:15912930

  9. Detection of Clonal T-Cell Receptor γ Gene Rearrangements in Paraffin-Embedded Tissue by Polymerase Chain Reaction and Nonradioactive Single-Strand Conformational Polymorphism Analysis

    PubMed Central

    Signoretti, Sabina; Murphy, Michael; Cangi, Maria Giulia; Puddu, Pietro; Kadin, Marshall E.; Loda, Massimo

    1999-01-01

    The diagnosis of T-cell lymphoproliferative disorders, which frequently involve the skin and other extranodal sites, is often problematic because of the difficulty in establishing clonality in paraffin-embedded tissue. To this end, we developed a simple, nonradioactive method to detect T-cell receptor γ (TCR-γ) gene rearrangements by polymerase chain reaction single-strand conformational polymorphism (PCR-SSCP) in paraffin-embedded tissue. Jurkat and HSB-2 cell lines and peripheral blood samples from normal individuals were used as monoclonal and polyclonal controls, respectively. DNA was extracted from 24 biopsies of T-cell lymphomas, 12 biopsies of reactive lymphoid infiltrates, and 2 biopsies of primary cutaneous large B-cell lymphomas. Vγ1–8, Vγ9, Vγ10, Vγ11, and Jγ1/Jγ2 consensus primers were used for TCR-γ gene rearrangement amplification and PCR products were analyzed by nonradioactive SSCP. Monoclonal controls yielded a well-defined banded pattern, whereas all polyclonal T-cell controls showed a reproducible pattern of smears. We detected monoclonality in 20/21 (95%) T-cell lymphoma cases, whereas no dominant T-cell clones were found in any of the reactive lymphoid infiltrates or B-cell lymphomas. Sensitivity of 1–5% was demonstrated by serially diluting Jurkat cells in mononuclear blood cells from normal individuals. We conclude that nonradioactive PCR-SSCP for TCR-γ gene rearrangement analysis is a useful adjunct to routine histological and immunophenotypic methods in the diagnosis of T-cell lymphoproliferative disorders in paraffin-embedded tissue. PMID:9916920

  10. Rapid Identification of Bacteria from Positive Blood Cultures by Fluorescence-Based PCR–Single-Strand Conformation Polymorphism Analysis of the 16S rRNA Gene

    PubMed Central

    Turenne, Christine Y.; Witwicki, Evelyn; Hoban, Daryl J.; Karlowsky, James A.; Kabani, Amin M.

    2000-01-01

    Bacteremia continues to result in significant morbidity and mortality, particularly in patients who are immunocompromised. Currently, patients with suspected bacteremia are empirically administered broad-spectrum antibiotics, as definitive diagnosis relies upon the use of blood cultures, which impose significant delays in and limitations to pathogen identification. To address the limitations of growth-based identification, the sequence variability of the 16S rRNA gene of bacteria was targeted for rapid identification of bacterial pathogens isolated directly from blood cultures using a fluorescence-based PCR–single-strand conformation polymorphism (SSCP) protocol. Species-specific SSCP patterns were determined for 25 of the most common bacterial species isolated from blood cultures; these isolates subsequently served as a reference collection for bacterial identification for new cases of bacteremia. A total of 272 blood-culture-positive patient specimens containing bacteria were tested. A previously determined SSCP pattern was observed for 251 (92%) specimens, with 21 (8%) specimens demonstrating SSCP patterns distinct from those in the reference collection. Time to identification from blood culture positivity ranged from 1 to 8 days with biochemical testing, whereas identification by fluorescence-based capillary electrophoresis was obtained as early as 7 h at a calculated cost of $10 (U.S. currency) per specimen when tested in batches of 10. Limitations encountered included the inability to consistently detect mixed cultures as well as some species demonstrating identical SSCP patterns. This method can be applied directly to blood cultures or whole-blood specimens, where early pathogen identification would result in a timely diagnosis with possible implications for patient management costs and the mortality and morbidity of infections. PMID:10655337

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

    SciTech Connect

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

    2012-06-28

    Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, but an effective vaccine has not yet been developed. An ideal vaccine would elicit protective antibodies while avoiding virus-specific T-cell responses, which have been implicated in vaccine-enhanced disease with previous RSV vaccines. We propose that heterologous proteins designed to present RSV-neutralizing antibody epitopes and to elicit cognate antibodies have the potential to fulfill these vaccine requirements, as they can be fashioned to be free of viral T-cell epitopes. Here we present the design and characterization of three epitope-scaffolds that present the epitope of motavizumab, a 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 {angstrom} resolution) was similar to the computationally designed model, with all hydrogen-bond interactions critical for binding to motavizumab preserved. Immunization of mice with this epitope-scaffold failed to elicit neutralizing antibodies but did elicit sera with F binding activity. The elicitation of F binding antibodies suggests that some of the design criteria for eliciting protective antibodies without virus-specific T-cell responses are being met, but additional optimization of these novel immunogens is required.

  12. Further exploration of the conformational space of α-synuclein fibrils: solid-state NMR assignment of a high-pH polymorph.

    PubMed

    Verasdonck, Joeri; Bousset, Luc; Gath, Julia; Melki, Ronald; Böckmann, Anja; Meier, Beat H

    2016-04-01

    Polymorphism is a common and important phenomenon for protein fibrils which has been linked to the appearance of strains in prion and other neurodegenerative diseases. Parkinson disease is a frequently occurring neurodegenerative pathology, tightly associated with the formation of Lewy bodies. These deposits mainly consist of α-synuclein in fibrillar, β-sheet-rich form. α-synuclein is known to form numerous different polymorphs, which show distinct structural features. Here, we describe the chemical shift assignments, and derive the secondary structure, of a polymorph that was fibrillized at higher-than-physiological pH conditions. The fibrillar core contains residues 40-95, with both the C- and N-terminus not showing any ordered, rigid parts. The chemical shifts are similar to those recorded previously for an assigned polymorph that was fibrillized at neutral pH. PMID:26318307

  13. Determination of B-Cell Epitopes in Patients with Celiac Disease: Peptide Microarrays

    PubMed Central

    Choung, Rok Seon; Marietta, Eric V.; Van Dyke, Carol T.; Brantner, Tricia L.; Rajasekaran, John; Pasricha, Pankaj J.; Wang, Tianhao; Bei, Kang; Krishna, Karthik; Krishnamurthy, Hari K.; Snyder, Melissa R.; Jayaraman, Vasanth; Murray, Joseph A.

    2016-01-01

    Background Most antibodies recognize conformational or discontinuous epitopes that have a specific 3-dimensional shape; however, determination of discontinuous B-cell epitopes is a major challenge in bioscience. Moreover, the current methods for identifying peptide epitopes often involve laborious, high-cost peptide screening programs. Here, we present a novel microarray method for identifying discontinuous B-cell epitopes in celiac disease (CD) by using a silicon-based peptide array and computational methods. Methods Using a novel silicon-based microarray platform with a multi-pillar chip, overlapping 12-mer peptide sequences of all native and deamidated gliadins, which are known to trigger CD, were synthesized in situ and used to identify peptide epitopes. Results Using a computational algorithm that considered disease specificity of peptide sequences, 2 distinct epitope sets were identified. Further, by combining the most discriminative 3-mer gliadin sequences with randomly interpolated3- or 6-mer peptide sequences, novel discontinuous epitopes were identified and further optimized to maximize disease discrimination. The final discontinuous epitope sets were tested in a confirmatory cohort of CD patients and controls, yielding 99% sensitivity and 100% specificity. Conclusions These novel sets of epitopes derived from gliadin have a high degree of accuracy in differentiating CD from controls, compared with standard serologic tests. The method of ultra-high-density peptide microarray described here would be broadly useful to develop high-fidelity diagnostic tests and explore pathogenesis. PMID:26824466

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

    PubMed

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

    2012-01-01

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

  15. A novel multi-epitope peptide vaccine against cancer: an in silico approach.

    PubMed

    Nezafat, Navid; Ghasemi, Younes; Javadi, Gholamreza; Khoshnoud, Mohammad Javad; Omidinia, Eskandar

    2014-05-21

    Cancer immunotherapy has an outstanding position in cancer prevention and treatment. In this kind of therapy, the immune system is activated to eliminate cancerous cells. Multi-epitope peptide cancer vaccines are manifesting as the next generation of cancer immunotherapy. In the present study, we have implemented various strategies to design an efficient multi-epitope vaccine. CD8+ cytolytic T lymphocytes (CTLs) epitopes, which have a pivotal role in cellular immune responses, helper epitopes and adjuvant, are three crucial components of peptide vaccine. CTL epitopes were determined from two high immunogenic protein Wilms tumor-1 (WT1) and human papillomavirus (HPV) E7 by various servers, which apply different algorithms. CTL epitopes were linked together by AAY and HEYGAEALERAG motifs to enhance epitope presentation. Pan HLA DR-binding epitope (PADRE) peptide sequence and helper epitopes, which have defined from Tetanus toxin fragment C (TTFrC) by various servers, were used to induce CD4+ helper T lymphocytes (HTLs) responses. Additionally, helper epitopes were conjugated together via GPGPG motifs that stimulate HTL immunity. Heparin-Binding Hemagglutinin (HBHA), a novel TLR4 agonist was employed as an adjuvant to polarize CD4+ T cells toward T-helper 1 to induce strong CTL responses. Moreover, the EAAAK linker was introduced to N and C terminals of HBHA for efficient separation. 3D model of protein was generated and predicted B cell epitopes were determined from the surface of built structure. Our protein contains several linear and conformational B cell epitopes, which suggests the antibody triggering property of this novel vaccine. Hence, our final protein can be used for prophylactic or therapeutic usages, because it can potentially stimulate both cellular and humoral immune responses. PMID:24512916

  16. Computer simulations of a tumor surface octapeptide epitope.

    PubMed

    Reid, R H; Hooper, C A; Brooks, B R

    1989-01-01

    Molecular dynamics using CHARMM and GEMM programs with the Star Technologies ST 100 array processor functioning at the speed of super computers was used as a searching algorithm for conformational exploration of the octapeptide Gly-Asn-Thr-Ile-Val-Ala-Glu. This poorly soluble octapeptide is the N-terminal epitope of an 11 KD glycoprotein antigen residing on human ductal carcinoma (breast) cells. Very long (nanoseconds) simulations were required. Both an alpha-helix and the N-acetyl-N1-methylamide derived minimized starting structures gave the same lowest potential energy conformation with simulations at 600 K. The same conformation was found only when using the latter starting conformation with simulations at 300 K. The lowest potential energy conformation was stabilized by 4 hydrophobic contacts and 13 H bonds completing one turn of a left-handed helix. PMID:2470439

  17. Monoclonal antibodies to conformational epitopes of the surface glycoprotein of caprine arthritis-encephalitis virus: potential application to competitive-inhibition enzyme-linked immunosorbent assay for detecting antibodies in goat sera.

    PubMed

    Ozyörük, F; Cheevers, W P; Hullinger, G A; McGuire, T C; Hutton, M; Knowles, D P

    2001-01-01

    Four immunoglobulin G1 monoclonal antibodies (MAbs) to the gp135 surface envelope glycoprotein (SU) of the 79-63 isolate of caprine arthritis-encephalitis virus (CAEV), referred to as CAEV-63, were characterized and evaluated for their ability to compete with antibody from CAEV-infected goats. Three murine MAbs (MAbs GPB16A, 29A, and 74A) and one caprine MAb (MAb F7-299) were examined. All MAbs reacted in nitrocellulose dot blots with native CAEV-63 SU purified by MAb F7-299 affinity chromatography, whereas none reacted with denatured and reduced SU. All MAbs reacted in Western blots with purified CAEV-63 SU or the SU component of whole-virus lysate following denaturation in the absence of reducing agent, indicating that intramolecular disulfide bonding was essential for epitope integrity. Peptide-N-glycosidase F digestion of SU abolished the reactivities of MAbs 74A and F7-299, whereas treatment of SU with N-acetylneuraminate glycohydrolase (sialidase A) under nonreducing conditions enhanced the reactivities of all MAbs as well as polyclonal goat sera. MAbs 29A and F7-299 were cross-reactive with the SU of an independent strain of CAEV (CAEV-Co). By enzyme-linked immunosorbent assay (ELISA), the reactivities of horseradish peroxidase (HRP)-conjugated MAbs 16A and 29A with homologous CAEV-63 SU were <10% of that of HRP-conjugated MAb 74A. The reactivity of HRP-conjugated MAb 74A was blocked by sera from goats immunized with CAEV-63 SU or infected with CAEV-63. The reactivity of MAb 74A was also blocked by sera from goats infected with a CAEV-Co molecular clone, although MAb 74A did not react with CAEV-Co SU in Western blots. Thus, goats infected with either CAEV-63 or CAEV-Co make antibodies that inhibit binding of MAb 74A to CAEV-63 SU. A competitive-inhibition ELISA based on displacement of MAb 74A reactivity has potential applicability for the serologic diagnosis of CAEV infection. PMID:11139194

  18. Single-strand conformation polymorphism analysis coupled with stratified DNA sequencing reveals reduced sequence variation in the su(s) and su(wa) regions of the Drosophila melanogaster X chromosome.

    PubMed Central

    Aguadé, M; Meyers, W; Long, A D; Langley, C H

    1994-01-01

    Single-strand conformation polymorphism (SSCP) analysis followed by DNA sequencing of stratified sub-samples was used to survey DNA polymorphism in the su(s) and su(wa) regions in a natural population of Drosophila melanogaster. su(s) and su(wa) are located near the telomere of the X chromosome, where the rate of crossing over per kilobase of DNA monotonically decreases toward the tip. SSCP was assessed in 12 noncoding segments amplified from the su(s) region (3213 bp) and in 8 noncoding segments amplified from the su(wa) region (1955 bp). Sets of segments were multiplexed in a single electrophoretic lane to increase the number of base pairs assayed per lane. Eight segments were monomorphic, and the other 12 segments exhibited two to four SSCP classes. Only four within-SSCP-class DNA sequence differences (a single nucleotide substitution) were observed among 24,360 bp compared within classes. The between-SSCP-class DNA sequence comparisons revealed 27 substitutions and 9 insertion/deletion polymorphisms. The average numbers of substitutional differences per site were 0.0010 and 0.0021 for su(s) and su(wa), respectively. These values are intermediate between those reported for the more distal y-ASC region (0.0004) and the more proximal Pgd locus (0.0024). This observation is consistent with the prediction of the hitchhiking-effect model-i.e., a monotonic increase in polymorphism as a function of crossing over per kilobase. Images PMID:8197115

  19. Vaccine Focusing to Cross-Subtype HIV-1 gp120 Variable Loop Epitopes

    PubMed Central

    Cardozo, Timothy; Wang, Shixia; Jiang, Xunqing; Kong, Xiang-Peng; Hioe, Catarina; Krachmarov, Chavdar

    2014-01-01

    We designed synthetic, epitope-focused immunogens that preferentially display individual neutralization epitopes targeted by cross-subtype anti-HIV V3 loop neutralizing monoclonal antibodies (mAbs). Vaccination of rabbits with these immunogens resulted in the elicitation of distinct polyclonal serum Abs that exhibit cross-subtype neutralization specificities mimicking the mAbs that guided the design. Our results prove the principle that a predictable range of epitope-specific polyclonal cross-subtype HIV-1 neutralizing Abs can be intentionally elicited in mammals by vaccination. The precise boundaries of the epitopes and conformational flexibility in the presentation of the epitopes in the immunogen appeared to be important for successful elicitation. This work may serve as a starting point for translating the activities of human broadly neutralizing anti-HIV-1 monoclonal antibodies (bNAbs) into matched immunogens that can contribute to an efficacious HIV-1 vaccine. PMID:25045827

  20. Thyrotropin Receptor Epitope and Human Leukocyte Antigen in Graves' Disease.

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  2. Mapping Epitopes on a Protein Antigen by the Proteolysis of Antigen-Antibody Complexes

    NASA Astrophysics Data System (ADS)

    Jemmerson, Ronald; Paterson, Yvonne

    1986-05-01

    A monoclonal antibody bound to a protein antigen decreases the rate of proteolytic cleavage of the antigen, having the greatest effect on those regions involved in antibody contact. Thus, an epitope can be identified by the ability of the antibody to protect one region of the antigen more than others from proteolysis. By means of this approach, two distinct epitopes, both conformationally well-ordered, were characterized on horse cytochrome c.

  3. Epitope mapping and functional analysis of sigma A and sigma NS proteins of avian reovirus

    SciTech Connect

    Huang, Pi H.; Li, Ying J.; Su, Yu P.; Lee, Long H.; Liu, Hung J. . E-mail: hjliu@mail.npust.edu.tw

    2005-02-20

    We have previously shown that avian reovirus (ARV) {sigma}A and {sigma}NS proteins possess dsRNA and ssRNA binding activity and suggested that there are two epitopes on {sigma}A (I and II) and three epitopes (A, B, and C) on {sigma}NS. To further define the location of epitopes on {sigma}A and {sigma}NS proteins and to further elucidate the biological functions of these epitopes by using monoclonal antibodies (MAbs) 62, 1F9, H1E1, and 4A123 against the ARV S1133 strain, the full-length and deletion fragments of S2 and S4 genes of ARV generated by polymerase chain reaction (PCR) were cloned into pET32 expression vectors and the fusion proteins were overexpressed in Escherichia coli BL21 strain. Epitope mapping using MAbs and E. coli-expressed deletion fragments of {sigma}A and {sigma}NS of the ARV S1133 strain, synthetic peptides, and the cross reactivity of MAbs to heterologous ARV strains demonstrated that epitope II on {sigma}A was located at amino acid residues {sup 340}QWVMAGLVSAA{sup 350} and epitope B on {sigma}NS at amino acid residues {sup 180}MLDMVDGRP{sup 188}. The MAbs (62, 1F9, and H1E1) directed against epitopes II and B did not require the native conformation of {sigma}A and {sigma}NS, suggesting that their binding activities were conformation-independent. On the other hand, MAb 4A123 only reacted with complete {sigma}NS but not with truncated {sigma}NS fusion proteins in Western blot, suggesting that the binding activity of MAb to epitope A on {sigma}NS was conformation-dependent. Amino acid sequence analysis and the binding assays of MAb 62 to heterologous ARV strains suggested that epitope II on {sigma}A was highly conserved among ARV strains and that this epitope is suitable as a serological marker for the detection of ARV antibodies following natural infection in chickens. On the contrary, an amino acid substitution at position 183 (M to V) in epitope B of ARV could hinder the reactivity of the {sigma}NS with MAb 1F9. The {sigma}NS of ARV with ss

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  6. Prediction of Antibody Epitopes.

    PubMed

    Nielsen, Morten; Marcatili, Paolo

    2015-01-01

    Antibodies recognize their cognate antigens in a precise and effective way. In order to do so, they target regions of the antigenic molecules that have specific features such as large exposed areas, presence of charged or polar atoms, specific secondary structure elements, and lack of similarity to self-proteins. Given the sequence or the structure of a protein of interest, several methods exploit such features to predict the residues that are more likely to be recognized by an immunoglobulin. Here, we present two methods (BepiPred and DiscoTope) to predict linear and discontinuous antibody epitopes from the sequence and/or the three-dimensional structure of a target protein. PMID:26424260

  7. Human self-protein CD8+ T-cell epitopes are both positively and negatively selected.

    PubMed

    Almani, Michal; Raffaeli, Shai; Vider-Shalit, Tal; Tsaban, Lea; Fishbain, Vered; Louzoun, Yoram

    2009-04-01

    The cellular immune system recognizes self-epitopes in the context of MHC-I molecules. The immunological general view presumes that these self-epitopes are just a background, both positively and negatively selecting T cells. We here estimate the number of epitopes in each human protein for many frequent HLA alleles, and a score representing over or under presentation of epitopes on these proteins. We further show that there is a clear selection for the presentation of specific self-protein types. Proteins presenting many epitopes include, for example, autoimmune regulator (AIRE) upregulated tissue-specific antigens, immune system receptors and proteins with a high expression level. On the other hand, proteins that may be considered less "useful" for the immune system, such as low expression level proteins, are under-presented. We combine our epitope estimate with single nucleotide polymorphism (SNP) measures to show that this selection can be directly observed through the fraction of non-synonymous SNP (replacement fraction), which is significantly higher inside epitopes than outside. PMID:19291702

  8. Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands

    PubMed Central

    Encarnación, Medina-Carmona; Palomino-Morales, Rogelio J.; Fuchs, Julian E.; Esperanza, Padín-Gonzalez; Noel, Mesa-Torres; Salido, Eduardo; Timson, David J.; Pey, Angel L.

    2016-01-01

    Protein dynamics is essential to understand protein function and stability, even though is rarely investigated as the origin of loss-of-function due to genetic variations. Here, we use biochemical, biophysical, cell and computational biology tools to study two loss-of-function and cancer-associated polymorphisms (p.R139W and p.P187S) in human NAD(P)H quinone oxidoreductase 1 (NQO1), a FAD-dependent enzyme which activates cancer pro-drugs and stabilizes several oncosuppressors. We show that p.P187S strongly destabilizes the NQO1 dimer in vitro and increases the flexibility of the C-terminal domain, while a combination of FAD and the inhibitor dicoumarol overcome these alterations. Additionally, changes in global stability due to polymorphisms and ligand binding are linked to the dynamics of the dimer interface, whereas the low activity and affinity for FAD in p.P187S is caused by increased fluctuations at the FAD binding site. Importantly, NQO1 steady-state protein levels in cell cultures correlate primarily with the dynamics of the C-terminal domain, supporting a directional preference in NQO1 proteasomal degradation and the use of ligands binding to this domain to stabilize p.P187S in vivo. In conclusion, protein dynamics are fundamental to understanding loss-of-function in p.P187S, and to develop new pharmacological therapies to rescue this function. PMID:26838129

  9. Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands

    NASA Astrophysics Data System (ADS)

    Encarnación, Medina-Carmona; Palomino-Morales, Rogelio J.; Fuchs, Julian E.; Esperanza, Padín-Gonzalez; Noel, Mesa-Torres; Salido, Eduardo; Timson, David J.; Pey, Angel L.

    2016-02-01

    Protein dynamics is essential to understand protein function and stability, even though is rarely investigated as the origin of loss-of-function due to genetic variations. Here, we use biochemical, biophysical, cell and computational biology tools to study two loss-of-function and cancer-associated polymorphisms (p.R139W and p.P187S) in human NAD(P)H quinone oxidoreductase 1 (NQO1), a FAD-dependent enzyme which activates cancer pro-drugs and stabilizes several oncosuppressors. We show that p.P187S strongly destabilizes the NQO1 dimer in vitro and increases the flexibility of the C-terminal domain, while a combination of FAD and the inhibitor dicoumarol overcome these alterations. Additionally, changes in global stability due to polymorphisms and ligand binding are linked to the dynamics of the dimer interface, whereas the low activity and affinity for FAD in p.P187S is caused by increased fluctuations at the FAD binding site. Importantly, NQO1 steady-state protein levels in cell cultures correlate primarily with the dynamics of the C-terminal domain, supporting a directional preference in NQO1 proteasomal degradation and the use of ligands binding to this domain to stabilize p.P187S in vivo. In conclusion, protein dynamics are fundamental to understanding loss-of-function in p.P187S, and to develop new pharmacological therapies to rescue this function.

  10. Structural basis for epitope sharing between group 1 allergens of cedar pollen.

    PubMed

    Midoro-Horiuti, Terumi; Schein, Catherine H; Mathura, Venkatarajan; Braun, Werner; Czerwinski, Edmund W; Togawa, Akihisa; Kondo, Yasuto; Oka, Tetsuo; Watanabe, Masanao; Goldblum, Randall M

    2006-02-01

    The group 1 allergens are a major cause of cedar pollen hypersensitivity in several geographic areas. Allergens from several taxa have been shown to cross-react. The goal of these studies was to compare the structural features of the shared and unique epitopes of the group 1 allergen from mountain cedar (Jun a 1) and Japanese cedar (Cry j 1). An array of overlapping peptides from the sequence of Jun a 1 and a panel of monoclonal anti-Cry j 1 antibodies were used to identify the IgE epitopes recognized by cedar-sensitive patients from Texas and Japan. IgE from Japanese patients reacted with peptides representing one of the two linear epitopes within the highly conserved beta-helical core structure and both epitopes within less ordered loops and turns near the N- and C-termini of Jun a 1. A three-dimensional (3D) model of the Cry j 1, based on the crystal structure of Jun a 1, indicated a similar surface exposure for the four described epitopes of Jun a 1 and the homologous regions of Cry j 1. The monoclonal antibodies identified another shared epitope, which is most likely conformational and a unique Cry j 1 epitope that may be the previously recognized glycopeptide IgE epitope. Defining the structural basis for shared and unique epitopes will help to identify critical features of IgE epitopes that can be used to develop mimotopes or identify allergen homologues for vaccine development. PMID:15975657

  11. Structural basis for epitope sharing between group 1 allergens of cedar pollen

    PubMed Central

    Midoro-Horiuti, Terumi; Schein, Catherine H.; Mathura, Venkatarajan; Braun, Werner; Czerwinski, Edmund W.; Togawa, Akihisa; Kondo, Yasuto; Oka, Tetsuo; Watanabe, Masanao; Goldblum, Randall M.

    2008-01-01

    The group 1 allergens are a major cause of cedar pollen hypersensitivity in several geographic areas. Allergens from several taxa have been shown to cross-react. The goal of these studies was to compare the structural features of the shared and unique epitopes of the group 1 allergen from mountain cedar (Jun a 1) and Japanese cedar (Cry j 1). An array of overlapping peptides from the sequence of Jun a 1 and a panel of monoclonal anti-Cry j 1 antibodies were used to identify the IgE epitopes recognized by cedar-sensitive patients from Texas and Japan. IgE from Japanese patients reacted with peptides representing one of the two linear epitopes within the highly conserved β-helical core structure and both epitopes within less ordered loops and turns near the N- and C-termini of Jun a 1. A three-dimensional (3D) model of the Cry j 1, based on the crystal structure of Jun a 1, indicated a similar surface exposure for the four described epitopes of Jun a 1 and the homologous regions of Cry j 1. The monoclonal antibodies identified another shared epitope, which is most likely conformational and a unique Cry j 1 epitope that may be the previously recognized glycopeptide IgE epitope. Defining the structural basis for shared and unique epitopes will help to identify critical features of IgE epitopes that can be used to develop mimotopes or identify allergen homologues for vaccine development. PMID:15975657

  12. Mercury-induced DNA polymorphism: Probing the conformation of Hg(II)-DNA via Staphylococcal nuclease digestion and circular dichroism measurements

    SciTech Connect

    Gruenwedel, D.W.; Cruikshank, M.K. )

    1990-02-27

    Exposing native calf thymus DNA to increasing concentrations of Hg(ClO{sub 4}){sub 2} not only produces dramatic changes in its circular dichroism (CD) but results also in the decrease, and ultimate cessation, of endonucleolytic DNA cleavage by staphylococcal nuclease. DNA cleavage proceeds at or near the rates exhibited by untreated DNA. At Hg(II) levels of 0.08 < r < 0.5, the rate of DNA hydrolysis decreases monotonically with increasing Hg(II) concentrations, and at r > 0.4, DNA cleavage ceases. Both the CD changes and the changes in the rate of DNA digestion are totally reversible upon the removal of Hg(II). For comparison purposes, native calf thymus DNA was also treated with methylmercury (CH{sub 3}Hg(II)), an agent known to disrupt the secondary structure of DNA. The treatment yielded single-stranded methylmercurated DNA with preserved right-handed helix screwness. The authors interpret the Hg(II)-induced alterations in the CD of native calf thymus DNA, and the hydrolysis rate changes observed with staphylococcal nuclease, to indicate that Hg(II) either produces in DNA reversible B {leftrightarrow} Z transitions, passing transiently through C-like conformations, or generates non-B-conformational structures of presumably left-handed geometry.

  13. Characterization of epitopes involved in the neutralization of Pasteurella haemolytica serotype A1 leukotoxin.

    PubMed

    Lainson, F A; Murray, J; Davies, R C; Donachie, W

    1996-09-01

    Defined segments of the leukotoxin A gene (lktA) from an A1 serotype of Pasteurella haemolytica were cloned into a plasmid vector and expressed as LacZ alpha fusion proteins. These fusion proteins were electrophoresed in SDS-PAGE gels and their immunoblotting reactivities with several monoclonal antibodies characterized. The epitope recognized by a strongly neutralizing monoclonal antibody was localized to a 32 amino acid region near the C terminus of the leukotoxin A (LktA) molecule. The epitope recognized by a non-neutralizing antibody was localized to a 33 amino acid region immediately adjacent. Smaller recombinant peptides containing these epitopes were not antigenic, but a polypeptide encompassing 229 amino acids at the C terminus evoked neutralizing antibodies when used to immunize specific-pathogen-free lambs. The distributions of linear epitopes recognized by this antiserum and by antisera raised to full-length recombinant LktA and to native LktA produced by P. haemolytica serotype A1 were determined by their reactivities with a set of overlapping 10 amino acid synthetic peptides. This revealed a complex distribution of linear epitopes at the C-terminal end of LktA. Toxin-neutralizing antibodies in convalescent sheep serum were shown to be directed against conformational epitopes by selective absorption of antibodies directed against linear epitopes. PMID:8828217

  14. Palivizumab epitope-displaying virus-like particles protect rodents from RSV challenge.

    PubMed

    Schickli, Jeanne H; Whitacre, David C; Tang, Roderick S; Kaur, Jasmine; Lawlor, Heather; Peters, Cory J; Jones, Joyce E; Peterson, Darrell L; McCarthy, Michael P; Van Nest, Gary; Milich, David R

    2015-04-01

    Respiratory syncytial virus (RSV) is the most common cause of serious viral bronchiolitis in infants, young children, and the elderly. Currently, there is not an FDA-approved vaccine available for RSV, though the mAb palivizumab is licensed to reduce the incidence of RSV disease in premature or at-risk infants. The palivizumab epitope is a well-characterized, approximately 24-aa helix-loop-helix structure on the RSV fusion (F) protein (F254-277). Here, we genetically inserted this epitope and multiple site variants of this epitope within a versatile woodchuck hepadnavirus core-based virus-like particle (WHcAg-VLP) to generate hybrid VLPs that each bears 240 copies of the RSV epitope in a highly immunogenic arrayed format. A challenge of such an epitope-focused approach is that to be effective, the conformational F254-277 epitope must elicit antibodies that recognize the intact virus. A number of hybrid VLPs containing RSV F254-277 were recognized by palivizumab in vitro and elicited high-titer and protective neutralizing antibody in rodents. Together, the results from this proof-of-principle study suggest that the WHcAg-VLP technology may be an applicable approach to eliciting a response to other structural epitopes. PMID:25751145

  15. The CEA/CD3-Bispecific Antibody MEDI-565 (MT111) Binds a Nonlinear Epitope in the Full-Length but Not a Short Splice Variant of CEA

    PubMed Central

    Huang, Jiaqi; Brohawn, Philip; Morehouse, Chris; Lekstrom, Kristen; Baeuerle, Patrick A.; Wu, Herren; Yao, Yihong; Coats, Steven R.; Dall’Acqua, William; Damschroder, Melissa; Hammond, Scott A.

    2012-01-01

    MEDI-565 (also known as MT111) is a bispecific T-cell engager (BiTE®) antibody in development for the treatment of patients with cancers expressing carcinoembryonic antigen (CEA). MEDI-565 binds CEA on cancer cells and CD3 on T cells to induce T-cell mediated killing of cancer cells. To understand the molecular basis of human CEA recognition by MEDI-565 and how polymorphisms and spliced forms of CEA may affect MEDI-565 activity, we mapped the epitope of MEDI-565 on CEA using mutagenesis and homology modeling approaches. We found that MEDI-565 recognized a conformational epitope in the A2 domain comprised of amino acids 326–349 and 388–410, with critical residues F326, T328, N333, V388, G389, P390, E392, I408, and N410. Two non-synonymous single-nucleotide polymorphisms (SNPs) (rs10407503, rs7249230) were identified in the epitope region, but they are found at low homozygosity rates. Searching the National Center for Biotechnology Information GenBank® database, we further identified a single, previously uncharacterized mRNA splice variant of CEA that lacks a portion of the N-terminal domain, the A1 and B1 domains, and a large portion of the A2 domain. Real-time quantitative polymerase chain reaction analysis of multiple cancers showed widespread expression of full-length CEA in these tumors, with less frequent but concordant expression of the CEA splice variant. Because the epitope was largely absent from the CEA splice variant, MEDI-565 did not bind or mediate T-cell killing of cells solely expressing this form of CEA. In addition, the splice variant did not interfere with MEDI-565 binding or activity when co-expressed with full-length CEA. Thus MEDI-565 may broadly target CEA-positive tumors without regard for expression of the short splice variant of CEA. Together our data suggest that MEDI-565 activity will neither be impacted by SNPs nor by a splice variant of CEA. PMID:22574157

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

    PubMed Central

    Sandomenico, Annamaria; Leonardi, Antonio; Berisio, Rita; Sanguigno, Luca; Focà, Giuseppina; Focà, Annalia; Ruggiero, Alessia; Doti, Nunzianna; Muscariello, Livio; Barone, Daniela; Farina, Claudio; Owsianka, Ania; Vitagliano, Luigi

    2016-01-01

    ABSTRACT The hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a β-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412 to 422 (C-epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-epitope I and one of our MAbs (C2) showed that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody-combining site is rotated by 180° compared to the orientations of the other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high degree of selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape. IMPORTANCE Hepatitis C virus (HCV) remains a major health care burden, affecting almost 3% of the global population. The conserved epitope comprising residues 412 to 423 of the viral E2 glycoprotein is a valid vaccine candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope adopts a β-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic peptides mimicking this structure to elicit

  17. Immunogenicity of Stabilized HIV-1 Envelope Trimers with Reduced Exposure of Non-neutralizing Epitopes.

    PubMed

    de Taeye, Steven W; Ozorowski, Gabriel; Torrents de la Peña, Alba; Guttman, Miklos; Julien, Jean-Philippe; van den Kerkhof, Tom L G M; Burger, Judith A; Pritchard, Laura K; Pugach, Pavel; Yasmeen, Anila; Crampton, Jordan; Hu, Joyce; Bontjer, Ilja; Torres, Jonathan L; Arendt, Heather; DeStefano, Joanne; Koff, Wayne C; Schuitemaker, Hanneke; Eggink, Dirk; Berkhout, Ben; Dean, Hansi; LaBranche, Celia; Crotty, Shane; Crispin, Max; Montefiori, David C; Klasse, P J; Lee, Kelly K; Moore, John P; Wilson, Ian A; Ward, Andrew B; Sanders, Rogier W

    2015-12-17

    The envelope glycoprotein trimer mediates HIV-1 entry into cells. The trimer is flexible, fluctuating between closed and more open conformations and sometimes sampling the fully open, CD4-bound form. We hypothesized that conformational flexibility and transient exposure of non-neutralizing, immunodominant epitopes could hinder the induction of broadly neutralizing antibodies (bNAbs). We therefore modified soluble Env trimers to stabilize their closed, ground states. The trimer variants were indeed stabilized in the closed conformation, with a reduced ability to undergo receptor-induced conformational changes and a decreased exposure of non-neutralizing V3-directed antibody epitopes. In rabbits, the stabilized trimers induced similar autologous Tier-1B or Tier-2 NAb titers to those elicited by the corresponding wild-type trimers but lower levels of V3-directed Tier-1A NAbs. Stabilized, closed trimers might therefore be useful components of vaccines aimed at inducing bNAbs. PMID:26687358

  18. Immune recognition of citrullinated epitopes.

    PubMed

    Nguyen, Hai; James, Eddie A

    2016-10-01

    Conversion of arginine into citrulline is a post-translational modification that is observed in normal physiological processes. However, abnormal citrullination can provoke autoimmunity by generating altered self-epitopes that are specifically targeted by autoantibodies and T cells. In this review we discuss the recognition of citrullinated antigens in human autoimmune diseases and the role that this modification plays in increasing antigenic diversity and circumventing tolerance mechanisms. Early published work demonstrated that citrullinated proteins are specifically targeted by autoantibodies in rheumatoid arthritis and that citrullinated peptides are more readily presented to T cells by arthritis-susceptible HLA class II 'shared epitope' proteins. Emerging data support the relevance of citrullinated epitopes in other autoimmune diseases, including type 1 diabetes and multiple sclerosis, whose susceptible HLA haplotypes also preferentially present citrullinated peptides. In these settings, autoimmune patients have been shown to have elevated responses to citrullinated epitopes derived from tissue-specific antigens. Contrasting evidence implicates autophagy or perforin and complement-mediated membrane attack as inducers of ectopic citrullination. In either case, the peptidyl deiminases responsible for citrullination are activated in response to inflammation or insult, providing a mechanistic link between this post-translational modification and interactions with the environment and infection. As such, it is likely that immune recognition of citrullinated epitopes also plays a role in pathogen clearance. Indeed, our recent data suggest that responses to citrullinated peptides facilitate recognition of novel influenza strains. Therefore, increased understanding of responses to citrullinated epitopes may provide important insights about the initiation of autoimmunity and recognition of heterologous viruses. PMID:27531825

  19. Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-γ Using Human-Bovine Interferon-γ Chimeras

    PubMed Central

    Zuber, Bartek; Rudström, Karin; Ehrnfelt, Cecilia

    2016-01-01

    Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-γ. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-γ in ELISA, 2 epitope clusters with 5 mAbs in each were defined; 2 mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-γ, epitopes were identified using 7 h/bIFN-γ chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-γ residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2 mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN-γ-mediated activation of human cells, in line with the involvement of region A in the IFN-γ receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes. PMID:27336613

  20. Phage display revisited: Epitope mapping of a monoclonal antibody directed against Neisseria meningitidis adhesin A using the PROFILER technology.

    PubMed

    Cariccio, Veronica Lanza; Domina, Maria; Benfatto, Salvatore; Venza, Mario; Venza, Isabella; Faleri, Agnese; Bruttini, Marco; Bartolini, Erika; Giuliani, Marzia Monica; Santini, Laura; Brunelli, Brunella; Norais, Nathalie; Borgogni, Erica; Midiri, Angelina; Galbo, Roberta; Romeo, Letizia; Biondo, Carmelo; Masignani, Vega; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2016-01-01

    There is a strong need for rapid and reliable epitope mapping methods that can keep pace with the isolation of increasingly larger numbers of mAbs. We describe here the identification of a conformational epitope using Phage-based Representation OF ImmunoLigand Epitope Repertoire (PROFILER), a recently developed high-throughput method based on deep sequencing of antigen-specific lambda phage-displayed libraries. A novel bactericidal monoclonal antibody (mAb 9F11) raised against Neisseria meningitidis adhesin A (NadA), an important component of the Bexsero(®) anti-meningococcal vaccine, was used to evaluate the technique in comparison with other epitope mapping methods. The PROFILER technology readily identified NadA fragments that were capable of fully recapitulating the reactivity of the entire antigen against mAb 9F11. Further analysis of these fragments using mutagenesis and hydrogen-deuterium exchange mass-spectrometry allowed us to identify the binding site of mAb 9F11 (A250-D274) and an adjoining sequence (V275-H312) that was also required for the full functional reconstitution of the epitope. These data suggest that, by virtue of its ability to detect a great variety of immunoreactive antigen fragments in phage-displayed libraries, the PROFILER technology can rapidly and reliably identify epitope-containing regions and provide, in addition, useful clues for the functional characterization of conformational mAb epitopes. PMID:26963435

  1. Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-γ Using Human-Bovine Interferon-γ Chimeras.

    PubMed

    Zuber, Bartek; Rudström, Karin; Ehrnfelt, Cecilia; Ahlborg, Niklas

    2016-09-01

    Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-γ. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-γ in ELISA, 2 epitope clusters with 5 mAbs in each were defined; 2 mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-γ, epitopes were identified using 7 h/bIFN-γ chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-γ residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2 mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN-γ-mediated activation of human cells, in line with the involvement of region A in the IFN-γ receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes. PMID:27336613

  2. Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

    PubMed Central

    Keck, Zhen-Yong; Enterlein, Sven G.; Howell, Katie A.; Vu, Hong; Shulenin, Sergey; Warfield, Kelly L.; Froude, Jeffrey W.; Araghi, Nazli; Douglas, Robin; Biggins, Julia; Lear-Rooney, Calli M.; Wirchnianski, Ariel S.; Lau, Patrick; Wang, Yong; Herbert, Andrew S.; Dye, John M.; Glass, Pamela J.; Holtsberg, Frederick W.; Foung, Steven K. H.

    2015-01-01

    ABSTRACT Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCE Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus

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

    PubMed

    Chen, Edwin; Salinas, Nichole D; Huang, Yining; Ntumngia, Francis; Plasencia, Manolo D; Gross, Michael L; Adams, John H; Tolia, Niraj Harish

    2016-05-31

    Plasmodium vivax Duffy Binding Protein (PvDBP) is the most promising vaccine candidate for P. vivax malaria. The polymorphic nature of PvDBP induces strain-specific immune responses, however, and the epitopes of broadly neutralizing antibodies are unknown. These features hamper the rational design of potent DBP-based vaccines and necessitate the identification of globally conserved epitopes. Using X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange mass spectrometry, and mutational mapping, we have defined epitopes for three inhibitory mAbs (mAbs 2D10, 2H2, and 2C6) and one noninhibitory mAb (3D10) that engage DBP. These studies expand the currently known inhibitory epitope repertoire by establishing protective motifs in subdomain three outside the receptor-binding and dimerization residues of DBP, and introduce globally conserved protective targets. All of the epitopes are highly conserved among DBP alleles. The identification of broadly conserved epitopes of inhibitory antibodies provides critical motifs that should be retained in the next generation of potent vaccines for P. vivax malaria. PMID:27194724

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

    PubMed

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

    2016-07-01

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

  5. Novel CD8(+) cytotoxic T cell epitopes in bovine leukemia virus with cattle.

    PubMed

    Bai, Lanlan; Takeshima, Shin-Nosuke; Isogai, Emiko; Kohara, Junko; Aida, Yoko

    2015-12-16

    Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human T cell leukemia virus (HTLV). The cytotoxic T lymphocyte (CTL) plays a key role in suppressing the progression of disease caused by BLV. T and B cell epitopes in BLV have been studied, but CD8(+) CTL epitopes remain poorly understood. We used a library of 115 synthetic peptides covering the entirety of the Env proteins (gp51 and gp30), the Gag proteins (p15, p24, and p12), and the Tax protein of BLV to identify 11 novel CD8(+) T cell epitopes (gp51N5, gp51N11, gp51N12, gp30N5, gp30N6, gp30N8, gp30N16, tax16, tax18, tax19, and tax20) in four calves experimentally infected with BLV. The number of CD8(+) T cell epitopes that could be identified in each calf correlated with the BLV proviral load. Interestingly, among the 11 epitopes identified, only gp51N11 was capable of inducing CD8(+) T cell-mediated cytotoxicity in all four calves, but it is not a suitable vaccine target because it shows a high degree of polymorphism according to the Wu-Kabat variability index. By contrast, no CTL epitopes were identified from the Gag structural protein. In addition, several epitopes were obtained from gp30 and Tax, indicating that cellular immunity against BLV is strongly targeted to these proteins. CD8(+) CTL epitopes from gp30 and Tax were less polymorphic than epitopes from. Indeed, peptides tax16, tax18, tax19, and tax20 include a leucine-rich activation domain that encompasses a transcriptional activation domain, and the gp30N16 peptide contains a proline-rich region that interacts with a protein tyrosine phosphatase SHP1 to regulate B cell activation. Moreover, at least one CD8(+) CTL epitope derived from gp30 was identified in each of the four calves. These results indicate that BLV gp30 may be the best candidate for the development of a BLV vaccine. PMID:26552001

  6. Anti-beta-2 glycoprotein I epitope specificity: from experimental models to diagnostic tools.

    PubMed

    Meroni, P L

    2016-07-01

    Beta-2 glycoprotein I (β2GPI) is the main antigenic target for anti-phospholipid antibodies (aPL), the serological markers of anti-phospholipid syndrome (APS). Conformational changes of the molecule seem to be essential for exposing the cryptic epitope for aPL binding and to trigger pathogenic pathways. There is increasing evidence that a conformational epitope located in the Domain I (DI) of the molecule is the main epitope targeted by human autoantibodies. The pathogenic role of the DI epitope has been recently supported by in vivo models and by immuno-histopathological findings in APS patients. Antibodies targeting β2GPI-DI are more frequently detected in patients with full-blown APS compared to asymptomatic aPL carriers or patients with infectious diseases who have antibodies directed against the whole molecule. Anti-DI antibodies are positively correlated with medium to high titres of aPL, with the presence of lupus anticoagulant and thrombotic and pregnancy manifestations, enabling identification of patients at higher risk of clinical events. However, some APS patients develop antibodies reacting against β2GPI epitopes other than DI, suggesting that other anti-β2GPI antibody subsets may be clinically relevant. Although preliminary results suggest that anti-DI antibodies can be detected by different assays in a comparable manner, further prospective studies are needed to support their use in the clinical setting and their predictive value. PMID:27252268

  7. Polymorphs and Versatile Solvates of 7-Hydroxyisoflavone.

    PubMed

    Gong, Ningbo; Zhang, Guoshun; Jin, Guimin; Du, Guanhua; Lu, Yang

    2016-04-01

    7-hydroxyisoflavone has been crystallized, identified, and characterized as 2 solvent-free conformational polymorphs and 5 solvates, which differ from each other in the mode of packing and in molecular conformation. All the 7 crystal structures were previously unreported. The conformational polymorphs and solvates were compared by Hirshfeld surface and fingerprint plot analysis and were spectroscopically characterized by powder X-ray diffraction, differential scanning calorimetry, and thermal gravimetric analysis. Hydrogen bond played an important role in the formation of polymorphs. From this study, we can predict that more solvates could be cultivated in other polarity solvents such as isopropanol or 2-butanol at appropriate conditions. PMID:26935882

  8. Mapping epitopes and antigenicity by site-directed masking

    NASA Astrophysics Data System (ADS)

    Paus, Didrik; Winter, Greg

    2006-06-01

    Here we describe a method for mapping the binding of antibodies to the surface of a folded antigen. We first created a panel of mutant antigens (-lactamase) in which single surface-exposed residues were mutated to cysteine. We then chemically tethered the cysteine residues to a solid phase, thereby masking a surface patch centered on each cysteine residue and blocking the binding of antibodies to this region of the surface. By these means we mapped the epitopes of several mAbs directed to -lactamase. Furthermore, by depleting samples of polyclonal antisera to the masked antigens and measuring the binding of each depleted sample of antisera to unmasked antigen, we mapped the antigenicity of 23 different epitopes. After immunization of mice and rabbits with -lactamase in Freund's adjuvant, we found that the antisera reacted with both native and denatured antigen and that the antibody response was mainly directed to an exposed and flexible loop region of the native antigen. By contrast, after immunization in PBS, we found that the antisera reacted only weakly with denatured antigen and that the antibody response was more evenly distributed over the antigenic surface. We suggest that denatured antigen (created during emulsification in Freund's adjuvant) elicits antibodies that bind mainly to the flexible regions of the native protein and that this explains the correlation between antigenicity and backbone flexibility. Denaturation of antigen during vaccination or natural infections would therefore be expected to focus the antibody response to the flexible loops. backbone flexibility | Freund's adjuvant | conformational epitope | antisera

  9. Utilities for high throughput use of the single strand conformational polymorphism method: screening of 791 patients with familial hypercholesterolaemia for mutations in exon 3 of the low density lipoprotein receptor gene.

    PubMed Central

    Whittall, R; Gudnason, V; Weavind, G P; Day, L B; Humphries, S E; Day, I N

    1995-01-01

    We have modified several aspects of the single strand conformational polymorphism (SSCP) method to increase the speed with which the technique can be used for mutation detection. The methods attain high resolution of small mobility differences using long (30 cm) gels and use a modified polymerase reaction to maximise detection sensitivity using a minimised quantity of 32P. By using custom cut "sharktooth" combs (4.5 mm between teeth) as the slot formers, commercially available multichannel pipettes (9 mm tip to tip) can be used to load eight or 12 samples at a time from standard microtitre plates. PCR products that have been prepared and radiolabelled using simplified protocols are loaded on to the gel, and after a precalculated time of electrophoresis another set of samples can be loaded, either with combs moved across 2.25 mm or onto the same gel tracks. The run conditions are calculated so that there is no overlap between the bands produced by the two loadings, thus doubling the amount of information that can be gained from one gel. A computer program has been developed to solve equations to determine suitable timings for repetitive loadings. Finally, a modification of the gel pouring system is described so that two gels can be poured between three standard glass plates, with both gels run simultaneously. Of the order of 1000 PCR reactions can be prepared and analysed in 24 man hours using five 40 cm x 30 cm gel tanks. The application of these techniques is described to detect SSCPs in exon 3 of the low density lipoprotein receptor (LDLR) gene in 791 patients with familial hypercholesterolaemia (FH). Eight different SSCP patterns were seen, one of which was caused by the previously described E80K mutation, which was present in 11 patients (1.4%). In total, 32 patients (4%) were identified with exon 3 mutations. Images PMID:7562961

  10. The genetics of amphibian declines: Population substructure and molecular differentiation in the Yosemite Toad, Bufo canorus (Anura, Bufonidae) based on single-strand conformation polymorphism analysis (SSCP) and mitochondrial DNA sequence data

    USGS Publications Warehouse

    Bradley, Shaffer H.; Fellers, G.M.; Magee, A.; Randal, Voss S.

    2000-01-01

    We present a comprehensive survey of genetic variation across the range of the narrowly distributed endemic Yosemite toad Bufo canorus, a declining amphibian restricted to the Sierra Nevada of California. Based on 322 bp of mitochondrial cytochrome b sequence data, we found limited support for the monophyly of B. canorus and its closely related congener B. exsul to the exclusion of the widespread western toad B. boreas. However, B. exsul was always phylogenetically nested within B. canorus, suggesting that the latter may not be monophyletic. SSCP (single-strand conformation polymorphism) analysis of 372 individual B. canorus from 28 localities in Yosemite and Kings Canyon National Parks revealed no shared haplotypes among these two regions and lead us to interpret these two parks as distinct management units for B. canorus. Within Yosemite, we found significant genetic substructure both at the level of major drainages and among breeding ponds. Kings Canyon samples show a different pattern, with substantial variation among breeding sites, but no substructure among drainages. Across the range of B. canorus as well as among Yosemite ponds, we found an isolation-by-distance pattern suggestive of a stepping stone model of migration. However, in Kings Canyon we found no hint of such a pattern, suggesting that movement patterns of toads may be quite different in these nearby parklands. Our data imply that management for B. canorus should focus at the individual pond level, and effective management may necessitate reintroductions if local extirpations occur. A brief review of other pond-breeding anurans suggests that highly structured populations are often the case, and thus that our results for B. canorus may be general for other species of frogs and toads.

  11. A unique mutation in the vitamin D receptor gene in three Japanese patients with vitamin D-dependent rickets type II: utility of single-strand conformation polymorphism analysis for heterozygous carrier detection.

    PubMed Central

    Saijo, T; Ito, M; Takeda, E; Huq, A H; Naito, E; Yokota, I; Sone, T; Pike, J W; Kuroda, Y

    1991-01-01

    Vitamin D-dependent rickets type II is a hereditary disease resulting from a defective vitamin D receptor. In three Japanese patients with vitamin D-dependent rickets type II whose fibroblasts displayed normal cytosol binding and impaired nuclear uptake of 1,25-dihydroxyvitamin D3, western, Southern, and northern analyses failed to disclose any abnormalities in vitamin D3 receptor protein and its gene. Exons 2 and 3 of the vitamin D receptor cDNA, which encode the DNA-binding domain consisting of two zinc fingers, were amplified by PCR and sequenced to identify the specific mutations in the vitamin D receptor gene. In the three patients and one normal control a T-to-C transition was found in the putative initiation codon, while this transition was not observed in another normal control. This finding suggested that an original initiation codon was located at positions 10-12 in the human vitamin D receptor cDNA sequence reported previously. In contrast, a unique G-to-A transition at position 140 in exon 3, resulting in substitution of arginine by glutamine at residue 47, was revealed only in these three patients. The arginine at 47 is located between two zinc fingers and is conserved within all steroid hormone receptors. Therefore, it is highly conceivable that this amino acid substitution is responsible for the defect of the vitamin D receptor in the patients. Single-strand conformation polymorphism analysis of amplified DNA confirmed that all patients were homozygous and that parents from one family were heterozygous carriers for this mutation. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:1652893

  12. Identification and localization of minimal MHC-restricted CD8+ T cell epitopes within the Plasmodium falciparum AMA1 protein

    PubMed Central

    2010-01-01

    Background Plasmodium falciparum apical membrane antigen-1 (AMA1) is a leading malaria vaccine candidate antigen that is expressed by sporozoite, liver and blood stage parasites. Since CD8+ T cell responses have been implicated in protection against pre-erythrocytic stage malaria, this study was designed to identify MHC class I-restricted epitopes within AMA1. Methods A recombinant adenovirus serotype 5 vector expressing P. falciparum AMA1 was highly immunogenic when administered to healthy, malaria-naive adult volunteers as determined by IFN-γ ELISpot responses to peptide pools containing overlapping 15-mer peptides spanning full-length AMA1. Computerized algorithms (NetMHC software) were used to predict minimal MHC-restricted 8-10-mer epitope sequences within AMA1 15-mer peptides active in ELISpot. A subset of epitopes was synthesized and tested for induction of CD8+ T cell IFN-γ responses by ELISpot depletion and ICS assays. A 3-dimensional model combining Domains I + II of P. falciparum AMA1 and Domain III of P. vivax AMA1 was used to map these epitopes. Results Fourteen 8-10-mer epitopes were predicted to bind to HLA supertypes A01 (3 epitopes), A02 (4 epitopes), B08 (2 epitopes) and B44 (5 epitopes). Nine of the 14 predicted epitopes were recognized in ELISpot or ELISpot and ICS assays by one or more volunteers. Depletion of T cell subsets confirmed that these epitopes were CD8+ T cell-dependent. A mixture of the 14 minimal epitopes was capable of recalling CD8+ T cell IFN-γ responses from PBMC of immunized volunteers. Thirteen of the 14 predicted epitopes were polymorphic and the majority localized to the more conserved front surface of the AMA1 model structure. Conclusions This study predicted 14 and confirmed nine MHC class I-restricted CD8+ T cell epitopes on AMA1 recognized in the context of seven HLA alleles. These HLA alleles belong to four HLA supertypes that have a phenotypic frequency between 23% - 100% in different human populations. PMID

  13. T Cell Epitope Clustering in the Highly Immunogenic BZLF1 Antigen of Epstein-Barr Virus

    PubMed Central

    Rist, Melissa J.; Neller, Michelle A.; Burrows, Jacqueline M.

    2014-01-01

    ABSTRACT Polymorphism in the human leukocyte antigen (HLA) loci ensures that the CD8+ T cell response to viruses is directed against a diverse range of antigenic epitopes, thereby minimizing the impact of virus escape mutation across the population. The BZLF1 antigen of Epstein-Barr virus is an immunodominant target for CD8+ T cells, but the response has been characterized only in the context of a limited number of HLA molecules due to incomplete epitope mapping. We have now greatly expanded the number of defined CD8+ T cell epitopes from BZLF1, allowing the response to be evaluated in a much larger proportion of the population. Some regions of the antigen fail to be recognized by CD8+ T cells, while others include clusters of overlapping epitopes presented by different HLA molecules. These highly immunogenic regions of BZLF1 include polymorphic sequences, such that up to four overlapping epitopes are impacted by a single amino acid variation common in different regions of the world. This focusing of the immune response to limited regions of the viral protein could be due to sequence similarity to human proteins creating “immune blind spots” through self-tolerance. This study significantly enhances the understanding of the immune response to BZLF1, and the precisely mapped T cell epitopes may be directly exploited in vaccine development and adoptive immunotherapy. IMPORTANCE Epstein-Barr virus (EBV) is an important human pathogen, associated with several malignancies, including nasopharyngeal carcinoma and Hodgkin lymphoma. T lymphocytes are critical for virus control, and clinical trials aimed at manipulating this arm of the immune system have demonstrated efficacy in treating these EBV-associated diseases. These trials have utilized information on the precise location of viral epitopes for T cell recognition, for either measuring or enhancing responses. In this study, we have characterized the T cell response to the highly immunogenic BZLF1 antigen of EBV by

  14. Facts and fictions about polymorphism.

    PubMed

    Cruz-Cabeza, Aurora J; Reutzel-Edens, Susan M; Bernstein, Joel

    2015-12-01

    We present new facts about polymorphism based on (i) crystallographic data from the Cambridge Structural Database (CSD, a database built over 50 years of community effort), (ii) 229 solid form screens conducted at Hoffmann-La Roche and Eli Lilly and Company over the course of 8+ and 15+ years respectively and (iii) a dataset of 446 polymorphic crystals with energies and properties computed with modern DFT-d methods. We found that molecular flexibility or size has no correlation with the ability of a compound to be polymorphic. Chiral molecules, however, were found to be less prone to polymorphism than their achiral counterparts and compounds able to hydrogen bond exhibit only a slightly higher propensity to polymorphism than those which do not. Whilst the energy difference between polymorphs is usually less than 1 kcal mol(-1), conformational polymorphs are capable of differing by larger values (up to 2.5 kcal mol(-1) in our dataset). As overall statistics, we found that one in three compounds in the CSD are polymorphic whilst at least one in two compounds from the Roche and Lilly set display polymorphism with a higher estimate of up to three in four when compounds are screened intensively. Whilst the statistics provide some guidance of expectations, each compound constitutes a new challenge and prediction and realization of targeted polymorphism still remains a holy grail of materials sciences. PMID:26400501

  15. Epitope mapping by solution NMR spectroscopy.

    PubMed

    Bardelli, M; Livoti, E; Simonelli, L; Pedotti, M; Moraes, A; Valente, A P; Varani, L

    2015-06-01

    Antibodies play an ever more prominent role in basic research as well as in the biotechnology and pharmaceutical sectors. Characterizing their epitopes, that is, the region that they recognize on their target molecule, is useful for purposes ranging from molecular biology research to vaccine design and intellectual property protection. Solution NMR spectroscopy is ideally suited to the atomic level characterization of intermolecular interfaces and, as a consequence, to epitope discovery. Here, we illustrate how NMR epitope mapping can be used to rapidly and accurately determine protein antigen epitopes. The basic concept is that differences in the NMR signal of an antigen free or bound by an antibody will identify epitope residues. NMR epitope mapping provides more detailed information than mutagenesis or peptide mapping and can be much more rapid than X-ray crystallography. Advantages and drawbacks of this technique are discussed together with practical considerations. PMID:25726811

  16. Towards in silico prediction of immunogenic epitopes.

    PubMed

    Flower, Darren R

    2003-12-01

    As torrents of new data now emerge from microbial genomics, bioinformatic prediction of immunogenic epitopes remains challenging but vital. In silico methods often produce paradoxically inconsistent results: good prediction rates on certain test sets but not others. The inherent complexity of immune presentation and recognition processes complicates epitope prediction. Two encouraging developments - data driven artificial intelligence sequence-based methods for epitope prediction and molecular modeling methods based on three-dimensional protein structures - offer hope for the future. PMID:14644141

  17. Epitope characterization of an anti-PD-L1 antibody using orthogonal approaches.

    PubMed

    Hao, Gang; Wesolowski, John S; Jiang, Xuliang; Lauder, Scott; Sood, Vanita D

    2015-04-01

    The binding of programmed death ligand 1 protein (PD-L1) to its receptor programmed death protein 1 (PD-1) mediates immunoevasion in cancer and chronic viral infections, presenting an important target for therapeutic intervention. Several monoclonal antibodies targeting the PD-L1/PD-1 signaling axis are undergoing clinical trials; however, the epitopes of these antibodies have not been described. We have combined orthogonal approaches to localize and characterize the epitope of a monoclonal antibody directed against PD-L1 at good resolution and with high confidence. Limited proteolysis and mass spectrometry were applied to reveal that the epitope resides in the first immunoglobulin domain of PD-L1. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to identify a conformational epitope comprised of discontinuous strands that fold to form a beta sheet in the native structure. This beta sheet presents an epitope surface that significantly overlaps with the PD-1 binding interface, consistent with a desired PD-1 competitive mechanism of action for the antibody. Surface plasmon resonance screening of mutant PD-L1 variants confirmed that the region identified by HDX-MS is critical for the antibody interaction and further defined specific residues contributing to the binding energy. Taken together, the results are consistent with the observed inhibitory activity of the antibody on PD-L1-mediated immune evasion. This is the first report of an epitope for any antibody targeting PD-L1 and demonstrates the power of combining orthogonal epitope mapping techniques. PMID:25664688

  18. A novel monoclonal antibody to a defined peptide epitope in MUC16.

    PubMed

    Marcos-Silva, Lara; Ricardo, Sara; Chen, Kowa; Blixt, Ola; Arigi, Emma; Pereira, Daniela; Høgdall, Estrid; Mandel, Ulla; Bennett, Eric P; Vakhrushev, Sergey Y; David, Leonor; Clausen, Henrik

    2015-11-01

    The MUC16 mucin is overexpressed and aberrantly glycosylated in ovarian carcinomas. Immunodetection of circulating MUC16 is one of the most used cancer biomarker assays, but existing antibodies to MUC16 fail to distinguish normal and aberrant cancer glycoforms. Although all antibodies react 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 MUC16 immunity. Here, we developed a MUC16 vaccine based on a 1.7TR (264 aa) expressed in Escherichia coli and in vitro enzymatically glycosylated to generate the aberrant cancer-associated glycoform Tn. This vaccine elicited a potent serum IgG response in mice and we identified two major 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 of ovarian benign and cancer lesions, 5E11 showed similar reactivity as traditional MUC16 antibodies, suggesting that the epitope is not efficiently glycosylated. The study provides a vaccine design and immunodominant MUC16 TR epitopes. PMID:26201951

  19. Epitope Mapping of Anti-Interleukin-13 Neutralizing Antibody CNTO607

    SciTech Connect

    Teplyakov, Alexey; Obmolova, Galina; Wu, Sheng-Jiun; Luo, Jinquan; Kang, James; O'Neil, Karyn; Gilliland, Gary L.

    2009-06-24

    CNTO607 is a neutralizing anti-interleukin-13 (IL-13) human monoclonal antibody obtained from a phage display library. To determine how this antibody inhibits the biological effect of IL-13, we determined the binding epitope by X-ray crystallography. The crystal structure of the complex between CNTO607 Fab and IL-13 reveals the antibody epitope at the surface formed by helices A and D of IL-13. This epitope overlaps with the IL-4Ralpha/IL-13Ralpha1 receptor-binding site, which explains the neutralizing effect of CNTO607. The extensive antibody interface covers an area of 1000 A(2), which is consistent with the high binding affinity. The key features of the interface are the charge and shape complementarity of the molecules that include two hydrophobic pockets on IL-13 that accommodate Phe32 [complementarity-determining region (CDR) L2] and Trp100a (CDR H3) and a number of salt bridges between basic residues of IL-13 and acidic residues of the antibody. Comparison with the structure of the free Fab shows that the CDR residues do not change their conformation upon complex formation, with the exception of two residues in CDR H3, Trp100a and Asp100b, which change rotamer conformations. To evaluate the relative contribution of the epitope residues to CNTO607 binding, we performed alanine-scanning mutagenesis of the A-D region of IL-13. This study confirmed the primary role of electrostatic interactions for antigen recognition.

  20. Epitope specificity of human immunodeficiency virus-1 antibody dependent cellular cytotoxicity [ADCC] responses.

    PubMed

    Pollara, Justin; Bonsignori, Mattia; Moody, M Anthony; Pazgier, Marzena; Haynes, Barton F; Ferrari, Guido

    2013-07-01

    Antibody dependent cellular cytotoxicity [ADCC] has been suggested to play an important role in control of Human Immunodeficiency Virus-1 [HIV-1] viral load and protection from infection. ADCC antibody responses have been mapped to multiple linear and conformational epitopes within the HIV-1 envelope glycoproteins gp120 and gp41. Many epitopes targeted by antibodies that mediate ADCC overlap with those recognized by antibodies capable of virus neutralization. In addition, recent studies conducted with human monoclonal antibodies derived from HIV-1 infected individuals and HIV-1 vaccine-candidate vaccinees have identified a number of antibodies that lack the ability to capture primary HIV-1 isolates or mediate neutralizing activity, but are able to bind to the surface of infected CD4+ T cells and mediate ADCC. Of note, the conformational changes in the gp120 that may not exclusively relate to binding of the CD4 molecule are important in exposing epitopes recognized by ADCC responses. Here we discuss the HIV-1 envelope epitopes targeted by ADCC antibodies in the context of the potential protective capacities of ADCC. PMID:24191939

  1. Unconventional T-cell recognition of an arthritogenic epitope of proteoglycan aggrecan released from degrading cartilage.

    PubMed

    Falconer, Jane; Mahida, Rahul; Venkatesh, Divya; Pearson, Jeffrey; Robinson, John H

    2016-04-01

    It has been proposed that peptide epitopes bind to MHC class II molecules to form distinct structural conformers of the same MHC II-peptide complex termed type A and type B, and that the two conformers of the same peptide-MHC II complex are recognized by distinct CD4 T cells, termed type A and type B T cells. Both types recognize short synthetic peptides but only type A recognize endosomally processed intact antigen. Type B T cells that recognize self peptides from exogenously degraded proteins have been shown to escape negative selection during thymic development and so have the potential to contribute to the pathogenesis of autoimmunity. We generated and characterized mouse CD4 T cells specific for an arthritogenic epitope of the candidate joint autoantigen proteoglycan aggrecan. Cloned T-cell hybridomas specific for a synthetic peptide containing the aggrecan epitope showed two distinct response patterns based on whether they could recognize processed intact aggrecan. Fine mapping demonstrated that both types of T-cell recognized the same core epitope. The results are consistent with the generation of aggrecan-specific type A and type B T cells. Type B T cells were activated by supernatants released from degrading cartilage, indicating the presence of antigenic extracellular peptides or fragments of aggrecan. Type B T cells could play a role in the pathogenesis of proteoglycan-induced arthritis in mice, a model for rheumatoid arthritis, by recognizing extracellular peptides or protein fragments of joint autoantigens released by inflamed cartilage. PMID:26581676

  2. Epitope topography controls bioactivity in supramolecular nanofibers

    PubMed Central

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

    2015-01-01

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

  3. Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen.

    PubMed

    Malito, Enrico; Faleri, Agnese; Lo Surdo, Paola; Veggi, Daniele; Maruggi, Giulietta; Grassi, Eva; Cartocci, Elena; Bertoldi, Isabella; Genovese, Alessia; Santini, Laura; Romagnoli, Giacomo; Borgogni, Erica; Brier, Sébastien; Lo Passo, Carla; Domina, Maria; Castellino, Flora; Felici, Franco; van der Veen, Stijn; Johnson, Steven; Lea, Susan M; Tang, Christoph M; Pizza, Mariagrazia; Savino, Silvana; Norais, Nathalie; Rappuoli, Rino; Bottomley, Matthew J; Masignani, Vega

    2013-02-26

    Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen-antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of ∼1,000 Å(2) on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen-antibody interfaces are required to understand and design effective immunogens. PMID:23396847

  4. Epitope-Based Vaccine Target Screening against Highly Pathogenic MERS-CoV: An In Silico Approach Applied to Emerging Infectious Diseases

    PubMed Central

    Li, Sijin; Sun, Jing; Teng, Yumei; Wu, Meini; Li, Jianfan; Li, Yanhan; Hu, Ningzhu; Wang, Haixuan; Hu, Yunzhang

    2015-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) with pandemic potential is a major worldwide threat to public health. However, vaccine development for this pathogen lags behind as immunity associated with protection is currently largely unknown. In this study, an immunoinformatics-driven genome-wide screening strategy of vaccine targets was performed to thoroughly screen the vital and effective dominant immunogens against MERS-CoV. Conservancy and population coverage analysis of the epitopes were done by the Immune Epitope Database. The results showed that the nucleocapsid (N) protein of MERS-CoV might be a better protective immunogen with high conservancy and potential eliciting both neutralizing antibodies and T-cell responses compared with spike (S) protein. Further, the B-cell, helper T-cell and cytotoxic T lymphocyte (CTL) epitopes were screened and mapped to the N protein. A total of 15 linear and 10 conformal B-cell epitopes that may induce protective neutralizing antibodies were obtained. Additionally, a total of 71 peptides with 9-mer core sequence were identified as helper T-cell epitopes, and 34 peptides were identified as CTL epitopes. Based on the maximum HLA binding alleles, top 10 helper T-cell epitopes and CTL epitopes that may elicit protective cellular immune responses against MERS-CoV were selected as MERS vaccine candidates. Population coverage analysis showed that the putative helper T-cell epitopes and CTL epitopes could cover the vast majority of the population in 15 geographic regions considered where vaccine would be employed. The B- and T-cell stimulation potentials of the screened epitopes is to be further validated for their efficient use as vaccines against MERS-CoV. Collectively, this study provides novel vaccine target candidates and may prompt further development of vaccines against MERS-CoV and other emerging infectious diseases. PMID:26641892

  5. Identification of B- and T-Cell Epitopes of BB, a Carrier Protein Derived from the G Protein of Streptococcus Strain G148

    PubMed Central

    Goetsch, Liliane; Haeuw, Jean Francois; Champion, Thierry; Lacheny, Christine; N’Guyen, Thien; Beck, Alain; Corvaia, Nathalie

    2003-01-01

    Most conventional vaccines consist of killed organisms or purified antigenic proteins. Such molecules are generally poorly immunogenic and need to be coupled to carrier proteins. We have identified a new carrier molecule, BB, derived from the G protein of Streptococcus strain G148. We show that BB is able to induce strong antibody responses when conjugated to peptides or polysaccharides. In order to localize T and B cell epitopes in BB and match them with the albumin-binding region of the molecule, we immunized mice with BB, performed B and T pepscan analyses, and compared the results with pepscan done with sera and cells from humans. Our results indicate that BB has two distinct T helper epitopes, seven linear B-cell epitopes, and one conformational B-cell epitope in BALB/c mice. Four linear B-cell epitopes were identified from human sera, three of which overlapped mouse B-cell epitopes. Finally, three human T-cell epitopes were detected on the BB protein. One of these T-cell epitopes is common to BALB/c mice and humans and was localized in the region that contains the albumin-binding site. These data are of interest for the optimization of new carrier molecules derived from BB. PMID:12522050

  6. A new EV71 VP3 epitope in norovirus P particle vector displays neutralizing activity and protection in vivo in mice.

    PubMed

    Jiang, Liping; Fan, Rongjun; Sun, Shiyang; Fan, Peihu; Su, Weiheng; Zhou, Yan; Gao, Feng; Xu, Fei; Kong, Wei; Jiang, Chunlai

    2015-11-27

    Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16), as the main agents causing hand, foot and mouth disease (HFMD), have become a serious public health concern in the Asia-Pacific region. Recently, various neutralizing B cell epitopes of EV71 were identified as targets for promising vaccine candidates. Structural studies of Picornaviridae indicated that potent immunodominant epitopes typically lie in the hypervariable loop of capsid surfaces. However, cross-neutralizing antibodies and cross-protection between EV71 and CVA16 have not been observed. Therefore, we speculated that divergent sequences of the two viruses are key epitopes for inducing protective neutralizing responses. In this study, we selected 10 divergent epitope candidates based on alignment of the EV71 and CVA16 P1 amino acid sequences using the Multalin interface page, and these epitopes are conserved among all subgenotypes of EV71. Simultaneously, by utilizing the norovirus P particle as a novel vaccine delivery carrier, we identified the 71-6 epitope (amino acid 176-190 of VP3) as a conformational neutralizing epitope against EV71 in an in vitro micro-neutralization assay as well as an in vivo protection assay in mice. Altogether, these results indicated that the incorporation of the 71-6 epitope into the norovirus P domain can provide a promising candidate for an effective synthetic peptide-based vaccine against EV71. PMID:26529072

  7. The Immune Epitope Database 2.0

    PubMed Central

    Vita, Randi; Zarebski, Laura; Greenbaum, Jason A.; Emami, Hussein; Hoof, Ilka; Salimi, Nima; Damle, Rohini; Sette, Alessandro; Peters, Bjoern

    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 of 4 years, the data from 180 978 experiments were curated manually from the literature, which covers ∼99% of all publicly available information on peptide epitopes mapped in infectious agents (excluding HIV) and 93% of those mapped in allergens. In addition, data that would otherwise be unavailable to the public from 129 186 experiments were submitted directly by investigators. The curation of epitopes related to autoimmunity is expected to be completed by the end of 2010. The database can be queried by epitope structure, source organism, MHC restriction, assay type or host organism, among other criteria. The database structure, as well as its querying, browsing and reporting interfaces, was completely redesigned for the IEDB 2.0 release, which became publicly available in early 2009. PMID:19906713

  8. Conformational modulation mediated by polyglutamine expansion in CAG repeat expansion disease-associated proteins.

    PubMed

    Verani, Margherita; Bustamante, Maria; Martufi, Paola; Daldin, Manuel; Cariulo, Cristina; Azzollini, Lucia; Fodale, Valentina; Puglisi, Francesca; Weiss, Andreas; Macdonald, Douglas; Petricca, Lara; Caricasole, Andrea

    2016-09-16

    We have previously reported TR-FRET based immunoassays to detect a conformational change imparted on huntingtin protein by the polyglutamine expansion, which we confirmed using biophysical methodologies. Using these immunoassays, we now report that polyglutamine expansion influences the conformational properties of other polyglutamine disease proteins, exemplified by the androgen receptor (associated with spinal bulbar muscular atrophy) and TATA binding protein (associated with spinocerebellar ataxia 17). Using artificial constructs bearing short or long polyglutamine expansions or a multimerized, unrelated epitope (mimicking the increase in anti-polyglutamine antibody epitopes present in polyglutamine repeats of increasing length) we confirmed that the conformational TR-FRET based immunoassay detects an intrinsic conformational property of polyglutamine repeats. The TR-FRET based conformational immunoassay may represent a rapid, scalable tool to identify modulators of polyglutamine-mediated conformational change in different proteins associated with CAG triplet repeat disorders. PMID:27520369

  9. Disulfide-bonded discontinuous epitopes on the glycoprotein of vesicular stomatitis virus (New Jersey serotype).

    PubMed

    Grigera, P R; Keil, W; Wagner, R R

    1992-06-01

    ostensibly determines the conformational structure of the VSV-New Jersey G protein required for presentation of the major discontinuous epitopes recognized by neutralizing MAbs. PMID:1374811

  10. Identification of a highly conserved and surface exposed B-cell epitope on the nucleoprotein of influenza A virus.

    PubMed

    Gui, Xun; Ge, Pinghui; Wang, Xuliang; Yang, Kunyu; Yu, Hai; Zhao, Qinjian; Chen, Yixin; Xia, Ningshao

    2014-06-01

    Influenza virus still poses a major threat to human health worldwide. The nucleoprotein (NP) of influenza A virus plays an essential role in the viral replication and transcription and hence becomes a promising therapeutic target. NP forms a complicated conformation under native conditions and might denature when performing immunoassays such as western blot in the study of NP function. Therefore, it is useful to make an NP specific monoclonal antibody (mAb) that recognizes linear epitope instead of conformational epitope. In this study, a recombinant NP (rNP) of influenza A virus was over-expressed and used to generate a panel of anti-NP mAbs. These anti-NP mAbs were grouped into three classes based on their reactivity in Western blots. Only Class I mAb can react with linear rNP fragments. One of Class I mAb, 4D2, was characterized further by epitope mapping with a series of overlapping synthetic peptides, indicating that the 4D2 epitope is a surface exposed, linear epitope between amino acid residues 243 and 251. This epitope is highly conserved among different influenza A viruses with an identity of 98.4% (17,922/18,210). Western blot, co-immunoprecipitation, immunofluorescence, and immunohistochemistry experiments all indicated 4D2 is highly specific to NP of influenza A virus. The results demonstrated that 4D2 can be used as a research tool for functional study of NP in the replication cycle of influenza A virus. Further work is needed to understand the function and importance of this epitope. PMID:24136709

  11. Mapping of epitopes recognized by antibodies induced by immunization of mice with PspA and PspC.

    PubMed

    Vadesilho, Cintia F M; Ferreira, Daniela M; Gordon, Stephen B; Briles, David E; Moreno, Adriana T; Oliveira, Maria Leonor S; Ho, Paulo L; Miyaji, Eliane N

    2014-07-01

    Pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC) are important candidates for an alternative vaccine against pneumococcal infections. Since these antigens show variability, the use of variants that do not afford broad protection may lead to the selection of vaccine escape bacteria. Epitopes capable of inducing antibodies with broad cross-reactivities should thus be the preferred antigens. In this work, experiments using peptide arrays show that most linear epitopes recognized by antibodies induced in mice against different PspAs were located at the initial 44 amino acids of the mature protein and that antibodies against these linear epitopes did not confer protection against a lethal challenge. Conversely, linear epitopes recognized by antibodies to PspC included the consensus sequences involved in the interaction with human factor H and secretory immunoglobulin A (sIgA). Since linear epitopes of PspA were not protective, larger overlapping fragments containing 100 amino acids of PspA of strain Rx1 were constructed (fragments 1 to 7, numbered from the N terminus) to permit the mapping of antibodies with conformational epitopes not represented in the peptide arrays. Antibodies from mice immunized with fragments 1, 2, 4, and 5 were capable of binding onto the surface of pneumococci and mediating protection against a lethal challenge. The fact that immunization of mice with 100-amino-acid fragments located at the more conserved N-terminal region of PspA (fragments 1 and 2) induced protection against a pneumococcal challenge indicates that the induction of antibodies against conformational epitopes present at this region may be important in strategies for inducing broad protection against pneumococci. PMID:24807052

  12. Antibody Recognition of a Highly Conserved Influenza Virus Epitope

    SciTech Connect

    Ekiert, Damian C.; Bhabha, Gira; Elsliger, Marc-André; Friesen, Robert H.E.; Jongeneelen, Mandy; Throsby, Mark; Goudsmit, Jaap; Wilson, Ian A.; Scripps; Crucell

    2009-05-21

    Influenza virus presents an important and persistent threat to public health worldwide, and current vaccines provide immunity to viral isolates similar to the vaccine strain. High-affinity antibodies against a conserved epitope could provide immunity to the diverse influenza subtypes and protection against future pandemic viruses. Cocrystal structures were determined at 2.2 and 2.7 angstrom resolutions for broadly neutralizing human antibody CR6261 Fab in complexes with the major surface antigen (hemagglutinin, HA) from viruses responsible for the 1918 H1N1 influenza pandemic and a recent lethal case of H5N1 avian influenza. In contrast to other structurally characterized influenza antibodies, CR6261 recognizes a highly conserved helical region in the membrane-proximal stem of HA1 and HA2. The antibody neutralizes the virus by blocking conformational rearrangements associated with membrane fusion. The CR6261 epitope identified here should accelerate the design and implementation of improved vaccines that can elicit CR6261-like antibodies, as well as antibody-based therapies for the treatment of influenza.

  13. Purification of polyclonal anti-conformational antibodies for use in affinity selection from random peptide phage display libraries: A study using the hydatid vaccine EG95

    PubMed Central

    Read, A.J.; Gauci, C.G.; Lightowlers, M.W.

    2009-01-01

    The use of polyclonal antibodies to screen random peptide phage display libraries often results in the recognition of a large number of peptides that mimic linear epitopes on various proteins. There appears to be a bias in the use of this technology toward the selection of peptides that mimic linear epitopes. In many circumstances the correct folding of a protein immunogen is required for conferring protection. The use of random peptide phage display libraries to identify peptide mimics of conformational epitopes in these cases requires a strategy for overcoming this bias. Conformational epitopes on the hydatid vaccine EG95 have been shown to result in protective immunity in sheep, whereas linear epitopes are not protective. In this paper we describe a strategy that results in the purification of polyclonal antibodies directed against conformational epitopes while eliminating antibodies directed against linear epitopes. These affinity purified antibodies were then used to select a peptide from a random peptide phage display library that has the capacity to mimic conformational epitopes on EG95. This peptide was subsequently used to affinity purify monospecific antibodies against EG95. PMID:19349218

  14. Immunogenicity of stabilized HIV-1 envelope trimers with reduced exposure of non-neutralizing epitopes

    PubMed Central

    de Taeye, Steven W.; Ozorowski, Gabriel; de la Peña, Alba Torrents; Guttman, Miklos; Julien, Jean-Philippe; van den Kerkhof, Tom L.G.M.; Burger, Judith A.; Pritchard, Laura K.; Pugach, Pavel; Yasmeen, Anila; Crampton, Jordan; Hu, Joyce; Bontjer, Ilja; Torres, Jonathan L.; Arendt, Heather; DeStefano, Joanne; Koff, Wayne C.; Schuitemaker, Hanneke; Eggink, Dirk; Berkhout, Ben; Dean, Hansi; LaBranche, Celia; Crotty, Shane; Crispin, Max; Montefiori, David C.; Klasse, P. J.; Lee, Kelly K.; Moore, John P.; Wilson, Ian A.; Ward, Andrew B.; Sanders, Rogier W.

    2016-01-01

    Summary The envelope glycoprotein trimer mediates HIV-1 entry into cells. The trimer is flexible, fluctuating between closed and more open conformations and sometimes sampling the fully open, CD4-bound form. We hypothesized that conformational flexibility could hinder the induction of broadly neutralizing antibodies (bNAbs). We therefore modified soluble Env trimers to stabilize their closed, ground states. The trimer variants were indeed stabilized in the closed conformation, with a reduced ability to undergo receptor-induced conformational changes and a decreased exposure of non-neutralizing V3-directed antibody epitopes. In rabbits, the stabilized trimers induced similar autologous Tier-1B or Tier-2 NAb titers to those elicited by the corresponding wild-type trimers, but lower levels of V3-directed Tier-1A NAbs. Stabilized, closed trimers might therefore be useful components of vaccines aimed at inducing bNAbs. PMID:26687358

  15. The POM Monoclonals: A Comprehensive Set of Antibodies to Non-Overlapping Prion Protein Epitopes

    PubMed Central

    Polymenidou, Magdalini; Moos, Rita; Scott, Mike; Sigurdson, Christina; Shi, Yong-zhong; Yajima, Bill; Hafner-Bratkovič, Iva; Jerala, Roman; Hornemann, Simone; Wuthrich, Kurt; Bellon, Anne; Vey, Martin; Garen, Graciela; James, Michael N. G.; Kav, Nat; Aguzzi, Adriano

    2008-01-01

    PrPSc, a misfolded and aggregated form of the cellular prion protein PrPC, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrPC in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrPC and PrPSc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrPC. 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 PrPC. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrPSc. Other antibodies immunoprecipitate PrPC, but not PrPSc. 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 PrPC and PrPSc. Amino-proximal antibodies were found to react with repetitive PrPC 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. PMID:19060956

  16. Designing an efficient multi-epitope peptide vaccine against Vibrio cholerae via combined immunoinformatics and protein interaction based approaches.

    PubMed

    Nezafat, Navid; Karimi, Zeinab; Eslami, Mahboobeh; Mohkam, Milad; Zandian, Sanam; Ghasemi, Younes

    2016-06-01

    Cholera continues to be a major global health concern. Among different Vibrio cholerae strains, only O1 and O139 cause acute diarrheal diseases that are related to epidemic and pandemic outbreaks. The currently available cholera vaccines are mainly lived and attenuated vaccines consisting of V. cholerae virulence factors such as toxin-coregulated pili (TCP), outer membrane proteins (Omps), and nontoxic cholera toxin B subunit (CTB). Nowadays, there is a great interest in designing an efficient epitope vaccine against cholera. Epitope vaccines consisting of immunodominant epitopes and adjuvant molecules enhance the possibility of inciting potent protective immunity. In this study, V. cholerae protective antigens (OmpW, OmpU, TcpA and TcpF) and the CTB, which is broadly used as an immunostimulatory adjuvant, were analyzed using different bioinformatics and immunoinformatics tools. The common regions between promiscuous epitopes, binding to various HLA-II supertype alleles, and B-cell epitopes were defined based upon the aforementioned protective antigens. The ultimately selected epitopes and CTB adjuvant were fused together using proper GPGPG linkers to enhance vaccine immunogenicity. A three-dimensional model of the thus constructed vaccine was generated using I-TASSER. The model was structurally validated using the ProSA-web error-detection software and the Ramachandran plot. The validation results indicated that the initial 3D model needed refinement. Subsequently, a high-quality model obtained after various refinement cycles was used for defining conformational B-cell epitopes. Several linear and conformational B-cell epitopes were determined within the epitope vaccine, suggesting likely antibody triggering features of our designed vaccine. Next, molecular docking was performed between the 3D vaccine model and the tertiary structure of the toll like receptor 2 (TLR2). To gain further insight into the interaction between vaccine and TLR2, molecular dynamics

  17. Automatic Generation of Validated Specific Epitope Sets

    PubMed Central

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

    2015-01-01

    Accurate measurement of B and T cell responses is a valuable tool to study autoimmunity, allergies, immunity to pathogens, and host-pathogen interactions and assist in the design and evaluation of T cell vaccines and immunotherapies. In this context, it is desirable to elucidate a method to select validated reference sets of epitopes to allow detection of T and B cells. However, the ever-growing information contained in the Immune Epitope Database (IEDB) and the differences in quality and subjects studied between epitope assays make this task complicated. In this study, we develop a novel method to automatically select reference epitope sets according to a categorization system employed by the IEDB. From the sets generated, three epitope sets (EBV, mycobacteria and dengue) were experimentally validated by detection of T cell reactivity ex vivo from human donors. Furthermore, a web application that will potentially be implemented in the IEDB was created to allow users the capacity to generate customized epitope sets. PMID:26568965

  18. Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform

    PubMed Central

    Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; Venza, Mario; Venza, Isabella; Borgogni, Erica; Castellino, Flora; Midiri, Angelina; Galbo, Roberta; Romeo, Letizia; Biondo, Carmelo; Masignani, Vega; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2016-01-01

    We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete “hot spots” with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope. PMID:27530334

  19. Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform.

    PubMed

    Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; Venza, Mario; Venza, Isabella; Borgogni, Erica; Castellino, Flora; Midiri, Angelina; Galbo, Roberta; Romeo, Letizia; Biondo, Carmelo; Masignani, Vega; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2016-01-01

    We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete "hot spots" with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope. PMID:27530334

  20. Characterization of specific antigenic epitopes and the nuclear export signal of the Porcine circovirus 2 ORF3 protein.

    PubMed

    Gu, Jinyan; Wang, Lun; Jin, Yulan; Lin, Cui; Wang, Huijuan; Zhou, Niu; Xing, Gang; Liao, Min; Zhou, Jiyong

    2016-02-29

    Porcine circovirus 2 (PCV2) is the etiological agent of postweaning multisystemic wasting syndrome. PCV2 ORF3 protein is a nonstructural protein known to induce apoptosis, but little is known about the biological function of ORF3 protein. Therefore, we undertook this study to map ORF3 protein epitopes recognized by a panel of monoclonal antibodies (mAbs) and to characterize putative nuclear localization (NLS) and nuclear export (NES) sequences in ORF3. The linear epitopes targeted by two previously published mAbs 3B1 and 1H3 and a novel mouse mAb 3C3 were defined using overlapping pools of peptides. Here, we find that ORF3 in PCV2 infected cells contains a conformational epitope targeted by the antibody 3C3, which is distinct from linear epitopes recognized by the antibodies 3B1 and 1H3 in recombinant ORF3 protein. These results suggest that the linear epitope recognized by 3B1 and 1H3 is masked in PCV2 infected cells, and that the conformational epitope is unique to PCV2 infection. Furthermore, we find that ORF3 protein expressed in cytoplasm in early stages of PCV2 infection and then accumulated in nucleus over time. Moreover, we localize a NES at the N-terminus (residues 1-35aa) of ORF3 which plays critical role in nuclear export activity. These findings provide a novel insight that deepens our understanding of the biological function of PCV2 ORF3. PMID:26854343

  1. Persistent hydrogen bonding in polymorphic crystal structures.

    PubMed

    Galek, Peter T A; Fábián, László; Allen, Frank H

    2009-02-01

    The significance of hydrogen bonding and its variability in polymorphic crystal structures is explored using new automated structural analysis methods. The concept of a chemically equivalent hydrogen bond is defined, which may be identified in pairs of structures, revealing those types of bonds that may persist, or not, in moving from one polymorphic form to another. Their frequency and nature are investigated in 882 polymorphic structures from the Cambridge Structural Database. A new method to compare conformations of equivalent molecules is introduced and applied to derive distinct subsets of conformational and packing polymorphs. The roles of chemical functionality and hydrogen-bond geometry in persistent interactions are systematically explored. Detailed structural comparisons reveal a large majority of persistent hydrogen bonds that are energetically crucial to structural stability. PMID:19155561

  2. Design and Antigenic Epitopes Prediction of a New Trial Recombinant Multiepitopic Rotaviral Vaccine: In Silico Analyses.

    PubMed

    Jafarpour, Sima; Ayat, Hoda; Ahadi, Ali Mohammad

    2015-01-01

    Rotavirus is the major etiologic factor of severe diarrheal disease. Natural infection provides protection against subsequent rotavirus infection and diarrhea. This research presents a new vaccine designed based on computational models. In this study, three types of epitopes are considered-linear, conformational, and combinational-in a proposed model protein. Several studies on rotavirus vaccines have shown that VP6 and VP4 proteins are good candidates for vaccine production. In the present study, a fusion protein was designed as a new generation of rotavirus vaccines by bioinformatics analyses. This model-based study using ABCpred, BCPREDS, Bcepred, and Ellipro web servers showed that the peptide presented in this article has the necessary properties to act as a vaccine. Prediction of linear B-cell epitopes of peptides is helpful to investigate whether these peptides are able to activate humoral immunity. PMID:25965449

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

    PubMed

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

    2016-01-01

    Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100(mel)47-52/40-42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100(mel)47-52/40-42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8(+) T cell response. Importantly, we demonstrate that different gp100(mel)-derived spliced epitopes are generated and presented to CD8(+) T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100(mel)-derived spliced epitopes trigger activation of CD8(+) T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

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

    PubMed Central

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

    2016-01-01

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

  5. Mapping of neutralizing epitopes on Renibacterium salmoninarum p57 by use of transposon mutagenesis and synthetic peptides.

    PubMed

    Wiens, Gregory D; Owen, Jennifer

    2005-06-01

    Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish. PMID:15932983

  6. Mapping of Neutralizing Epitopes on Renibacterium salmoninarum p57 by Use of Transposon Mutagenesis and Synthetic Peptides

    PubMed Central

    Wiens, Gregory D.; Owen, Jennifer

    2005-01-01

    Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish. PMID:15932983

  7. Structure-Based Design of a Protein Immunogen that Displays an HIV-1 gp41 Neutralizing Epitope

    SciTech Connect

    Stanfield, Robyn L.; Julien, Jean-Philippe; Pejchal, Robert; Gach, Johannes S.; Zwick, Michael B.; Wilson, Ian A.

    2012-06-27

    Antibody Z13e1 is a relatively broadly neutralizing anti-human immunodeficiency virus type 1 antibody that recognizes the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Based on the crystal structure of an MPER epitope peptide in complex with Z13e1 Fab, we identified an unrelated protein, interleukin (IL)-22, with a surface-exposed region that is structurally homologous in its backbone to the gp41 Z13e1 epitope. By grafting the gp41 Z13e1 epitope sequence onto the structurally homologous region in IL-22, we engineered a novel protein (Z13-IL22-2) that contains the MPER epitope sequence for use as a potential immunogen and as a reagent for the detection of Z13e1-like antibodies. The Z13-IL22-2 protein binds Fab Z13e1 with a K{sub d} of 73 nM. The crystal structure of Z13-IL22-2 in complex with Fab Z13e1 shows that the epitope region is faithfully replicated in the Fab-bound scaffold protein; however, isothermal calorimetry studies indicate that Fab binding to Z13-IL22-2 is not a lock-and-key event, leaving open the question of whether conformational changes upon binding occur in the Fab, in Z13-IL-22, or in both.

  8. Discrimination and Variable Impact of ANCA Binding to Different Surface Epitopes on Proteinase 3, the Wegener’s Autoantigen

    PubMed Central

    Silva, Francisco; Hummel, Amber M.; Jenne, Dieter E.; Specks, Ulrich

    2010-01-01

    Proteinase 3 (PR3)-specific antineutrophil cytoplasmic antibodies (ANCA) are highly specific for the autoimmune small vessel vasculitis, Wegener’s granulomatosis (WG). PR3-ANCA have proven diagnostic value but their pathogenic potential and utility as a biomarker for disease activity remain unclear. PR3-ANCA recognize conformational epitopes, and epitope-specific PR3-ANCA subsets with variable impact on biological functions of PR3 have been postulated. The aims of this study were to identify specific PR3 surface epitopes recognized by monoclonal antibodies (moAbs) and to determine whether the findings can be used to measure the functional impact of epitope-specific PR3-ANCA and their potential relationship to disease activity. We used a novel flow cytometry assay based on TALON-beads coated with recombinant human (H) and murine (M) PR3 and 10 custom-designed chimeric human/mouse rPR3-variants (Hm1–5/Mh1–5) identifying 5 separate non-conserved PR3 surface epitopes. Anti-PR3 moAbs recognize 4 major surface epitopes, and we identified the specific surface location of 3 of these with the chimeric rPR3-variants. The ability of PR3-ANCA to inhibit the enzymatic activity of PR3 was measured indirectly using a capture-ELISA system based on the different epitopes recognized by capturing moAbs. Epitope-specific PR3-ANCA capture-ELISA results obtained from patient plasma (n=27) correlated with the inhibition of enzymatic activity of PR3 by paired IgG preparations (r=0.7, P<0.01). The capture-ELISA results also seem to reflect disease activity. In conclusion, insights about epitopes recognized by anti-PR3 moAbs can be applied to separate PR3-ANCA subsets with predictable functional qualities. The ability of PR3-ANCA to inhibit the enzymatic activity of PR3, a property linked to disease activity, can now be gauged using a simple epitope-based capture-ELISA system. PMID:20810247

  9. Sex influences on the penetrance of HLA shared-epitope genotypes for rheumatoid arthritis

    SciTech Connect

    Meyer, J.M.

    1996-02-01

    The association between rheumatoid arthritis (RA) and HLA DRB1 alleles may arise through linkage disequilibrium with a disease locus or the direct involvement of HLA alleles in RA. In support of the latter possibility, the shared-epitope hypothesis has been postulated, stating that conformationally similar DR{beta} chains encoded by several DRB1 alleles confer disease susceptibility. To examine these alternative hypotheses of marker-disease association and to investigate gender differences in RA susceptibility, we analyzed the distributions of PCR-based DRB1 genotypes of 309 Caucasian RA patients and 283 Caucasian controls. Initially, the marker-association-segregation {chi}{sup 2} method was used to evaluate evidence for linkage disequilibrium and the direct involvement of markers DR4 Dw4, DR4 Dw14, and DR1 in RA susceptibility. Additional shared-epitope models that grouped DRB1 alleles into five classes (*0401, *0404/*0102, *0405/*0408/*0101, *1001, and all others) and postulated relationships between genotypes and RA susceptibility were also fitted to observed genotypic distributions by the method of minimal {chi}{sup 2}. For females, a linkage-disequilibrium model provided a good fit to the data, as did a shared-epitope model with RA most penetrant among individuals with the *0401, *0401 genotype. For males, the best model indicated highest RA penetrance among shared-epitope compound heterozygotes. Clinically, male RA patients had more subcutaneous nodules and greater use of slowly acting antirheumatic drugs, while female RA patients had earlier disease onset. This study therefore suggests that sex-related factors influence the RA penetrance associated with DRB1 shared-epitope genotypes and that DRB1 effects on RA prognosis and pathogenesis should be considered separately for men and women. 67 refs., 7 tabs.

  10. Neutralizing Monoclonal Antibodies Directed against Defined Linear Epitopes on Domain 4 of Anthrax Protective Antigen▿

    PubMed Central

    Kelly-Cirino, Cassandra D.; Mantis, Nicholas J.

    2009-01-01

    The anthrax protective antigen (PA) is the receptor-binding subunit common to lethal toxin (LT) and edema toxin (ET), which are responsible for the high mortality rates associated with inhalational Bacillus anthracis infection. Although recombinant PA (rPA) is likely to be an important constituent of any future anthrax vaccine, evaluation of the efficacies of the various candidate rPA vaccines is currently difficult, because the specific B-cell epitopes involved in toxin neutralization have not been completely defined. In this study, we describe the identification and characterization of two murine monoclonal immunoglobulin G1 antibodies (MAbs), 1-F1 and 2-B12, which recognize distinct linear neutralizing epitopes on domain 4 of PA. 1-F1 recognized a 12-mer peptide corresponding to residues 692 to 703; this epitope maps to a region of domain 4 known to interact with the anthrax toxin receptor CMG-2 and within a conformation-dependent epitope recognized by the well-characterized neutralizing MAb 14B7. As expected, 1-F1 blocked PA's ability to associate with CMG-2 in an in vitro solid-phase binding assay, and it protected murine macrophage cells from intoxication with LT. 2-B12 recognized a 12-mer peptide corresponding to residues 716 to 727, an epitope located immediately adjacent to the core 14B7 binding site and a stretch of amino acids not previously identified as a target of neutralizing antibodies. 2-B12 was as effective as 1-F1 in neutralizing LT in vitro, although it only partially inhibited PA binding to its receptor. Mice passively administered 1-F1 or 2-B12 were partially protected against a lethal challenge with LT. These results advance our fundamental understanding of the mechanisms by which antibodies neutralize anthrax toxin and may have future application in the evaluation of candidate rPA vaccines. PMID:19703971

  11. Localization of immunodominant epitopes within the "a" determinant of hepatitis B surface antigen using monoclonal antibodies.

    PubMed

    Golsaz-Shirazi, Forough; Mohammadi, Hamed; Amiri, Mohammad Mehdi; Khoshnoodi, Jalal; Kardar, Gholam Ali; Jeddi-Tehrani, Mahmood; Shokri, Fazel

    2016-10-01

    The common "a" determinant is the major immunodominant region of hepatitis B surface antigen (HBsAg) shared by all serotypes and genotypes of hepatitis B virus (HBV). Antibodies against this region are thought to confer protection against HBV and are essential for viral clearance. Mutations within the "a" determinant may lead to conformational changes in this region, which can affect the binding of neutralizing antibodies. There is an increasing concern about identification and control of mutant viruses which is possible by comprehensive structural investigation of the epitopes located within this region. Anti-HBs monoclonal antibodies (mAbs) against different epitopes of HBsAg are a promising tool to meet this goal. In the present study, 19 anti-HBs mAbs were employed to map epitopes localized within the "a" determinant, using a panel of recombinant mutant HBsAgs. The topology of the epitopes was analyzed by competitive enzyme-linked immunosorbent assay (ELISA). Our results indicate that all of the mAbs seem to recognize epitopes within or in the vicinity of the "a" determinant of HBsAg. Different patterns of binding with mutant forms were observed with different mAbs. Amino acid substitutions at positions 123, 126, 129, 144, and 145 dramatically reduced the reactivity of antibodies with HBsAg. The T123N mutation had the largest impact on antibody binding to HBsAg. The reactivity pattern of our panel of mAbs with mutant forms of HBsAg could have important clinical implications for immunoscreening, diagnosis of HBV infection, design of a new generation of recombinant HB vaccines, and immunoprophylaxis of HBV infection as an alternative to therapy with hepatitis B immune globulin (HBIG). PMID:27439498

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

    PubMed

    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. PMID:26891811

  13. Mapping and characterization of antigenic epitopes of arginine kinase of Scylla paramamosain.

    PubMed

    Yang, Yang; Cao, Min-Jie; Alcocer, Marcos; Liu, Qing-Mei; Fei, Dan-Xia; Mao, Hai-Yan; Liu, Guang-Ming

    2015-06-01

    Arginine kinase (AK) is a panallergen present in crustaceans, which can induce an immunoglobulin (Ig) E-mediated immune response in humans. The aim of this work was to map and characterize the antigenic epitopes of Scylla paramamosain AK. Specific-protein-A-enriched IgG raised in rabbits against purified S. paramamosain AK was used to screen a phage display random peptide library. Five AK mimotope clones were identified among 20 random clones after biopanning. Four conformational epitopes D3A4K43M1A5T49T44I7, L31K33V35T32E11E18F14S34D37, V177G172M173D176Q178T174L181K175L187, and R202L170Y203E190P205W204L187T206Y145 were identified with the program LocaPep, and mapped to S. paramamosain AK. The key amino acids of these conformational epitopes were D3, K33, T174, and W204, respectively. On the basis of biopanning, six IgE-specific peptides were mapped with synthetic overlapping peptides using the sera from crab-allergic patients, and four seropositive peptides (amino acids 113-127, 127-141, 141-155, and 204-218) were confirmed as linear epitopes in a degranulation assay in RBL-2H3 cells. Stability experiments showed that the structural integrity of AK is essential for its allergenicity, and the intramolecular disulfide bond at Cys201-Cys271 is essential for its structural stability. PMID:25728640

  14. Calorimetric determinations and theoretical calculations of polymorphs of thalidomide

    NASA Astrophysics Data System (ADS)

    Lara-Ochoa, F.; Pérez, G. Espinosa; Mijangos-Santiago, F.

    2007-09-01

    The analysis of the thermograms of thalidomide obtained for the two reported polymorphs α and β by differential scanning calorimetry (DSC) shows some inconsistencies that are discussed in the present work. The conception of a new polymorph form, named β ∗, allowed us to explain the observed thermal behavior more satisfactorily. This new polymorph shows enantiotropy with both α and β polymorphs, reflected in the unique endotherm obtained in the DSC-thermograms, when a heating rate of 10 °C/min is applied. Several additional experiments, such as re-melting of both polymorph forms, showed that there is indeed a new polymorph with an endotherm located between the endotherms of α and β. IR, Raman, and powder X-ray permit us to characterize the isolated compound, resulting from the re-melting of both polymorph forms. Mechanical calculations were performed to elucidate the conformations of each polymorph, and ab initio quantum chemical calculations were performed to determine the energy of the more stable conformers and the spatial cell energy for both polymorphs α and β. These results suggested a possible conformation for the newly discovered polymorph β ∗.

  15. In silico predicted conserved B-cell epitopes in the Merozoite Surface Antigen -2 family of B. bovis are neutralization-sensitive

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Merozoite Surface Antigens-2 of Babesia bovis conform a family of GPI-anchored glycoproteins located at the parasite cell surface, that contain neutralization-sensitive B-cell epitopes, thus constituting putative vaccine candidates for bovine babesiosis. It was previously shown that (i) the MSA-...

  16. Identification of a novel overlapping sequential E epitope (E') on the bovine leukaemia virus SU glycoprotein and analysis of immunological data.

    PubMed

    Forti, Katia; Rizzo, Giorgia; Cagiola, Monica; Ferrante, Giovanna; Marini, Carla; Feliziani, Francesco; Pezzotti, Giovanni; De Giuseppe, Antonio

    2014-08-01

    Bovine leukaemia virus (BLV), an oncogenic C-type retrovirus, is the causative agent of enzootic bovine leucosis. Binding of BLV to its cellular receptor is mediated by the surface envelope glycoprotein subunit (SU). Previous studies have identified eight different epitopes (A through H) on the BLV SU. In this study, a new sequential epitope was identified using the monoclonal antibody 2G7 (MAb 2G7) on the C-terminal region of the BLV SU. To localise and refine the map of this epitope, a series of deleted forms in the C and N-terminal ends of the glycoprotein were made and synthesised in baculovirus and Escherichia coli expression systems. The synthetic proteins were analysed both in Western blot and MAb-capture ELISA assays. MAb 2G7 recognised a stretch of 11 amino acids, named epitope E', corresponding to residues 189-SDWVPSVRSWA-199 (comprising the 33 amino acids signal peptide) overlapping with the E epitope of the SU. The data obtained by Enzyme-Linked Immunosorbent Assay (ELISA) revealed that the E' epitope was hidden on whole BLV particles and that the variation in reactivity between epitope E' and MAb 2G7 depends on the glycosylation state of SU. Similarly, the analysis of immunological data evidenced that the failure of interaction between the MAb anti-DD' and its epitope was also due to a steric hindrance of the glycosylation. Finally, the ELISA assay analysis performed with the deleted and mutated forms of rSU evidenced that the conformational epitopes F, G and H lied into in the 34-173 amino-acids residues of N-terminal region of SU. PMID:24916842

  17. Maturation-Induced Cloaking of Neutralization Epitopes on HIV-1 Particles

    PubMed Central

    Joyner, Amanda S.; Willis, Jordan R.; Crowe, James E.; Aiken, Christopher

    2011-01-01

    To become infectious, HIV-1 particles undergo a maturation process involving proteolytic cleavage of the Gag and Gag-Pol polyproteins. Immature particles contain a highly stable spherical Gag lattice and are impaired for fusion with target cells. The fusion impairment is relieved by truncation of the gp41 cytoplasmic tail (CT), indicating that an interaction between the immature viral core and gp41 within the particle represses HIV-1 fusion by an unknown mechanism. We hypothesized that the conformation of Env on the viral surface is regulated allosterically by interactions with the HIV-1 core during particle maturation. To test this, we quantified the binding of a panel of monoclonal antibodies to mature and immature HIV-1 particles by immunofluorescence imaging. Surprisingly, immature particles exhibited markedly enhanced binding of several gp41-specific antibodies, including two that recognize the membrane proximal external region (MPER) and neutralize diverse HIV-1 strains. Several of the differences in epitope exposure on mature and immature particles were abolished by truncation of the gp41 CT, thus linking the immature HIV-1 fusion defect with altered Env conformation. Our results suggest that perturbation of fusion-dependent Env conformational changes contributes to the impaired fusion of immature particles. Masking of neutralization-sensitive epitopes during particle maturation may contribute to HIV-1 immune evasion and has practical implications for vaccine strategies targeting the gp41 MPER. PMID:21931551

  18. Activation of the NLRP3 inflammasome by vault nanoparticles expressing a chlamydial epitope

    PubMed Central

    Zhu, Ye; Jiang, Janina; Said-Sadier, Najwane; Boxx, Gale; Champion, Cheryl; Tetlow, Ashley; Kickhoefer, Valerie A.; Rome, Leonard H.; Ojcius, David M.; Kelly, Kathleen A.

    2014-01-01

    The full potential of vaccines relies on development of effective delivery systems and adjuvants and is critical for development of successful vaccine candidates. We have shown that recombinant vaults engineered to encapsulate microbial epitopes are highly stable structures and are an ideal vaccine vehicle for epitope delivery which does not require the inclusion of an adjuvant. We studied the ability of vaults which were engineered for use as a vaccine containing an immunogenic epitope of C. trachomatis, polymorphic membrane protein G (PmpG), to be internalized into human monocytes and behave as a “natural adjuvant”. We here show that incubation of monocytes with the PmpG-1-vaults activates caspase-1 and stimulates IL-1β secretion through a process requiring the NLRP3 inflammasome and that cathepsin B and Syk are involved in the inflammasome activation. We also observed that the PmpG-1-vaults are internalized through a pathway that is transiently acidic and leads to destabilization of lysosomes. In addition, immunization of mice with PmpG-1-vaults induced PmpG-1 responsive CD4+ cells upon re-stimulation with PmpG peptide in vitro, suggesting that vault vaccines can be engineered for specific adaptive immune responses. We conclude that PmpG-1-vault vaccines can stimulate NLRP3 inflammasomes and induce PmpG-specific T cell responses. PMID:25448112

  19. Attitudinal Conformity and Anonymity

    ERIC Educational Resources Information Center

    Tyson, Herbert; Kaplowitz, Stan

    1977-01-01

    Tested college students for conformity when conditions contributing to conformity were absent. Found that social pressures (responding in public, being surveyed by fellow group members) are necessary to produce conformity. (RL)

  20. Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein.

    PubMed

    Gilman, Morgan S A; Moin, Syed M; Mas, Vicente; Chen, Man; Patel, Nita K; Kramer, Kari; Zhu, Qing; Kabeche, Stephanie C; Kumar, Azad; Palomo, Concepción; Beaumont, Tim; Baxa, Ulrich; Ulbrandt, Nancy D; Melero, José A; Graham, Barney S; McLellan, Jason S

    2015-07-01

    Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens. PMID:26161532

  1. Characterization of a Prefusion-Specific Antibody That Recognizes a Quaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein

    PubMed Central

    Gilman, Morgan S. A.; Moin, Syed M.; Mas, Vicente; Chen, Man; Patel, Nita K.; Kramer, Kari; Zhu, Qing; Kabeche, Stephanie C.; Kumar, Azad; Palomo, Concepción; Beaumont, Tim; Baxa, Ulrich; Ulbrandt, Nancy D.; Melero, José A.; Graham, Barney S.; McLellan, Jason S.

    2015-01-01

    Prevention efforts for respiratory syncytial virus (RSV) have been advanced due to the recent isolation and characterization of antibodies that specifically recognize the prefusion conformation of the RSV fusion (F) glycoprotein. These potently neutralizing antibodies are in clinical development for passive prophylaxis and have also aided the design of vaccine antigens that display prefusion-specific epitopes. To date, prefusion-specific antibodies have been shown to target two antigenic sites on RSV F, but both of these sites are also present on monomeric forms of F. Here we present a structural and functional characterization of human antibody AM14, which potently neutralized laboratory strains and clinical isolates of RSV from both A and B subtypes. The crystal structure and location of escape mutations revealed that AM14 recognizes a quaternary epitope that spans two protomers and includes a region that undergoes extensive conformational changes in the pre- to postfusion F transition. Binding assays demonstrated that AM14 is unique in its specific recognition of trimeric furin-cleaved prefusion F, which is the mature form of F on infectious virions. These results demonstrate that the prefusion F trimer contains potent neutralizing epitopes not present on monomers and that AM14 should be particularly useful for characterizing the conformational state of RSV F-based vaccine antigens. PMID:26161532

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  4. Elicitation of HIV-1 neutralizing antibodies by presentation of 4E10 and 10E8 epitopes on Norovirus P particles.

    PubMed

    Yu, Yongjiao; Fu, Lu; Shi, Yuhua; Guan, Shanshan; Yang, Lan; Gong, Xin; Yin, He; He, Xiaoqiu; Liu, Dongni; Kuai, Ziyu; Shan, Yaming; Wang, Song; Kong, Wei

    2015-12-01

    Eliciting efficient broadly neutralizing antibodies (BnAbs) is a major goal in vaccine development against human immunodeficiency virus type 1 (HIV-1). Conserved epitopes in the membrane-proximal external region (MPER) of HIV-1 are a significant target. In this study, Norovirus P particles (NoV PPs) were used as carriers to display conformational 4E10 and 10E8 epitopes in different patterns with an appropriate linker. Immune responses to the recombinant NoV PPs were characterized in guinea pigs and Balb/c mice and could induce high levels of MPER-binding antibodies. Modest neutralizing activities could be detected in sera of guinea pigs but not of Balb/c mice. The 4E10 or 10E8 epitopes dispersed on three loops on the outermost surface of NoV PPs (4E10-loop123 PP or 10E8-loop123 PP) elicited higher neutralizing activities than the equivalent number of epitopes presented on loop 2 only (4E10-3loop2 PP or 10E8-3loop2 PP). The epitopes on different loops of the PP were well-exposed and likely formed an appropriate conformation to induce neutralizing antibodies. Although sera of immunized guinea pigs could neutralize several HIV envelope-pseudoviruses, a vaccine candidate for efficiently inducing HIV-1 BnAbs remains to be developed. PMID:26455781

  5. Molecular modeling and in-silico engineering of Cardamom mosaic virus coat protein for the presentation of immunogenic epitopes of Leptospira LipL32.

    PubMed

    Kumar, Vikram; Damodharan, S; Pandaranayaka, Eswari P J; Madathiparambil, Madanan G; Tennyson, Jebasingh

    2016-01-01

    Expression of Cardamom mosaic virus (CdMV) coat protein (CP) in E. coli forms virus-like particles. In this study, the structure of CdMV CP was predicted and used as a platform to display epitopes of the most abundant surface-associated protein, LipL32 of Leptospira at C, N, and both the termini of CdMV CP. In silico, we have mapped sequential and conformational B-cell epitopes from the crystal structure of LipL32 of Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130 using IEDB Elipro, ABCpred, BCPRED, and VaxiJen servers. Our results show that the epitopes displayed at the N-terminus of CdMV CP are promising vaccine candidates as compared to those displayed at the C-terminus or at both the termini. LipL32 epitopes, EP2, EP3, EP4, and EP6 are found to be promising B-cell epitopes for vaccine development. Based on the type of amino acids, length, surface accessibility, and docking energy with CdMV CP model, the order of antigenicity of the LipL32 epitopes was found to be EP4 > EP3 > EP2 > EP6. PMID:25692534

  6. Short epitope-based synthetic peptides for serodiagnosis of human strongyloidiasis.

    PubMed

    Feliciano, Nágilla D; Ribeiro, Vanessa S; Gonzaga, Henrique T; Santos, Fabiana A A; Fujimura, Patricia T; Goulart, Luiz R; Costa-Cruz, Julia M

    2016-04-01

    Strongyloidiasis is one of the major intestinal infections in humans, and a neglected tropical disease whose diagnosis still poses a challenge. We hypothesized that diagnostic tests based on short peptides containing major epitopes may represent a promising strategy to improve strongyloidiasis detection due to reduced cross-reactivity and higher sensitivity. Our aim was to evaluate two synthetic peptides selected by phage display (C10 and D3) as potential tools for serodiagnosis of strongyloidiasis, and to predict their putative antigen target. To investigate their diagnostic potential, we have tested different panels of serum samples (n=120) by enzyme linked immunosorbent assay (ELISA) to detect specific IgG, and their diagnostic parameters were calculated. Similarities with proteins from Strongyloides stercoralis were searched and conformational epitopes were predicted and aligned to known protein structures. Both C10 and D3 achieved sensitivity of 95%, and specificities were 89.2% and 92.5%, respectively. D3 presented the highest diagnostic efficiency (93.3%). Epitope prediction for both C10 and D3 led to the alignment with the cytochrome c oxidase subunit 1 structure. In brief, we propose two synthetic peptides as new biomarkers for serodiagnosis of strongyloidiasis, which can be promptly used for ELISA and in future field sensor platforms. PMID:26956434

  7. The Analysis of B-Cell Epitopes of Influenza Virus Hemagglutinin

    PubMed Central

    Shcherbinin, D.N.; Alekseeva, S.V.; Shmarov, M.M.; Smirnov, Yu.A.; Naroditskiy, B.S.; Gintsburg, A.L.

    2016-01-01

    Vaccination has been successfully used to prevent influenza for a long time. Influenza virus hemagglutinin (HA), which induces a humoral immune response in humans and protection against the flu, is the main antigenic component of modern influenza vaccines. However, new seasonal and pandemic influenza virus variants with altered structures of HA occasionally occur. This allows the pathogen to avoid neutralization with antibodies produced in response to previous vaccination. Development of a vaccine with the new variants of HA acting as antigens takes a long time. Therefore, during an epidemic, it is important to have passive immunization agents to prevent and treat influenza, which can be monoclonal or single-domain antibodies with universal specificity (broad-spectrum agents). We considered antibodies to conserved epitopes of influenza virus antigens as universal ones. In this paper, we tried to characterize the main B-cell epitopes of hemagglutinin and analyze our own and literature data on broadly neutralizing antibodies. We conducted a computer analysis of the best known conformational epitopes of influenza virus HAs using materials of different databases. The analysis showed that the core of the HA molecule, whose antibodies demonstrate pronounced heterosubtypic activity, can be used as a target for the search for and development of broad-spectrum antibodies to the influenza virus. PMID:27099781

  8. Scope for using plant viruses to present epitopes from animal pathogens.

    PubMed

    Porta; Lomonossoff

    1998-01-01

    Epitope presentation to the immune system for vaccination purposes can be achieved either via an inactivated or attenuated form of a pathogen or via its isolated antigenic sequences. When free, these peptides can adopt a variety of conformations, most of which will not exist in their native environment. Conjugation to carrier proteins restricts mobility of the peptides and increases their immunogenicity. A high local concentration of epitopes boosts the immune response further and can be generated by the use of self-aggregating carriers, such as the capsid proteins of viruses. In this regard plant viruses have in recent years started to make an impact as safer alternatives to the use of bacterial and attenuated animal viruses: the latter both require propagation in costly cell-culture systems where they can undergo reversion towards a virulent form and/or become contaminated by other pathogens. Plant virus-based vectors can be multiplied cheaply and to high yields (exceeding 1 mg/g plant tissue) in host plants. Both helical (tobacco mosaic virus, potato virus X, alfalfa mosaic virus) and icosahedral (cowpea mosaic virus, tomato bushy stunt virus) particles have been used to express a number of animal B-cell epitopes, whose immunogenic properties have been explored to varying degrees. Copyright 1998 John Wiley & Sons, Ltd. PMID:10398492

  9. The Analysis of B-Cell Epitopes of Influenza Virus Hemagglutinin.

    PubMed

    Shcherbinin, D N; Alekseeva, S V; Shmarov, M M; Smirnov, Yu A; Naroditskiy, B S; Gintsburg, A L

    2016-01-01

    Vaccination has been successfully used to prevent influenza for a long time. Influenza virus hemagglutinin (HA), which induces a humoral immune response in humans and protection against the flu, is the main antigenic component of modern influenza vaccines. However, new seasonal and pandemic influenza virus variants with altered structures of HA occasionally occur. This allows the pathogen to avoid neutralization with antibodies produced in response to previous vaccination. Development of a vaccine with the new variants of HA acting as antigens takes a long time. Therefore, during an epidemic, it is important to have passive immunization agents to prevent and treat influenza, which can be monoclonal or single-domain antibodies with universal specificity (broad-spectrum agents). We considered antibodies to conserved epitopes of influenza virus antigens as universal ones. In this paper, we tried to characterize the main B-cell epitopes of hemagglutinin and analyze our own and literature data on broadly neutralizing antibodies. We conducted a computer analysis of the best known conformational epitopes of influenza virus HAs using materials of different databases. The analysis showed that the core of the HA molecule, whose antibodies demonstrate pronounced heterosubtypic activity, can be used as a target for the search for and development of broad-spectrum antibodies to the influenza virus. PMID:27099781

  10. Characterization of a key neutralizing epitope on pertussis toxin recognized by the monoclonal antibody 1B7

    PubMed Central

    Sutherland, Jamie N; Maynard, Jennifer A

    2009-01-01

    Despite over five decades of research and vaccination, infection by Bordetella pertussis remains a serious disease with no specific treatments or validated correlates of protective immunity. Of the numerous monoclonal antibodies binding pertussis toxin (PTx) that have been produced and characterized, the murine IgG2a monoclonal antibody 1B7 is uniquely neutralizing in all in vitro assays and in vivo murine models of infection. 1B7 binds an epitope on the enzymatically active S1-subunit of PTx (PTx-S1) with some linear elements but previous work with S1 scanning peptides, phage displayed peptide libraries, and S1 truncation/deletion variants were unable to more precisely define the epitope. Using computational docking algorithms, alanine scanning mutagenesis, and surface plasmon resonance, we characterize the epitope bound by 1B7 on PTx-S1 in molecular detail and define energetically important interactions between residues at the interface. Six residues on PTx-S1 and six residues on 1B7 were identified which, when altered to alanine, resulted in variants with significantly reduced affinity for the native partner. Using this information, a model of the 1B7-S1 interaction was developed, indicating a predominantly conformational epitope located on the base of S1 near S4. The location of this epitope is consistent with previous data and is shown to be conserved across several naturally occurring strain variants including PTx-S1A, B (Tohama-I), D, and E (18-323) in addition to the catalytically inactive 9K/129G variant. This highly neutralizing but poorly immunogenic epitope may represent an important target for next generation vaccine development, identification of immune correlates and passive immunization strategies in pertussis. PMID:19899804

  11. Conserved Neutralizing Epitope at Globular Head of Hemagglutinin in H3N2 Influenza Viruses

    PubMed Central

    Iba, Yoshitaka; Fujii, Yoshifumi; Ohshima, Nobuko; Sumida, Tomomi; Kubota-Koketsu, Ritsuko; Ikeda, Mariko; Wakiyama, Motoaki; Shirouzu, Mikako; Okada, Jun; Okuno, Yoshinobu; Yokoyama, Shigeyuki

    2014-01-01

    ABSTRACT Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses. IMPORTANCE Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular

  12. Advances in the study of HLA-restricted epitope vaccines

    PubMed Central

    Zhao, Lingxiao; Zhang, Min; Cong, Hua

    2013-01-01

    Vaccination is a proven strategy for protection from disease. An ideal vaccine would include antigens that elicit a safe and effective protective immune response. HLA-restricted epitope vaccines, which include T-lymphocyte epitopes restricted by HLA alleles, represent a new and promising immunization approach. In recent years, research in HLA-restricted epitope vaccines for the treatment of tumors and for the prevention of viral, bacterial, and parasite-induced infectious diseases have achieved substantial progress. Approaches for the improvement of the immunogenicity of epitope vaccines include (1) improving the accuracy of the methods used for the prediction of epitopes, (2) making use of additional HLA-restricted CD8+ T-cell epitopes, (3) the inclusion of specific CD4+ T-cell epitopes, (4) adding B-cell epitopes to the vaccine construction, (5) finding more effective adjuvants and delivery systems, (6) using immunogenic carrier proteins, and (7) using multiple proteins as epitopes sources. In this manuscript, we review recent research into HLA-restricted epitope vaccines. PMID:23955319

  13. Epitopes expressed in different adenovirus capsid proteins induce different levels of epitope-specific immunity.

    PubMed

    Krause, Anja; Joh, Ju H; Hackett, Neil R; Roelvink, Peter W; Bruder, Joseph T; Wickham, Thomas J; Kovesdi, Imre; Crystal, Ronald G; Worgall, Stefan

    2006-06-01

    On the basis of the concept that the capsid proteins of adenovirus (Ad) gene transfer vectors can be genetically manipulated to enhance the immunogenicity of Ad-based vaccines, the present study compared the antiantigen immunogenicity of Ad vectors with a common epitope of the hemagglutinin (HA) protein of the influenza A virus incorporated into the outer Ad capsid protein hexon, penton base, fiber knob, or protein IX. Incorporation of the same epitope into the different capsid proteins provided insights into the correlation between epitope position and antiepitope immunity. Following immunization of three different strains of mice (C57BL/6, BALB/c, and CBA) with either an equal number of Ad particles (resulting in a different total HA copy number) or different Ad particle numbers (to achieve the same HA copy number), the highest primary (immunoglobulin M [IgM]) and secondary (IgG) anti-HA humoral and cellular CD4 gamma interferon and interleukin-4 responses against HA were always achieved with the Ad vector carrying the HA epitope in fiber knob. These observations suggest that the immune response against an epitope inserted into Ad capsid proteins is not necessarily dependent on the capsid protein number and imply that the choice of incorporation site in Ad capsid proteins in their use as vaccines needs to be compared in vivo. PMID:16699033

  14. Epitope location for two monoclonal antibodies against human cystatin C, representing opposite aggregation inhibitory properties.

    PubMed

    Behrendt, Izabela; Prądzińska, Martyna; Spodzieja, Marta; Kołodziejczyk, Aleksandra S; Rodziewicz-Motowidło, Sylwia; Szymańska, Aneta; Czaplewska, Paulina

    2016-07-01

    Human cystatin C (hCC), like many other amyloidogenic proteins, dimerizes and possibly makes aggregates by subdomain swapping. Inhibition of the process should suppress the fibrillogenesis leading to a specific amyloidosis (hereditary cystatin C amyloid angiopathy, HCCAA). It has been reported that exogenous agents like monoclonal antibodies against cystatin C are able to suppress formation of cystatin C dimers and presumably control the neurodegenerative disease. We have studied in detail two monoclonal antibodies (mAbs) representing very different aggregation inhibitory potency, Cyst10 and Cyst28, to find binding sites in hCC sequence responsible for the immunocomplex formation and pave the way for possible immunotherapy of HCCAA. We used the epitope extraction/excision mass spectrometry approach with the use of different enzymes complemented by affinity studies with synthetic hCC fragments as a basic technique for epitope identification. The results were analyzed in the context of hCC structure allowing us to discuss the binding sites for both antibodies. Epitopic sequences for clone Cyst28 which is a highly potent dimerization inhibitor were found in N-terminus, loop 1 and 2 (L1, L2) and fragments of β2 and β3 strands. The crucial difference between conformational epitope sequences found for both mAbs seems to be the lack of interactions with hCC via N-terminus and the loop 1 in the case of mAb Cyst10. Presumably the interactions of mAbs with hCC via L1 and β sheet fragments make the hCC structure rigid and unable to undergo the swapping process. PMID:27143169

  15. Epitope Mapping of Rhi o 1 and Generation of a Hypoallergenic Variant: A CANDIDATE MOLECULE FOR FUNGAL ALLERGY VACCINES.

    PubMed

    Sircar, Gaurab; Jana, Kuladip; Dasgupta, Angira; Saha, Sudipto; Gupta Bhattacharya, Swati

    2016-08-19

    Efficacy of allergen-specific immunotherapy is often severely impaired by detrimental IgE-mediated side effects of native allergen during vaccination. Here, we present the molecular determinants for IgE recognition of Rhi o 1 and eventually converting the allergen into a hypoallergenic immunogen to restrain health hazards during desensitization. Rhi o 1 is a respiratory fungal allergen. Despite having cross-reactivity with cockroach allergen, we observed that non-cross-reactive epitope predominantly determined IgE binding to Rhi o 1. Denaturation and refolding behavior of the allergen confirmed that its IgE reactivity was not essentially conformation-dependent. A combinatorial approach consisting of computational prediction and a peptide-based immunoassay identified two peptides ((44)TGEYLTQKYFNSQRNN and (311)GAEKNWAGQYVVDCNK) of Rhi o 1 that frequently reacted with IgE antibodies of sensitized patients. Interestingly, these peptides did not represent purely linear IgE epitopes but were presented in a conformational manner by forming a spatially clustered surface-exposed epitope conferring optimal IgE-binding capacity to the folded allergen. Site-directed alanine substitution identified four residues of the IgE epitope that were crucial for antibody binding. A multiple mutant (T49A/Y52A/K314A/W316A) showing 100-fold lower IgE binding and reduced allergenic activity was generated. The TYKW mutant retained T-cell epitopes, as evident from its lymphoproliferative capacity but down-regulated pro-allergic IL-5 secretion. The TYKW mutant induced enhanced focusing of blocking IgG antibodies specifically toward the IgE epitope of the allergen. Anti-TYKW mutant polyclonal IgG antibodies competitively inhibited binding of IgE antibodies to Rhi o 1 up to 70% and suppressed allergen-mediated histamine release by 10-fold. In conclusion, this is a simple yet rational strategy based on epitope mapping data to develop a genetically modified hypoallergenic variant showing

  16. Molecular evolution of a central region containing B cell epitopes in the gene encoding the p67 sporozoite antigen within a field population of Theileria parva.

    PubMed

    Obara, Isaiah; Ulrike, Seitzer; Musoke, Tony; Spooner, Paul R; Jabbar, Ahmed; Odongo, David; Kemp, Stephen; Silva, Joana C; Bishop, Richard P

    2015-05-01

    Protective immunity induced by the infective sporozoite stage of Theileria parva indicates a potential role for antibodies directed against conserved serologically reactive regions of the major sporozoite surface antigen p67 in vaccination to control the parasite. We have examined the allelic variation and determined the extent of B cell epitope polymorphism of the gene encoding p67 among field isolates originating from cattle exposed to infected ticks in the Marula area of the rift valley in central Kenya where the African cape buffalo (Syncerus caffer) and cattle co-graze. In the first of two closely juxtaposed epitope sequences in the central region of the p67 protein, an in-frame deletion of a seven-amino acid segment results in a truncation that was observed in parasites derived from cattle that co-grazed with buffalo. In contrast, the variation in the second epitope was primarily due to nonsynonymous substitutions, resulting in relatively low overall amino acid conservation in this segment of the protein. We also observed polymorphism in the region of the protein adjacent to the two defined epitopes, but this was not sufficient to provide statistically significant evidence for positive selection. The data indicates that B cell epitopes previously identified within the p67 gene are polymorphic within the Marula field isolates. Given the complete sequence identity of the p67 gene in all previously characterized T. parva isolates that are transmissible between cattle by ticks, the diversity observed in p67 from the Marula isolates in combination with the clinical reaction of the infected cattle is consistent with them originating from ticks that had acquired T. parva from buffalo. PMID:25673078

  17. Induced Secondary Structure and Polymorphism in an Intrinsically Disordered Structural Linker of the CNS: Solid-State NMR and FTIR Spectroscopy of Myelin Basic Protein Bound to Actin

    PubMed Central

    Ahmed, Mumdooh A.M.; Bamm, Vladimir V.; Shi, Lichi; Steiner-Mosonyi, Marta; Dawson, John F.; Brown, Leonid; Harauz, George; Ladizhansky, Vladimir

    2009-01-01

    Abstract The 18.5 kDa isoform of myelin basic protein (MBP) is a peripheral membrane protein that maintains the structural integrity of the myelin sheath of the central nervous system by conjoining the cytoplasmic leaflets of oligodendrocytes and by linking the myelin membrane to the underlying cytoskeleton whose assembly it strongly promotes. It is a multifunctional, intrinsically disordered protein that behaves primarily as a structural stabilizer, but with elements of a transient or induced secondary structure that represent binding sites for calmodulin or SH3-domain-containing proteins, inter alia. In this study we used solid-state NMR (SSNMR) and Fourier transform infrared (FTIR) spectroscopy to study the conformation of 18.5 kDa MBP in association with actin microfilaments and bundles. FTIR spectroscopy of fully 13C,15N-labeled MBP complexed with unlabeled F-actin showed induced folding of both protein partners, viz., some increase in β-sheet content in actin, and increases in both α-helix and β-sheet content in MBP, albeit with considerable extended structure remaining. Solid-state NMR spectroscopy revealed that MBP in MBP-actin assemblies is structurally heterogeneous but gains ordered secondary structure elements (both α-helical and β-sheet), particularly in the terminal fragments and in a central immunodominant epitope. The overall conformational polymorphism of MBP is consistent with its in vivo roles as both a linker (membranes and cytoskeleton) and a putative signaling hub. PMID:19134474

  18. Minute Time Scale Prolyl Isomerization Governs Antibody Recognition of an Intrinsically Disordered Immunodominant Epitope*

    PubMed Central

    Fassolari, Marisol; Chemes, Lucia B.; Gallo, Mariana; Smal, Clara; Sánchez, Ignacio E.; de Prat-Gay, Gonzalo

    2013-01-01

    Conformational rearrangements in antibody·antigen recognition are essential events where kinetic discrimination of isomers expands the universe of combinations. We investigated the interaction mechanism of a monoclonal antibody, M1, raised against E7 from human papillomavirus, a prototypic viral oncoprotein and a model intrinsically disordered protein. The mapped 12-amino acid immunodominant epitope lies within a “hinge” region between the N-terminal intrinsically disordered and the C-terminal globular domains. Kinetic experiments show that despite being within an intrinsically disordered region, the hinge E7 epitope has at least two populations separated by a high energy barrier. Nuclear magnetic resonance traced the origin of this barrier to a very slow (t½ ∼4 min) trans-cis prolyl isomerization event involving changes in secondary structure. The less populated (10%) cis isomer is the binding-competent species, thus requiring the 90% of molecules in the trans configuration to isomerize before binding. The association rate for the cis isomer approaches 6 × 107 m−1 s−1, a ceiling for antigen-antibody interactions. Mutagenesis experiments showed that Pro-41 in E7Ep was required for both binding and isomerization. After a slow postbinding unimolecular rearrangement, a consolidated complex with KD = 1.2 × 10−7 m is reached. Our results suggest that presentation of this viral epitope by the antigen-presenting cells would have to be “locked” in the cis conformation, in opposition to the most populated trans isomer, in order to select the specific antibody clone that goes through affinity and kinetic maturation. PMID:23504368

  19. The mepsMAP server. Mapping epitopes on protein surface: mining annotated proteins.

    PubMed

    Carrabino, D; D'Onorio De Meo, P; Sanna, N; Castrignanò, T; Orsini, M; Floris, M; Tramontano, A

    2007-06-01

    For a growing number of biologists DNA or protein data are typically retrieved and managed on the Web, and not in the laboratory. A large number of bioinformatics datasets from primary and (thousands of) secondary databases are scattered on the Web in various formats. A biologist end-user might need to access and use tens of databases and tools every day. For this reason, the bioinformatics community is developing more and more service-oriented architectures (SOAs): software architecture of loosely coupled software services that can be accessed without knowledge of, or control over, their internal architecture. Data-processing and analysis tasks can be automated by having free access to bioinformatics Web services (WSs) that are the building blocks of the SOAs. In this paper we introduce a new bioinformatics Web server, mepsMAP (mapping epitopes on protein surface: Mining Annotated Proteins), developed to identify the recognition sites between antibodies and their cognate antigens. In some cases, the recognition site is represented by a continuous segment of the antigen sequence, but much more often the epitope is "conformational," i.e., the antibody recognizes the location and type of exposed antigen side chains that are not necessarily contiguous in the antigen's sequence, but brought together by its three-dimensional structure. A facility on the server allows the user to search putative conformational epitopes on protein surface, querying the system for proteins with a given annotation. The mepsMAP server has been implemented as a SOA composed by a database and a set of four WSs. We present here the software architecture of the system with a detailed description of the WS dataflow that has been optimized to provide the best computing performance while maintaining the easiest end-user access to the system via a Web interface. PMID:17695751

  20. A Novel Antibody Humanization Method Based on Epitopes Scanning and Molecular Dynamics Simulation

    PubMed Central

    Zhao, Bin-Bin; Gong, Lu-Lu; Jin, Wen-Jing; Liu, Jing-Jun; Wang, Jing-Fei; Wang, Tian-Tian; Yuan, Xiao-Hui; He, You-Wen

    2013-01-01

    1-17-2 is a rat anti-human DEC-205 monoclonal antibody that induces internalization and delivers antigen to dendritic cells (DCs). The potentially clinical application of this antibody is limited by its murine origin. Traditional humanization method such as complementarity determining regions (CDRs) graft often leads to a decreased or even lost affinity. Here we have developed a novel antibody humanization method based on computer modeling and bioinformatics analysis. First, we used homology modeling technology to build the precise model of Fab. A novel epitope scanning algorithm was designed to identify antigenic residues in the framework regions (FRs) that need to be mutated to human counterpart in the humanization process. Then virtual mutation and molecular dynamics (MD) simulation were used to assess the conformational impact imposed by all the mutations. By comparing the root-mean-square deviations (RMSDs) of CDRs, we found five key residues whose mutations would destroy the original conformation of CDRs. These residues need to be back-mutated to rescue the antibody binding affinity. Finally we constructed the antibodies in vitro and compared their binding affinity by flow cytometry and surface plasmon resonance (SPR) assay. The binding affinity of the refined humanized antibody was similar to that of the original rat antibody. Our results have established a novel method based on epitopes scanning and MD simulation for antibody humanization. PMID:24278299

  1. Translation of HLA-HIV associations to the cellular level: HIV adapts to inflate CD8 T cell responses against Nef and HLA-adapted variant epitopes.

    PubMed

    Almeida, Coral-Ann M; Bronke, Corine; Roberts, Steven G; McKinnon, Elizabeth; Keane, Niamh M; Chopra, Abha; Kadie, Carl; Carlson, Jonathan; Haas, David W; Riddler, Sharon A; Haubrich, Richard; Heckerman, David; Mallal, Simon; John, Mina

    2011-09-01

    Strong statistical associations between polymorphisms in HIV-1 population sequences and carriage of HLA class I alleles have been widely used to identify possible sites of CD8 T cell immune selection in vivo. However, there have been few attempts to prospectively and systematically test these genetic hypotheses arising from population-based studies at a cellular, functional level. We assayed CD8 T cell epitope-specific IFN-γ responses in 290 individuals from the same cohort, which gave rise to 874 HLA-HIV associations in genetic analyses, taking into account autologous viral sequences and individual HLA genotypes. We found immunological evidence for 58% of 374 associations tested as sites of primary immune selection and identified up to 50 novel HIV-1 epitopes using this reverse-genomics approach. Many HLA-adapted epitopes elicited equivalent or higher-magnitude IFN-γ responses than did the nonadapted epitopes, particularly in Nef. At a population level, inclusion of all of the immunoreactive variant CD8 T cell epitopes in Gag, Pol, Nef, and Env suggested that HIV adaptation leads to an inflation of Nef-directed immune responses relative to other proteins. We concluded that HLA-HIV associations mark viral epitopes subject to CD8 T cell selection. These results can be used to guide functional studies of specific epitopes and escape mutations, as well as to test, train, and evaluate analytical models of viral escape and fitness. The inflation of Nef and HLA-adapted variant responses may have negative effects on natural and vaccine immunity against HIV and, therefore, has implications for diversity coverage approaches in HIV vaccine design. PMID:21821798

  2. Differential basal-to-apical accessibility of lamin A/C epitopes in the nuclear lamina regulated by changes in cytoskeletal tension

    NASA Astrophysics Data System (ADS)

    Ihalainen, Teemu O.; Aires, Lina; Herzog, Florian A.; Schwartlander, Ruth; Moeller, Jens; Vogel, Viola

    2015-12-01

    Nuclear lamins play central roles at the intersection between cytoplasmic signalling and nuclear events. Here, we show that at least two N- and C-terminal lamin epitopes are not accessible at the basal side of the nuclear envelope under environmental conditions known to upregulate cell contractility. The conformational epitope on the Ig-domain of A-type lamins is more buried in the basal than apical nuclear envelope of human mesenchymal stem cells undergoing osteogenesis (but not adipogenesis), and in fibroblasts adhering to rigid (but not soft) polyacrylamide hydrogels. This structural polarization of the lamina is promoted by compressive forces, emerges during cell spreading, and requires lamin A/C multimerization, intact nucleoskeleton-cytoskeleton linkages (LINC), and apical-actin stress-fibre assembly. Notably, the identified Ig-epitope overlaps with emerin, DNA and histone binding sites, and comprises various laminopathy mutation sites. Our findings should help decipher how the physical properties of cellular microenvironments regulate nuclear events.

  3. Fake conformal symmetry in conformal cosmological models

    NASA Astrophysics Data System (ADS)

    Jackiw, R.; Pi, So-Young

    2015-03-01

    We examine the local conformal invariance (Weyl invariance) in tensor-scalar theories used in recently proposed conformal cosmological models. We show that the Noether currents associated with Weyl invariance in these theories vanish. We assert that the corresponding Weyl symmetry does not have any dynamical role.

  4. Using patient serum to epitope map soybean glycinins reveals common epitopes shared with many legumes and tree nuts.

    PubMed

    Saeed, Hanaa; Gagnon, Christine; Cober, Elroy; Gleddie, Steve

    2016-02-01

    Soybean consumption is increasing in many Western diets; however, recent reviews suggest that the prevalence of soy allergy can be as high as 0.5% for the general population and up to 13% for children. The immunoglobulin-E (IgE) binding of sera from six soy-sensitive adult human subjects to soybean proteins separated by 2D gel electrophoresis was studied. Synthetic peptide sets spanning the mature glycinin subunit A2 and A3 primary sequences were used to map the IgE-binding regions. Putative epitopes identified in this study were also localized on glycinin hexamer models using bioinformatics software. We identified linear IgE-binding epitopes of the major storage protein Gly m 6 by screening individual soy-sensitive patient sera. These epitopes were then further analysed by 3D in silico model localization and compared to other plant storage protein epitopes. Web-based software applications were also used to study the ability to accurately predict epitopes with mixed results. A total of nine putative IgE-binding epitopes were identified in the glycinin A3 (A3.1-A3.3) and A2 (A2.1-A2.6) subunits. Most patients' sera IgE bound to only one or two epitopes, except for one patient's serum which bound to four different A2 epitopes. Two epitopes (A3.2 and A2.4) overlapped with a previously identified epitope hot spot of 11S globulins from other plant species. Most epitopes were predicted to be exposed on the surface of the 3D model of the glycinin hexamer. Amino acid sequence alignments of soybean acidic glycinins and other plant globulins revealed one dominant epitope hot spot among the four reported hot spots. This study may be helpful for future development of soy allergy immunotherapy and diagnosis. PMID:26766775

  5. Conformations of rat brain hexokinase: studies with monoclonal antibodies and peptide mapping techniques

    SciTech Connect

    Smith, A.D.; Wilson, J.E.

    1987-05-01

    Brain hexokinase (HK) consists of a single polypeptide chain with M/sub r/ approx. 100,000. Limited proteolysis of native HK with trypsin yields three major fragments, thought to correspond to discrete structural domains, with molecular masses of approximately 10, 50, and 40 kDa, and derived from the N-terminal, central, and C-terminal regions, respectively. Additional tryptic cleavage sites become susceptible in conformations induced by binding of specific ligands. A library of monoclonal antibodies has been developed for use as probes in examining the structure and function of domains present in HK. Epitopes for several of these have been mapped to specific structural regions in HK. Immunoblotting experiments with these antibodies have been useful in defining the location of susceptible proteolytic cleavage sites in various ligand-induced conformations of HK. Effects of ligand binding on epitope recognition have indicated that several of the antibodies interact with epitopes perturbed by ligand-induced conformational changes. Effects of antibody binding on function have also provided a basis for establishing structure-function relationships. For example, several of the monoclonal antibodies inhibit binding of hexokinase to mitochondria and all recognize epitopes located in the 10 kDa N-terminal domain, consistent with other results indicating that this region is critical to the binding function of hexokinase.

  6. Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

    PubMed Central

    Sarkar, Pampi; Labranche, Celia; Go, Eden P.; Clark, Daniel F.; Sun, Yide; Nandi, Avishek; Hartog, Karin; Desaire, Heather; Montefiori, David; Carfi, Andrea; Srivastava, Indresh K.; Barnett, Susan W.

    2013-01-01

    Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will ‘snap’ Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to ‘lock’ gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules. PMID:24146829

  7. Quantitative and epitope-specific antigenicity analysis of the human papillomavirus 6 capsid protein in aqueous solution or when adsorbed on particulate adjuvants.

    PubMed

    Li, Min; Wang, Xin; Cao, Lu; Lin, Zhijie; Wei, Minxi; Fang, Mujin; Li, Shaowei; Zhang, Jun; Xia, Ningshao; Zhao, Qinjian

    2016-08-17

    Human papillomavirus (HPV) 6 is a human pathogen which causes genital warts. Recombinant virus-like particle (VLP) based antigens are the active components in prophylactic vaccines to elicit functional antibodies. The binding and functional characteristics of a panel of 15 murine monoclonal antibodies (mAbs) against HPV6 was quantitatively assessed. Elite conformational indicators, recognizing the conformational epitopes, are also elite viral neutralizers as demonstrated with their viral neutralization efficiency (5 mAbs with neutralization titer below 4ng/mL) in a pseudovirion (PsV)-based system. The functionality of a given mAb is closely related to the nature of the corresponding epitope, rather than the apparent binding affinity to antigen. The epitope-specific antigenicity assays can be used to assess the binding activity of PsV or VLP preparations to neutralizing mAbs. These mAb-based assays can be used for process monitoring and for product release and characterization to confirm the existence of functional epitopes in purified antigen preparations. Due to the particulate nature of the alum adjuvants, the vaccine antigen adsorbed on adjuvants was considered largely as "a black box" due to the difficulty in analysis and visualization. Here, a novel method with fluorescence-based high content imaging for visualization and quantitating the immunoreactivity of adjuvant-adsorbed VLPs with neutralizing mAbs was developed, in which antigen desorption was not needed. The facile and quantitative in situ antigenicity analysis was amendable for automation. The integrity of a given epitope or two non-overlapping epitopes on the recombinant VLPs in their adjuvanted form can be assessed in a quantitative manner for cross-lot or cross-product comparative analysis with minimal manipulation of samples. PMID:27426626

  8. Identification of an epitope in the C terminus of normal prion protein whose expression is modulated by binding events in the N terminus.

    PubMed

    Li, R; Liu, T; Wong, B S; Pan, T; Morillas, M; Swietnicki, W; O'Rourke, K; Gambetti, P; Surewicz, W K; Sy, M S

    2000-08-18

    We have characterized the epitopes of a panel of 12 monoclonal antibodies (Mabs) directed to normal human cellular prion protein (PrP(C)) using ELISA and Western blotting of recombinant PrP or synthetic peptide fragments of PrP. The first group of antibodies, which is represented by Mabs 5B2 and 8B4, reacts with PrP(23-145), indicating that the epitopes for these Mabs are located in the 23 to 145 N-terminal region of human PrP. The second group includes Mabs 1A1, 6H3, 7A9, 8C6, 8H4, 9H7 and 2G8. These antibodies bind to epitopes localized within N-terminally truncated recombinant PrP(90-231). Finally, Mabs 5C3, 2C9 and 7A12 recognize both PrP(23-145) and PrP(90-231), suggesting that the epitopes for this group are located in the region encompassing residues 90 to 145. By Western blotting with PepSpot(TM), only three of Mabs studied (5B2, 8B4 and 2G8) bind to linear epitopes that are present in 13-residue long synthetic peptides corresponding to human PrP fragments. The remaining nine Mabs appear to recognize conformational epitopes. Two N terminus-specific Mabs were found to prevent the binding of the C terminus-specific Mab 6H3. This observation suggests that the unstructured N-terminal region may influence the local conformation within the folded C-terminal domain of prion protein. PMID:10966770

  9. Polymorphic light eruption

    MedlinePlus

    ... outdoors. Wear a sun hat. Wear sunglasses with UV protection. Use a lip balm with sunscreen. Alternative Names Polymorphic light eruption; Photodermatosis; PMLE Images Polymorphic light eruption on ...

  10. A Phosphorylation-Induced Turn Defines the Alzheimer's Disease AT8 Antibody Epitope on the Tau Protein.

    PubMed

    Gandhi, Neha S; Landrieu, Isabelle; Byrne, Cillian; Kukic, Predrag; Amniai, Laziza; Cantrelle, François-Xavier; Wieruszeski, Jean-Michel; Mancera, Ricardo L; Jacquot, Yves; Lippens, Guy

    2015-06-01

    Post mortem biochemical staging of Alzheimer's disease is currently based on immunochemical analysis of brain slices with the AT8 antibody. The epitope of AT8 is described around the pSer202/pThr205 region of the hyperphosphorylated form of the neuronal protein tau. In this study, NMR spectroscopy was used to precisely map the AT8 epitope on phosphorylated tau, and derive its defining structural features by a combination of NMR analyses and molecular dynamics. A particular turn conformation is stabilized by a hydrogen bond of the phosphorylated Thr205 residue to the amide proton of Gly207, and is further stabilized by the two Arg residues opposing the pSer202/pThr205. PMID:25881502

  11. A single mouse monoclonal antibody, E58 modulates multiple IgE epitopes on group 1 cedar pollen allergens.

    PubMed

    Goldblum, Randall M; Ning, Bo; Judy, Barbara M; Holthauzen, Luis Marcelo F; van Bavel, Julius; Kamijo, Atsushi; Midoro-Horiuti, Terumi

    2016-06-01

    We recently described a dominant role for conformational epitopes on the group 1 allergen of the mountain cedar (Juniperus ashei, Cupressaceae), Jun a 1, in pollen hypersensitivity in South Central U.S.A. Since these epitopes are surface exposed and are likely to be flexible, they may be susceptible to molecular or physical perturbations. This may make Jun a 1 a potential target for new forms of therapy for cedar pollinosis. Here, we describe a mouse monoclonal antibody, termed E58, which binds to the group 1 allergens of the cedar pollens from three highly populated regions of the world (central U.S.A., France and Japan). Upon binding to these allergens, E58 strongly reduces the binding of patient's IgE antibodies to these dominant allergens. This characteristic of E58, and potentially other similar antibodies, suggests an opportunity to develop preventative or therapeutic agents that may inhibit cedar pollen sensitization or prevent their allergic reactions. PMID:27174188

  12. Copper induces conformational changes in the N-terminal part of cell-surface PrPC.

    PubMed

    Leclerc, E; Serban, H; Prusiner, S B; Burton, D R; Williamson, R A

    2006-11-01

    Prion diseases are caused by misfolding of the cellular prion protein, PrPC. In vitro studies have shown that PrP binds copper via the octarepeat region lying within the unstructured N-terminal segment of the protein, but the significance of copper in PrP metabolism remains unclear. Here, six specific antibodies recognizing different epitope regions of PrP were used to measure the effect of copper on the conformation of the molecule at the cell surface. Binding of an antibody, E149, to an epitope within the octarepeat domain of PrP is halved in the presence of copper, whereas binding of antibodies recognizing epitope motifs C-terminal to residue 90 of PrP remain relatively unaltered under equivalent conditions. These experiments strongly suggest that copper induces localized conformational change within the N-terminal portion of cell-surface PrPC. PMID:16791441

  13. Identification of surface-exposed linear B-cell epitopes of the nonfimbrial adhesin CS31A of Escherichia coli by using overlapping peptides and antipeptide antibodies.

    PubMed Central

    Méchin, M C; Rousset, E; Girardeau, J P

    1996-01-01

    As a first step toward the design of an epitope vaccine, by using the nonfimbrial adhesin CS31A of Escherichia coli as a carrier, a low-resolution topological and epitope map of the CS31A subunit was developed by using solid-phase peptide synthesis and polyclonal rabbit antibodies raised against both native and denatured proteins. Peptides constituting antigenic epitopes on the major subunit (ClpG) of the multimeric CS31A antigen were identified by examining the binding of the antibodies to 249 overlapping nonapeptides covering the amino acid sequence of ClpG. With antibodies raised against denatured ClpG subunit, seven major epitope regions, corresponding to residues 10 to 18, 45 to 58, 88 to 107, 148 to 172, 187 to 196, 212 to 219, and 235 to 241, were located. Most of the epitopes were hydrophilic and were located in variable regions, residing largely in loop regions at the boundaries of secondary structural elements of ClpG. In contrast, antibodies raised against native CS31A antigen reacted only with the peptide AVNPNA (positions 179 to 184), demonstrating that this peptide was the only linear B-cell epitope of the native protein. The different immunogenic profiles of native CS31A antigen and denatured ClpG indicated that the denaturation process resulted in marked conformational changes in the protein, which could expose epitopes hidden or absent in native CS31A. To identify the surface-exposed epitopes, nine peptides covering the dominant antigenic regions of ClpG were synthesized and used to prepare site-specific antibodies. Antipeptide antibodies were tested, in a competitive enzyme-linked immunosorbent assay (ELISA), for cross-reactivity with native CS31A and denatured ClpG subunit. Four of these antipeptide antibodies bound to the native protein in an accessibility ELISA, indicating that residues 44 to 56, 174 to 190, 185 to 199, and 235 to 249 were surface exposed on CS31A. These data indicate that an immunodominant surface-exposed linear epitope was

  14. Deletion of fusion peptide or destabilization of fusion core of HIV gp41 enhances antigenicity and immunogenicity of 4E10 epitope

    SciTech Connect

    Li Jing; Chen Xi; Jiang Shibo Chen Yinghua

    2008-11-07

    The human monoclonal antibody 4E10 against the membrane-proximal external region (MPER) of HIV-1 gp41 demonstrates broad neutralizing activity across various strains, and makes its epitope an attractive target for HIV-1 vaccine development. Although the contiguous epitope of 4E10 has been identified, attempts to re-elicit 4E10-like antibodies have failed, possibly due to the lack of proper conformation of the 4E10 epitope. Here we used pIg-tail expression system to construct a panel of eukaryotic cell-surface expression plasmids encoding the extracellular domain of gp41 with deletion of fusion peptide and/or introduction of L568P mutation that may disrupt the gp41 six-helix bundle core conformation as DNA vaccines for immunization of mice. We found that these changes resulted in significant increase of the antigenicity and immunogenicity of 4E10 epitope. This information is thus useful for rational design of vaccines targeting the HIV-1 gp41 MPER.

  15. High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome

    PubMed Central

    Casina, Veronica C.; Hu, Wenbing; Mao, Jian-Hua; Lu, Rui-Nan; Hanby, Hayley A.; Pickens, Brandy; Kan, Zhong-Yuan; Lim, Woon K.; Mayne, Leland; Ostertag, Eric M.; Kacir, Stephen; Siegel, Don L.; Englander, S. Walter; Zheng, X. Long

    2015-01-01

    Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen–deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention. PMID:26203127

  16. Structure-Function Analysis of the Epitope for 4E10, a Broadly Neutralizing Human Immunodeficiency Virus Type 1 Antibody†

    PubMed Central

    Brunel, Florence M.; Zwick, Michael B.; Cardoso, Rosa M. F.; Nelson, Josh D.; Wilson, Ian A.; Burton, Dennis R.; Dawson, Philip E.

    2006-01-01

    The human immunodeficiency virus type 1 (HIV-1) neutralizing antibody 4E10 binds to a linear, highly conserved epitope within the membrane-proximal external region of the HIV-1 envelope glycoprotein gp41. We have delineated the peptide epitope of the broadly neutralizing 4E10 antibody to gp41 residues 671 to 683, using peptides with different lengths encompassing the previously suggested core epitope (NWFDIT). Peptide binding to the 4E10 antibody was assessed by competition enzyme-linked immunosorbent assay, and the Kd values of selected peptides were determined using surface plasmon resonance. An Ala scan of the epitope indicated that several residues, W672, F673, and T676, are essential (>1,000-fold decrease in binding upon replacement with alanine) for 4E10 recognition. In addition, five other residues, N671, D674, I675, W680, and L679, make significant contributions to 4E10 binding. In general, the Ala scan results agree well with the recently reported crystal structure of 4E10 in complex with a 13-mer peptide and with our circular dichroism analyses. Neutralization competition assays confirmed that the peptide NWFDITNWLWYIKKKK-NH2 could effectively inhibit 4E10 neutralization. Finally, to limit the conformational flexibility of the peptides, helix-promoting 2-aminoisobutyric acid residues and helix-inducing tethers were incorporated. Several peptides have significantly improved affinity (>1,000-fold) over the starting peptide and, when used as immunogens, may be more likely to elicit 4E10-like neutralizing antibodies. Hence, this study represents the first stage toward iterative development of a vaccine based on the 4E10 epitope. PMID:16439525

  17. High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome.

    PubMed

    Casina, Veronica C; Hu, Wenbing; Mao, Jian-Hua; Lu, Rui-Nan; Hanby, Hayley A; Pickens, Brandy; Kan, Zhong-Yuan; Lim, Woon K; Mayne, Leland; Ostertag, Eric M; Kacir, Stephen; Siegel, Don L; Englander, S Walter; Zheng, X Long

    2015-08-01

    Acquired thrombotic thrombocytopenic purpura (TTP), a thrombotic disorder that is fatal in almost all cases if not treated promptly, is primarily caused by IgG-type autoantibodies that inhibit the ability of the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) metalloprotease to cleave von Willebrand factor (VWF). Because the mechanism of autoantibody-mediated inhibition of ADAMTS13 activity is not known, the only effective therapy so far is repeated whole-body plasma exchange. We used hydrogen-deuterium exchange mass spectrometry (HX MS) to determine the ADAMTS13 binding epitope for three representative human monoclonal autoantibodies, isolated from TTP patients by phage display as tethered single-chain fragments of the variable regions (scFvs). All three scFvs bind the same conformationally discontinuous epitopic region on five small solvent-exposed loops in the spacer domain of ADAMTS13. The same epitopic region is also bound by most polyclonal IgG autoantibodies in 23 TTP patients that we tested. The ability of ADAMTS13 to proteolyze VWF is impaired by the binding of autoantibodies at the epitopic loops in the spacer domain, by the deletion of individual epitopic loops, and by some local mutations. Structural considerations and HX MS results rule out any disruptive structure change effect in the distant ADAMTS13 metalloprotease domain. Instead, it appears that the same ADAMTS13 loop segments that bind the autoantibodies are also responsible for correct binding to the VWF substrate. If so, the autoantibodies must prevent VWF proteolysis simply by physically blocking normal ADAMTS13 to VWF interaction. These results point to the mechanism for autoantibody action and an avenue for therapeutic intervention. PMID:26203127

  18. Extensive HLA class I allele promiscuity among viral CTL epitopes

    PubMed Central

    Frahm, Nicole; Yusim, Karina; Suscovich, Todd J.; Adams, Sharon; Sidney, John; Hraber, Peter; Hewitt, Hannah S.; Linde, Caitlyn H.; Kavanagh, Daniel G.; Woodberry, Tonia; Henry, Leah M.; Faircloth, Kellie; Listgarten, Jennifer; Kadie, Carl; Jojic, Nebojsa; Sango, Kaori; Brown, Nancy V.; Pae, Eunice; Zaman, M. Tauheed; Bihl, Florian; Khatri, Ashok; John, Mina; Mallal, Simon; Marincola, Francesco M.; Walker, Bruce D.; Sette, Alessandro; Heckerman, David; Korber, Bette T.; Brander, Christian

    2008-01-01

    Summary Promiscuous binding of T helper epitopes to MHC class II molecules has been well established, but few examples of promiscuous class I restricted epitopes exist. To address the extent of promiscuity of HLA class I peptides, responses to 242 well-defined viral epitopes were tested in 100 subjects regardless of the individuals’ HLA type. Surprisingly, half of all detected responses were seen in the absence of the originally reported restricting HLA class I allele, and only 3% of epitopes were recognized exclusively in the presence of their original allele. Functional assays confirmed the frequent recognition of HLA class I-restricted T cell epitopes on several alternative alleles across HLA class I supertypes and encoded on different class I loci. These data have significant implications for the understanding of MHC class I restricted antigen presentation and vaccine development. PMID:17705138

  19. Autoantibody recognition mechanisms of p53 epitopes

    NASA Astrophysics Data System (ADS)

    Phillips, J. C.

    2016-06-01

    There is an urgent need for economical blood based, noninvasive molecular biomarkers to assist in the detection and diagnosis of cancers in a cost-effective manner at an early stage, when curative interventions are still possible. Serum autoantibodies are attractive biomarkers for early cancer detection, but their development has been hindered by the punctuated genetic nature of the ten million known cancer mutations. A landmark study of 50,000 patients (Pedersen et al., 2013) showed that a few p53 15-mer epitopes are much more sensitive colon cancer biomarkers than p53, which in turn is a more sensitive cancer biomarker than any other protein. The function of p53 as a nearly universal "tumor suppressor" is well established, because of its strong immunogenicity in terms of not only antibody recruitment, but also stimulation of autoantibodies. Here we examine dimensionally compressed bioinformatic fractal scaling analysis for identifying the few sensitive epitopes from the p53 amino acid sequence, and show how it could be used for early cancer detection (ECD). We trim 15-mers to 7-mers, and identify specific 7-mers from other species that could be more sensitive to aggressive human cancers, such as liver cancer. Our results could provide a roadmap for ECD.

  20. Classification epitopes in groups based on their protein family

    PubMed Central

    2015-01-01

    Background The humoral immune system response is based on the interaction between antibodies and antigens for the clearance of pathogens and foreign molecules. The interaction between these proteins occurs at specific positions known as antigenic determinants or B-cell epitopes. The experimental identification of epitopes is costly and time consuming. Therefore the use of in silico methods, to help discover new epitopes, is an appealing alternative due the importance of biomedical applications such as vaccine design, disease diagnostic, anti-venoms and immune-therapeutics. However, the performance of predictions is not optimal been around 70% of accuracy. Further research could increase our understanding of the biochemical and structural properties that characterize a B-cell epitope. Results We investigated the possibility of linear epitopes from the same protein family to share common properties. This hypothesis led us to analyze physico-chemical (PCP) and predicted secondary structure (PSS) features of a curated dataset of epitope sequences available in the literature belonging to two different groups of antigens (metalloproteinases and neurotoxins). We discovered statistically significant parameters with data mining techniques which allow us to distinguish neurotoxin from metalloproteinase and these two from random sequences. After a five cross fold validation we found that PCP based models obtained area under the curve values (AUC) and accuracy above 0.9 for regression, decision tree and support vector machine. Conclusions We demonstrated that antigen's family can be inferred from properties within a single group of linear epitopes (metalloproteinases or neurotoxins). Also we discovered the characteristics that represent these two epitope groups including their similarities and differences with random peptides and their respective amino acid sequence. These findings open new perspectives to improve epitope prediction by considering the specific antigen

  1. Exceptionally long CDR3H of bovine scFv antigenized with BoHV-1 B-epitope generates specific immune response against the targeted epitope.

    PubMed

    Pasman, Yfke; Soliman, Caroline; Ramsland, Paul A; Kaushik, Azad K

    2016-09-01

    We discovered that some bovine antibodies are amongst the largest known to exist due to the presence of an exceptionally long CDR3H (≥49 amino acids) with multiple cysteines that provide a unique knob and stalk structure to the antigen binding site. The large CDR3H size, unlike mouse and human, provides a suitable platform for antigenization with large configurational B-epitopes. Here we report the identification of a B-epitope on the gC envelope protein of bovine herpes virus type-1 (BoHV-1) recognized by a bovine IgG1 antibody. The identified 156 amino acid long gC fragment (gC156) was expressed as a recombinant protein. Subsequently, a functional scFv fragment with a 61 amino-acid long CDR3H (scFv1H12) was expressed such that gC156 was grafted into the CDR3H, replacing the "knob" region (gC156scFv1H12 or Ag-scFv). Importantly, the Ag-scFv could be recognized by a neutralizing antibody fragment (scFv3-18L), which suggests that the engraftment of gC156 into the CDR3H of 1H12 maintained the native conformation of the BoHV-1 B-epitope. A 3D model of gC156 was generated using fold-recognition approaches and this was grafted onto the CDR3H stalk of the 1H12 Fab crystal structure to predict the 3D structure of the Ag-scFv. The grafted antigen in Ag-scFv is predicted to have a compact conformation with the ability to protrude into the solvent. Upon immunization of bovine calves, the antigenized scFv (gC156scFv1H12) induced a higher antibody response as compared to free recombinant gC156. These observations suggest that antigenization of bovine scFv with an exceptionally long CDR3H provides a novel approach to developing the next generation of vaccines against infectious agents that require induction of protective humoral immunity. PMID:27497190

  2. Identification of T. gondii epitopes, adjuvants, & host genetic factors that influence protection of mice & humans

    PubMed Central

    Tan, Tze Guan; Mui, Ernest; Cong, Hua; Witola, William; Montpetit, Alexandre; Muench, Stephen P.; Sidney, John; Alexander, Jeff; Sette, Alessandro; Grigg, Michael; Maewal, Ajesh; McLeod, Rima

    2010-01-01

    Toxoplasma gondii is an intracellular parasite that causes severe neurologic and ocular disease in immune-compromised and congenitally infected individuals. There is no vaccine protective against human toxoplasmosis. Herein, immunization of Ld mice with HF10 (HPGSVNEFDF) with palmitic acid moieties or a monophosphoryl lipid A derivative elicited potent IFN-γ production from Ld-restricted CD8+ T cells in vitro and protected mice. CD8+ T cell peptide epitopes from T. gondii dense granule proteins GRA 3, 6, 7, and Sag 1, immunogenic in humans for HLA-A02+, HLA-A03+, and HLA-B07+ cells were identified. Since peptide repertoire presented by MHC class I molecules to CD8+ T cells is shaped by endoplasmic reticulum-associated aminopeptidase (ERAAP), polymorphisms in the human ERAAP gene ERAP1 were studied and associate with susceptibility to human congenital toxoplasmosis (p<0.05). These results have important implications for vaccine development. PMID:20347630

  3. Proximal glycans outside of the epitopes regulate the presentation of HIV-1 envelope gp120 helper epitopes1

    PubMed Central

    Li, Hualin; Xu, Chong-Feng; Blais, Steven; Wan, Qi; Zhang, Hui-Tang; Landry, Samuel J.; Hioe, Catarina E.

    2010-01-01

    Glycosylation of HIV-1 envelope gp120 determines not only the proper structure, but also the immune responses against this antigen. While glycans may be part of specific epitopes or shield other epitopes from T cells and antibodies, this study provides evidence for a different immunomodulatory function of glycans associated with gp120 residues N230 and N448. These glycans are required for efficient MHC class II-restricted presentation of nearby CD4 T-cell epitopes, even though they are not part of the epitopes. The glycans do not affect CD4 T cell recognition of more distant epitopes, and are not essential for the proper folding and function of gp120. Data on CD4 T-cell recognition of N448 mutants combined with proteolysis analyses and surface electrostatic potential calculation around residue N448 support the notion that N448-glycan near the epitope's C-terminus renders the site to be surface accessible and allows its efficient processing. In contrast, the N230-glycan contributes to the nearby epitope presentation at a step other than the proteolytic processing of the epitope. Hence, N-glycans can determine CD4 T-cell recognition of nearby gp120 epitopes by regulating the different steps in the MHC class II processing and presentation pathway after APCs acquire the intact gp120 antigen exogenously. Modifications of amino acids bearing glycans at the C termini of gp120 helper epitopes may prove to be a useful strategy for enhancing the immunogenicity of HIV-1 envelope gp120. PMID:19414790

  4. Pep-3D-Search: a method for B-cell epitope prediction based on mimotope analysis

    PubMed Central

    Huang, Yan Xin; Bao, Yong Li; Guo, Shu Yan; Wang, Yan; Zhou, Chun Guang; Li, Yu Xin

    2008-01-01

    Background 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. Results 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 oefficient (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

  5. Conformational landscape and low lying excited states of imatinib.

    PubMed

    Vinţeler, Emil; Stan, Nicoleta-Florina; Luchian, Raluca; Căinap, Călin; Ramalho, João P Prates; Chiş, Vasile

    2015-04-01

    The conformational changes of imatinib (IMT) are crucial for understanding the ligand-receptor interaction and its mechanism of action [Agofonov et al. (2014) Nature Struct Mol Biol 21:848-853]. Therefore, here we investigated the free energy conformational landscape of the free IMT base, aiming to describe the three-dimensional structures and energetic stability of its conformers. Forty-five unique conformers, within an energy window of 4.8 kcal mol(-1) were identified by a conformational search in gas-phase, at the B3LYP/6-31G(d) theoretical level. Among these, the 20 most stable, as well as 4 conformers resulting from optimization of experimental structures found in the two known polymorphs of IMT and in the c-Abl complex were further refined using the 6-31+G(d,p) basis set and the polarizable continuum solvation model. The most stable conformers in gas-phase and water exhibit a V-shaped structure. The major difference between the most stable free conformers and the bioactive conformers consists in the relative orientation of the pyrimidine-pyridine groups responsible for hydrogen bonding interactions in the ATP-binding pocket. The ratio of mole fractions corresponding to the two known (α and β) polymorphic forms of IMT was estimated from the calculated thermochemical data, in quantitative agreement with the existing experimental data related to their solubility. The electronic absorption spectrum of this compound was investigated in water and explained based on the theoretical TD-DFT results, considering the Boltzmann population-averaged computed data at CAM-B3LYP/6-31+G(d,p) level of theory for the nine most stable conformers. PMID:25764326

  6. Epitope located N-glycans impair the MHC-I epitope generation and presentation.

    PubMed

    Chiritoiu, Gabriela N; Jandus, Camilla; Munteanu, Cristian V A; Ghenea, Simona; Gannon, Philippe O; Romero, Pedro; Petrescu, Stefana M

    2016-06-01

    The degradation process of the antigens specific to MHC-I presentation depends mainly on the proteasomal proteases in the cytosol. However, since many antigens are glycoproteins, including tumor antigens or viruses envelope proteins, their glycosylation status could also affect their processing and presentation. Here, we investigate the processing of tyrosinase, a multiple glycosylated tumor antigen overexpressed in human malignant melanoma. By LC-MS/MS analysis of human tyrosinase expressed in a melanoma cell, we show that all seven sites of tyrosinase are at least partially N-glycosylated. Using human CD8+ T-cell clones specific for the tyrosinase epitope YMDGTMSQV (369-377), including an N-glycosylation site, we found that transfectants of single and triple N-glycosylation mutants are recognized by specific T cells. Importantly, single, triple, and the aglycosylated tyrosinase mutants lacking the epitope located N-glycosylation site (N371D) were able to trigger higher CD8+ T-cell activation. The LC/MS analysis showed significant increase of the amount of YMDGTMSQV peptide resulted from accelerated oligomerization and degradation of aglycosylated mutants. The generation of the antigenic peptide by the antigen processing machinery is therefore largely independent of tyrosinase N-glycosylation. However, while distal N-glycans had no effect on the epitope generation, the mutants lacking the N371 glycan generated the antigenic peptide more efficiently. We conclude that epitope located N-glycans limit the ability of human tyrosinase to provide HLA-A2-restricted antigen for recognition by specific CD8+ T cells. PMID:26701645

  7. Recombinant infectious bursal disease virus carrying hepatitis C virus epitopes.

    PubMed

    Upadhyay, Chitra; Ammayappan, Arun; Patel, Deendayal; Kovesdi, Imre; Vakharia, Vikram N

    2011-02-01

    The delivery of foreign epitopes by a replicating nonpathogenic avian infectious bursal disease virus (IBDV) was explored. The aim of the study was to identify regions in the IBDV genome that are amenable to the introduction of a sequence encoding a foreign peptide. By using a cDNA-based reverse genetics system, insertions or substitutions of sequences encoding epitope tags (FLAG, c-Myc, or hepatitis C virus epitopes) were engineered in the open reading frames of a nonstructural protein (VP5) and the capsid protein (VP2). Attempts were also made to generate recombinant IBDV that displayed foreign epitopes in the exposed loops (P(BC) and P(HI)) of the VP2 trimer. We successfully recovered recombinant IBDVs expressing c-Myc and two different virus-neutralizing epitopes of human hepatitis C virus (HCV) envelope glycoprotein E in the VP5 region. Western blot analyses with anti-c-Myc and anti-HCV antibodies provided positive identification of both the c-Myc and HCV epitopes that were fused to the N terminus of VP5. Genetic analysis showed that the recombinants carrying the c-Myc/HCV epitopes maintained the foreign gene sequences and were stable after several passages in Vero and 293T cells. This is the first report describing efficient expression of foreign peptides from a replication-competent IBDV and demonstrates the potential of this virus as a vector. PMID:21106739

  8. Chimeric Epitope Vaccine from Multistage Antigens for Lymphatic Filariasis.

    PubMed

    Anugraha, G; Madhumathi, J; Prince, P R; Prita, P J Jeya; Khatri, V K; Amdare, N P; Reddy, M V R; Kaliraj, P

    2015-10-01

    Lymphatic filariasis, a mosquito-borne parasitic disease, affects more than 120 million people worldwide. Vaccination for filariasis by targeting different stages of the parasite will be a boon to the existing MDA efforts of WHO which required repeated administration of the drug to reduce the infection level and sustained transmission. Onset of a filaria-specific immune response achieved through antigen vaccines can act synergistically with these drugs to enhance the parasite killing. Multi-epitope vaccine approach has been proved to be successful against several parasitic diseases as it overcomes the limitations associated with the whole antigen vaccines. Earlier results from our group suggested the protective efficacy of multi-epitope vaccine comprising two immunodominant epitopes from Brugia malayi antioxidant thioredoxin (TRX), several epitopes from transglutaminase (TGA) and abundant larval transcript-2 (ALT-2). In this study, the prophylactic efficacy of the filarial epitope protein (FEP), a chimera of selective epitopes identified from our earlier study, was tested in a murine model (jird) of filariasis with L3 larvae. FEP conferred a significantly (P < 0.0001) high protection (69.5%) over the control in jirds. We also observed that the multi-epitope recombinant construct (FEP) induces multiple types of protective immune responses, thus ensuring the successful elimination of the parasite; this poses FEP as a potential vaccine candidate. PMID:26179420

  9. Identification of Autoantigen Epitopes in Alopecia Areata.

    PubMed

    Wang, Eddy H C; Yu, Mei; Breitkopf, Trisia; Akhoundsadegh, Noushin; Wang, Xiaojie; Shi, Feng-Tao; Leung, Gigi; Dutz, Jan P; Shapiro, Jerry; McElwee, Kevin J

    2016-08-01

    Alopecia areata (AA) is believed to be a cell-mediated autoimmune hair loss disease. Both CD4 and cytotoxic CD8 T cells (CTLs) are important for the onset and progression of AA. Hair follicle (HF) keratinocyte and/or melanocyte antigen epitopes are suspected potential targets of autoreactive CTLs, but the specific epitopes have not yet been identified. We investigated the potential for a panel of known epitopes, expressed by HF keratinocytes and melanocytes, to induce activation of CTL populations in peripheral blood mononuclear cells. Specific synthetic epitopes derived from HF antigens trichohyalin and tyrosinase-related protein-2 induced significantly higher frequencies of response in AA CTLs compared with healthy controls (IFN-gamma secretion). Apoptosis assays revealed conditioned media from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides elevated the expression of apoptosis markers in primary HF keratinocytes. A cytokine array revealed higher expression of IL-13 and chemokine ligand 5 (CCL5, RANTES) from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides compared with controls. The data indicate that AA affected subjects present with an increased frequency of CTLs responsive to epitopes originating from keratinocytes and melanocytes; the activated CTLs secreted soluble factors that induced apoptosis in HF keratinocytes. Potentially, CTL response to self-antigen epitopes, particularly trichohyalin epitopes, could be a prognostic marker for human AA. PMID:27094591

  10. Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen.

    PubMed Central

    Shaw, David M; Woods, Andrew M; Myers, Kevin A; Westwater, Caroline; Rahi-Saund, Veena; Davies, Michael J; Renouf, David V; Hounsell, Elizabeth F; Stern, Peter L

    2002-01-01

    The human 5T4 oncofoetal antigen is a focus for development of several antibody-directed therapies on the basis of the murine monoclonal antibody against 5T4 (mAb5T4), which recognizes a conformational epitope. 5T4 molecules are highly N-glycosylated transmembrane glycoproteins whose extracellular domain contains two regions of leucine-rich repeats (LRRs) and associated flanking regions, separated by an intervening hydrophilic sequence. Using a series of deletion and mutated cDNA constructs as well as chimaeras with the murine homologue, we have mapped the mAb5T4 epitope to the more membrane-proximal LRR2 or its flanking region. Analysis of the glycosylation of the seven consensus Asp-Xaa-Ser/Thr sites was consistent with all of the sites being glycosylated. A combination of two high-mannose chains (predominantly octasaccharide) and five mostly sialylated bi-, tri- and tetra-antennary complex chains with minor quantities of core fucose were detected. The two glycosylation sites, which are the most likely to have predominantly high-mannose chains, are in the only two regions that show significant differences between the human and the 81% identical mouse sequence. A site-directed mutation, which abolished glycosylation at one of these sites (position 192), did not alter antigenicity. The other, which is nearest to the N-terminus in the human, has an Asn-Leu-Thr to Asn-Leu-Leu conversion in the mouse, so cannot be glycosylated in the latter species. The large complex glycosylation at the other sites is likely to influence the antigenicity and tertiary structure generating the 5T4 epitope. PMID:11903056

  11. B-Cell Epitopes in GroEL of Francisella tularensis

    PubMed Central

    Lu, Zhaohua; Rynkiewicz, Michael J.; Madico, Guillermo; Li, Sheng; Yang, Chiou-Ying; Perkins, Hillary M.; Sompuram, Seshi R.; Kodela, Vani; Liu, Tong; Morris, Timothy; Wang, Daphne; Roche, Marly I.; Seaton, Barbara A.; Sharon, Jacqueline

    2014-01-01

    The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60), is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft), the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs) specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA. In a mouse model of respiratory tularemia with the highly pathogenic Ft type A strain SchuS4, the Ab64 and N200 IgG2a mAbs, which block each other’s binding to and are sensitive to the same two point mutations in FtGroEL, reduced bacterial burden indicating that they target protective GroEL B-cell epitopes. The Ab64 and N200 epitopes, as well as those of three other mAbs with different crossblocking profiles, Ab53, N3, and N30, were mapped by hydrogen/deuterium exchange–mass spectrometry (DXMS) and visualized on a homology model of FtGroEL. This model was further supported by its experimentally-validated computational docking to the X-ray crystal structures of Ab64 and Ab53 Fabs. The structural analysis and DXMS profiles of the Ab64 and N200 mAbs suggest that their protective effects may be due to induction or stabilization of a conformational change in FtGroEL. PMID:24968190

  12. Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen.

    PubMed

    Shaw, David M; Woods, Andrew M; Myers, Kevin A; Westwater, Caroline; Rahi-Saund, Veena; Davies, Michael J; Renouf, David V; Hounsell, Elizabeth F; Stern, Peter L

    2002-04-01

    The human 5T4 oncofoetal antigen is a focus for development of several antibody-directed therapies on the basis of the murine monoclonal antibody against 5T4 (mAb5T4), which recognizes a conformational epitope. 5T4 molecules are highly N-glycosylated transmembrane glycoproteins whose extracellular domain contains two regions of leucine-rich repeats (LRRs) and associated flanking regions, separated by an intervening hydrophilic sequence. Using a series of deletion and mutated cDNA constructs as well as chimaeras with the murine homologue, we have mapped the mAb5T4 epitope to the more membrane-proximal LRR2 or its flanking region. Analysis of the glycosylation of the seven consensus Asp-Xaa-Ser/Thr sites was consistent with all of the sites being glycosylated. A combination of two high-mannose chains (predominantly octasaccharide) and five mostly sialylated bi-, tri- and tetra-antennary complex chains with minor quantities of core fucose were detected. The two glycosylation sites, which are the most likely to have predominantly high-mannose chains, are in the only two regions that show significant differences between the human and the 81% identical mouse sequence. A site-directed mutation, which abolished glycosylation at one of these sites (position 192), did not alter antigenicity. The other, which is nearest to the N-terminus in the human, has an Asn-Leu-Thr to Asn-Leu-Leu conversion in the mouse, so cannot be glycosylated in the latter species. The large complex glycosylation at the other sites is likely to influence the antigenicity and tertiary structure generating the 5T4 epitope. PMID:11903056

  13. An orthorhombic polymorph of 10,11-dihydrocarbamazepine.

    PubMed

    Harrison, William T A; Yathirajan, H S; Anilkumar, H G

    2006-05-01

    The title compound (systematic name: 10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide), C15H14N2O, is shown to crystallize as an orthorhombic polymorph to complement the known monoclinic form. The molecular conformations of both forms are very similar, involving a bent conformation for the seven-membered azepine ring and an overall ;butterfly' shape. The molecules assemble into chains by way of N-H...O bonds and N-H...pi interactions in both crystal modifications. The two polymorphs appear to form due to different van der Waals interactions between the layer-like sheets of molecules. PMID:16679591

  14. Characterization of neutralization epitopes in the V2 region of human immunodeficiency virus type 1 gp120: role of glycosylation in the correct folding of the V1/V2 domain.

    PubMed Central

    Wu, Z; Kayman, S C; Honnen, W; Revesz, K; Chen, H; Vijh-Warrier, S; Tilley, S A; McKeating, J; Shotton, C; Pinter, A

    1995-01-01

    A number of monoclonal antibodies (MAbs) with various levels of neutralizing activity that recognize epitopes in the V1/V2 domain of LAI-related gp120s have been described. These include rodent antibodies directed against linear and conformational epitopes and a chimpanzee MAb, C108G, with extremely potent neutralizing activity directed against a glycan-dependent epitope. A fusion glycoprotein expression system that expressed the isolated V1/V2 domain of gp120 in native form was used to analyze the structural characteristics of these epitopes. A number of MAbs (C108G, G3-4, 684-238, SC258, 11/68b, 38/66a, 38/66c, 38/62c, and CRA3) that did not bind with high affinity to peptides immunoprecipitated a fusion glycoprotein expressing the V1/V2 domain of HXB2 gp120 in the absence of other human immunodeficiency virus sequences, establishing that their epitopes were fully specified within this region. Biochemical analyses indicated that in the majority of V1/V2 fusion molecules only five of the six glycosylation signals in the V1/V2 domain were utilized, and the glycoforms were found to be differentially recognized by particular MAbs. Both C108G and MAbs directed against conformational epitopes reacted with large fractions of the fully glycosylated molecules but with only small fractions of the incompletely glycosylated molecules. Mutational analysis of the V1 and V2 glycosylation signals indicated that in most cases the unutilized site was located either at position 156 or at position 160, suggesting the occurrence of competition for glycan addition at these neighboring positions. Mutation of glycosylation site 160 destroyed the C108G epitope but increased the fraction of the molecules that presented the conformational epitopes, while mutation of the highly conserved glycosylation site at position 156 greatly diminished the expression of the conformational epitopes and increased expression of the C108G epitope. Similar heterogeneity in glycosylation was also observed

  15. Galilean conformal electrodynamics

    NASA Astrophysics Data System (ADS)

    Bagchi, Arjun; Basu, Rudranil; Mehra, Aditya

    2014-11-01

    Maxwell's Electrodynamics admits two distinct Galilean limits called the Electric and Magnetic limits. We show that the equations of motion in both these limits are invariant under the Galilean Conformal Algebra in D = 4, thereby exhibiting non-relativistic conformal symmetries. Remarkably, the symmetries are infinite dimensional and thus Galilean Electrodynamics give us the first example of an infinitely extended Galilean Conformal Field Theory in D > 2. We examine details of the theory by looking at purely non-relativistic conformal methods and also use input from the limit of the relativistic theory.

  16. Measles Virus Epitope Presentation by HLA: Novel Insights into Epitope Selection, Dominance, and Microvariation

    PubMed Central

    Schellens, Ingrid M.; Meiring, Hugo D.; Hoof, Ilka; Spijkers, Sanne N.; Poelen, Martien C. M.; van Gaans-van den Brink, Jacqueline A. M.; Costa, Ana I.; Vennema, Harry; Keşmir, Can; van Baarle, Debbie; van Els, Cécile A. C. M.

    2015-01-01

    Immunity to infections with measles virus (MV) can involve vigorous human leukocyte antigen (HLA) class I-restricted CD8+ cytotoxic T cell (CTL) responses. MV, albeit regarded monotypic, is known to undergo molecular evolution across its RNA genome. To address which regions of the MV proteome are eligible for recognition by CD8+ CTLs and how different HLA class I loci contribute to the epitope display, we interrogated the naturally processed and presented MV peptidome extracted from cell lines expressing in total a broad panel of 16 different common HLA-A, -B, and -C molecules. The repertoire and abundance of MV peptides were bona fide identified by nanoHPLC–MS/MS. ­Eighty-nine MV peptides were discovered and assignment to an HLA-A, -B, or -C allele, based on HLA-peptide affinity prediction, was in most cases successful. Length variation and presentation by multiple HLA class I molecules was common in the MV peptidome. More than twice as many unique MV epitopes were found to be restricted by HLA-B than by HLA-A, while MV peptides with supra-abundant expression rates (>5,000 cc) were rather associated with HLA-A and HLA-C. In total, 59 regions across the whole MV proteome were identified as targeted by HLA class I. Sequence coverage by epitopes was highest for internal proteins transcribed from the MV-P/V/C and -M genes and for hemagglutinin. At the genome level, the majority of the HLA class I-selected MV epitopes represented codons having a higher non-synonymous mutation rate than silent mutation rate, as established by comparison of a set of 58 unique full length MV genomes. Interestingly, more molecular variation was seen for the epitopes expressed at rates ≥1,000 cc. These data for the first time indicate that HLA class I broadly samples the MV proteome and that CTL pressure may contribute to the genomic evolution of MV. PMID:26579122

  17. Identification of cytotoxic T lymphocyte epitopes on swine viruses: multi-epitope design for universal T cell vaccine.

    PubMed

    Liao, Yu-Chieh; Lin, Hsin-Hung; Lin, Chieh-Hua; Chung, Wen-Bin

    2013-01-01

    Classical swine fever (CSF), foot-and-mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) are the primary diseases affecting the pig industry globally. Vaccine induced CD8(+) T cell-mediated immune response might be long-lived and cross-serotype and thus deserve further attention. Although large panels of synthetic overlapping peptides spanning the entire length of the polyproteins of a virus facilitate the detection of cytotoxic T lymphocyte (CTL) epitopes, it is an exceedingly costly and cumbersome approach. Alternatively, computational predictions have been proven to be of satisfactory accuracy and are easily performed. Such a method enables the systematic identification of genome-wide CTL epitopes by incorporating epitope prediction tools in analyzing large numbers of viral sequences. In this study, we have implemented an integrated bioinformatics pipeline for the identification of CTL epitopes of swine viruses including the CSF virus (CSFV), FMD virus (FMDV) and PRRS virus (PRRSV) and assembled these epitopes on a web resource to facilitate vaccine design. Identification of epitopes for cross protections to different subtypes of virus are also reported in this study and may be useful for the development of a universal vaccine against such viral infections among the swine population. The CTL epitopes identified in this study have been evaluated in silico and possibly provide more and wider protection in compared to traditional single-reference vaccine design. The web resource is free and open to all users through http://sb.nhri.org.tw/ICES. PMID:24358361

  18. Crystal Polymorphism in a Carbamazepine Derivative: Oxcarbazepine

    PubMed Central

    LUTKER, KATIE M.; MATZGER, ADAM J.

    2011-01-01

    Although crystal polymorphism of carbamazepine (CBZ), an anticonvulsant used to treat epilepsy, has been known for decades, the phenomenon has only recently been noted for its keto-derivative oxcarbazepine (OCB). Here it is demonstrated that OCB possesses at least three anhydrous polymorphs. Although all forms are morphologically similar, making differentiation between crystal modifications by optical microscopy difficult, powder X-ray diffraction, Raman spectroscopy, and thermomicroscopy show distinctive differences. These techniques provide an efficient method of distinguishing between the three polymorphs. The crystal structure of form II of OCB is reported for the first time and the structure of form I has been redetermined at low temperature. Remarkably, both the molecular conformation and crystal packing of form II are in excellent agreement with the blind prediction made in 2007. PMID:19603503

  19. Fragment-based discovery of novel thymidylate synthase leads by NMR screening and group epitope mapping

    PubMed Central

    Begley, Darren W.; Zheng, Suxin; Varani, Gabriele

    2010-01-01

    Solution state nuclear magnetic resonance (NMR1) is a versatile tool for the study of binding interactions between small molecules and macromolecular targets. We applied ligand-based NMR techniques to the study of human thymidylate synthase (hTS) using known nanomolar inhibitors and a library of small molecule fragments. Screening by NMR led to the rapid identification of ligand pairs that bind in proximal sites within the co-factor binding pocket of hTS. Screening hits were used as search criteria within commercially available sources, and a subset of catalog analogs were tested for potency by in vitro assay and binding affinity by quantitative saturation transfer difference (STD)-NMR titration. Two compounds identified by this approach possess low micromolar affinity and potency, as well as excellent binding efficiency against hTS. Relative binding orientations for both leads were modeled using AutoDock, and the most likely bound conformations validated using experimentally-derived STD-NMR binding epitope data. These ligands represent novel starting points for fragment-based drug design of non-canonical TS inhibitors, and their binding epitopes highlight important and previously unexploited interactions with conserved residues in the cofactor binding site. PMID:20626411

  20. Predicting Interaction Sites from the Energetics of Isolated Proteins: A New Approach to Epitope Mapping

    PubMed Central

    Scarabelli, Guido; Morra, Giulia; Colombo, Giorgio

    2010-01-01

    Abstract An increasing number of functional studies of proteins have shown that sequence and structural similarities alone may not be sufficient for reliable prediction of their interaction properties. This is particularly true for proteins recognizing specific antibodies, where the prediction of antibody-binding sites, called epitopes, has proven challenging. The antibody-binding properties of an antigen depend on its structure and related dynamics. Aiming to predict the antibody-binding regions of a protein, we investigate a new approach based on the integrated analysis of the dynamical and energetic properties of antigens, to identify nonoptimized, low-intensity energetic interaction networks in the protein structure isolated in solution. The method is based on the idea that recognition sites may correspond to localized regions with low-intensity energetic couplings with the rest of the protein, which allows them to undergo conformational changes, to be recognized by a binding partner, and to tolerate mutations with minimal energetic expense. Upon analyzing the results on isolated proteins and benchmarking against antibody complexes, it is found that the method successfully identifies binding sites located on the protein surface that are accessible to putative binding partners. The combination of dynamics and energetics can thus discriminate between epitopes and other substructures based only on physical properties. We discuss implications for vaccine design. PMID:20441761

  1. Conformal bootstrap in embedding space

    NASA Astrophysics Data System (ADS)

    Fortin, Jean-François; Skiba, Witold

    2016-05-01

    It is shown how to obtain conformal blocks from embedding space with the help of the operator product expansion. The minimal conformal block originates from scalar exchange in a four-point correlation function of four scalars. All remaining conformal blocks are simple derivatives of the minimal conformal block. With the help of the orthogonality properties of the conformal blocks, the analytic conformal bootstrap can be implemented directly in embedding space, leading to a Jacobi-like definition of conformal field theories.

  2. Structure-Based Design of Inhibitors of Protein–Protein Interactions: Mimicking Peptide Binding Epitopes

    PubMed Central

    Pelay-Gimeno, Marta; Glas, Adrian; Koch, Oliver; Grossmann, Tom N

    2015-01-01

    Protein–protein interactions (PPIs) are involved at all levels of cellular organization, thus making the development of PPI inhibitors extremely valuable. The identification of selective inhibitors is challenging because of the shallow and extended nature of PPI interfaces. Inhibitors can be obtained by mimicking peptide binding epitopes in their bioactive conformation. For this purpose, several strategies have been evolved to enable a projection of side chain functionalities in analogy to peptide secondary structures, thereby yielding molecules that are generally referred to as peptidomimetics. Herein, we introduce a new classification of peptidomimetics (classes A–D) that enables a clear assignment of available approaches. Based on this classification, the Review summarizes strategies that have been applied for the structure-based design of PPI inhibitors through stabilizing or mimicking turns, β-sheets, and helices. PMID:26119925

  3. Structural Basis of Differential Neutralization of DENV-1 Genotypes by an Antibody that Recognizes a Cryptic Epitope

    PubMed Central

    Austin, S. Kyle; Dowd, Kimberly A.; Shrestha, Bimmi; Nelson, Christopher A.; Edeling, Melissa A.; Johnson, Syd; Pierson, Theodore C.; Diamond, Michael S.; Fremont, Daved H.

    2012-01-01

    We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC′ loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC′ loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development. PMID:23055922

  4. Identification of the epitope for a monoclonal antibody that blocks platelet aggregation induced by type III collagen.

    PubMed Central

    Glattauer, V; Werkmeister, J A; Kirkpatrick, A; Ramshaw, J A

    1997-01-01

    A library of eight conformation-dependent monoclonal antibodies that react with distinct epitopes on native human type III collagen has been examined for the ability of these antibodies to inhibit platelet aggregation induced by this collagen. Six of these antibodies had no effects; one, 1E7-D7/Col3, delayed the onset and slowed the rate of platelet aggregation, while another, 2G8-B1/Col3, completely inhibited aggregation. In order to identify the epitope recognized by this inhibitory antibody, a series of peptides that could fold to form triple-helical fragments was examined. Each peptide included six Gly-Xaa-Yaa triplets from the human type III collagen sequence, where Xaa and Yaa represent the particular amino acids in the sequence, and a C-terminal (Gly-Pro-Hyp)4 sequence to enhance triple-helical stability. Using these peptides we have identified the epitope as a nine-amino-acid sequence, GLAGAOGLR (where O is the one-letter code for 4-hydroxyproline), starting at position 520 in the human type III collagen helical domain. This sequence is proximal to the site proposed for the interaction of type III collagen with alpha2beta1-integrin of platelets. PMID:9173900

  5. Identification of the Neutralizing Epitopes of Merkel Cell Polyomavirus Major Capsid Protein within the BC and EF Surface Loops

    PubMed Central

    Fleury, Maxime J. J.; Nicol, Jérôme T. J.; Samimi, Mahtab; Arnold, Françoise; Cazal, Raphael; Ballaire, Raphaelle; Mercey, Olivier; Gonneville, Hélène; Combelas, Nicolas; Vautherot, Jean-Francois; Moreau, Thierry; Lorette, Gérard; Coursaget, Pierre; Touzé, Antoine

    2015-01-01

    Merkel cell polyomavirus (MCPyV) is the first polyomavirus clearly associated with a human cancer, i.e. the Merkel cell carcinoma (MCC). Polyomaviruses are small naked DNA viruses that induce a robust polyclonal antibody response against the major capsid protein (VP1). However, the polyomavirus VP1 capsid protein epitopes have not been identified to date. The aim of this study was to identify the neutralizing epitopes of the MCPyV capsid. For this goal, four VP1 mutants were generated by insertional mutagenesis in the BC, DE, EF and HI loops between amino acids 88-89, 150-151, 189-190, and 296-297, respectively. The reactivity of these mutants and wild-type VLPs was then investigated with anti-VP1 monoclonal antibodies and anti-MCPyV positive human sera. The findings together suggest that immunodominant conformational neutralizing epitopes are present at the surface of the MCPyV VLPs and are clustered within BC and EF loops. PMID:25812141

  6. Anti-H1 histone antibodies in systemic lupus erythematosus: epitope localization after immunoblotting of chymotrypsin-digested H1.

    PubMed Central

    Costa, O; Tchouatcha-Tchouassom, J C; Roux, B; Monier, J C

    1986-01-01

    Using micro enzyme-linked immunosorbent assay (micro-ELISA) anti-H1 antibodies are most frequently seen in systemic lupus erythematosus (SLE) (61.4% of patients). Positive anti-H1 ELISA reactions are rare in rheumatoid arthritis (RA) (5.8% of cases), melanomas (16.7%), leukaemias (13.6%) and other cancers (5.6%). In SLE, the immunoglobulins (Ig) which constitute anti-H1 antibodies are, by order of importance, IgM, IgG and IgA. By means of immunoblotting using H1 solutions digested by alpha-1-chymotrypsin fixed on collagen membranes, we have shown that all the SLE sera containing anti-H1 antibodies recognize the sequential epitopes that are found on the carboxy terminal tail and, for 28% of anti-H1 sera, also the epitopes present on the aminoterminal half. The technique used did not make it possible to determine with certainty whether anti-H1 autoantibodies are also directed against the conformational epitopes of the globular part of the molecule. Images Fig. 2 PMID:2423279

  7. Homology modelling of the major peanut allergen Ara h 2 and surface mapping of IgE-binding epitopes.

    PubMed

    Barre, Annick; Borges, Jean-Philippe; Culerrier, Raphaël; Rougé, Pierre

    2005-09-15

    Three-dimensional models built for the peanut Ara h 2 allergen and other structurally-related 2S albumin allergens of dietary nuts exhibited an overall three-dimensional fold stabilized by disulphide bridges well conserved among all the members of the 2S albumin superfamily. Conformational analysis of the linear IgE-binding epitopes mapped on the molecular surface of Ara h 2 showed no structural homology with the corresponding regions of the walnut Jug r 1, the pecan nut Car i 1 or the Brazil nut Ber e 1 allergens. The absence of epitopic community does not support the allergenic cross-reactivity observed between peanut and walnut or Brazil nut, which presumably depends on other ubiquitous seed storage protein allergens, namely the vicilins. However, the major IgE-binding epitope identified on the molecular surface of the walnut Jug r 1 allergen shared a pronounced structural homology with the corresponding region of the pecan nut Car i 1 allergen. With the exception of peanut, 2S albumins could thus account for the IgE-binding cross-reactivity observed between some other dietary nuts, e.g. walnut and pecan nut. PMID:15899521

  8. Identification of the neutralizing epitopes of Merkel cell polyomavirus major capsid protein within the BC and EF surface loops.

    PubMed

    Fleury, Maxime J J; Nicol, Jérôme T J; Samimi, Mahtab; Arnold, Françoise; Cazal, Raphael; Ballaire, Raphaelle; Mercey, Olivier; Gonneville, Hélène; Combelas, Nicolas; Vautherot, Jean-Francois; Moreau, Thierry; Lorette, Gérard; Coursaget, Pierre; Touzé, Antoine

    2015-01-01

    Merkel cell polyomavirus (MCPyV) is the first polyomavirus clearly associated with a human cancer, i.e. the Merkel cell carcinoma (MCC). Polyomaviruses are small naked DNA viruses that induce a robust polyclonal antibody response against the major capsid protein (VP1). However, the polyomavirus VP1 capsid protein epitopes have not been identified to date. The aim of this study was to identify the neutralizing epitopes of the MCPyV capsid. For this goal, four VP1 mutants were generated by insertional mutagenesis in the BC, DE, EF and HI loops between amino acids 88-89, 150-151, 189-190, and 296-297, respectively. The reactivity of these mutants and wild-type VLPs was then investigated with anti-VP1 monoclonal antibodies and anti-MCPyV positive human sera. The findings together suggest that immunodominant conformational neutralizing epitopes are present at the surface of the MCPyV VLPs and are clustered within BC and EF loops. PMID:25812141

  9. Deletion modification enhances anthrax specific immunity and protective efficacy of a hepatitis B core particle-based anthrax epitope vaccine.

    PubMed

    Yin, Ying; Zhang, Sheng; Cai, Chenguang; Zhang, Jun; Dong, Dayong; Guo, Qiang; Fu, Ling; Xu, Junjie; Chen, Wei

    2014-02-01

    Protective antigen (PA) is one of the major virulence factors of anthrax and is also the major constituent of the current anthrax vaccine. Previously, we found that the 2β2-2β3 loop of PA contains a dominant neutralizing epitope, the SFFD. We successfully inserted the 2β2-2β3 loop of PA into the major immunodominant region (MIR) of hepatitis B virus core (HBc) protein. The resulting fusion protein, termed HBc-N144-PA-loop2 (HBcL2), can effectively produce anthrax specific protective antibodies in an animal model. However, the protective immunity caused by HBcL2 could still be improved. In this research, we removed amino acids 79-81 from the HBc MIR of the HBcL2. This region was previously reported to be the major B cell epitope of HBc, and in keeping with this finding, we observed that the short deletion in the MIR not only diminished the intrinsic immunogenicity of HBc but also stimulated a higher titer of anthrax specific immunity. Most importantly, this deletion led to the full protection of the immunized mice against a lethal dose anthrax toxin challenge. We supposed that the conformational changes which occurred after the short deletion and foreign insertion in the MIR of HBc were the most likely reasons for the improvement in the immunogenicity of the HBc-based anthrax epitope vaccine. PMID:24054942

  10. Novel HLA-B27-restricted Epitopes from Chlamydia trachomatis Generated upon Endogenous Processing of Bacterial Proteins Suggest a Role of Molecular Mimicry in Reactive Arthritis*

    PubMed Central

    Alvarez-Navarro, Carlos; Cragnolini, Juan J.; Dos Santos, Helena G.; Barnea, Eilon; Admon, Arie; Morreale, Antonio; López de Castro, José A.

    2013-01-01

    Reactive arthritis (ReA) is an HLA-B27-associated spondyloarthropathy that is triggered by diverse bacteria, including Chlamydia trachomatis, a frequent intracellular parasite. HLA-B27-restricted T-cell responses are elicited against this bacterium in ReA patients, but their pathogenetic significance, autoimmune potential, and relevant epitopes are unknown. High resolution and sensitivity mass spectrometry was used to identify HLA-B27 ligands endogenously processed and presented by HLA-B27 from three chlamydial proteins for which T-cell epitopes were predicted. Fusion protein constructs of ClpC, Na+-translocating NADH-quinone reductase subunit A, and DNA primase were expressed in HLA-B27+ cells, and their HLA-B27-bound peptidomes were searched for endogenous bacterial ligands. A non-predicted peptide, distinct from the predicted T-cell epitope, was identified from ClpC. A peptide recognized by T-cells in vitro, NQRA(330–338), was detected from the reductase subunit. This is the second HLA-B27-restricted T-cell epitope from C. trachomatis with relevance in ReA demonstrated to be processed and presented in live cells. A novel peptide from the DNA primase, DNAP(211–223), was also found. This was a larger variant of a known epitope and was highly homologous to a self-derived natural ligand of HLA-B27. All three bacterial peptides showed high homology with human sequences containing the binding motif of HLA-B27. Molecular dynamics simulations further showed a striking conformational similarity between DNAP(211–223) and its homologous and much more flexible human-derived HLA-B27 ligand. The results suggest that molecular mimicry between HLA-B27-restricted bacterial and self-derived epitopes is frequent and may play a role in ReA. PMID:23867464

  11. A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses

    SciTech Connect

    Boonsathorn, Naphatsawan; Panthong, Sumolrat; Chittaganpitch, Malinee; Phuygun, Siripaporn; Waicharoen, Sunthareeya; Prachasupap, Apichai; Yasugi, Mayo; Ono, Ken-ichiro; and others

    2014-09-26

    Highlights: • A human monoclonal antibody against influenza virus was produced from a volunteer. • The antibody was generated from the PBMCs of the volunteer using the fusion method. • The antibody neutralized heterosubtypically group 1 influenza A viruses (H1 and H9). • The antibody targeted a novel epitope in globular head region of the hemagglutinin. • Sequences of the identified epitope are highly conserved among H1 and H9 subtypes. - Abstract: Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.

  12. Benchmarking B cell epitope prediction: underperformance of existing methods.

    PubMed

    Blythe, Martin J; Flower, Darren R

    2005-01-01

    Sequence profiling is used routinely to predict the location of B-cell epitopes. In the postgenomic era, the need for reliable epitope prediction is clear. We assessed 484 amino acid propensity scales in combination with ranges of plotting parameters to examine exhaustively the correlation of peaks and epitope location within 50 proteins mapped for polyclonal responses. After examining more than 10(6) combinations, we found that even the best set of scales and parameters performed only marginally better than random. Our results confirm the null hypothesis: Single-scale amino acid propensity profiles cannot be used to predict epitope location reliably. The implication for studies using such methods is obvious. PMID:15576553

  13. Conformations of Substituted Ethanes.

    ERIC Educational Resources Information Center

    Kingsbury, Charles A.

    1979-01-01

    Reviews state-of-the-art of conformational analysis and factors which affect it. Emphasizes sp-3 hybridized acrylic molecules. Provides examples on the importance of certain factors in determining conformation. Purpose, is to provide examples for examination questions. (Author/SA)

  14. Synthesis of the TACO scaffold as a new selectively deprotectable conformationally restricted triazacyclophane based scaffold.

    PubMed

    Brouwer, Arwin J; van de Langemheen, Helmus; Ciaffoni, Adriano; Schilder, Kitty E; Liskamp, Rob M J

    2014-06-01

    The synthesis of a new triazacyclophane scaffold (TACO scaffold) containing three selectively deprotectable amines is described. The TACO scaffold is conformationally more constrained than our frequently used TAC scaffold, due to introduction of a substituent on the para position of the benzoic acid hinge, which prevents ring flipping and makes it more attractive than the TAC scaffold for preparation of artificial receptor molecules or for mimicking discontinuous epitopes toward protein mimics when more preorganization is required. PMID:24856258

  15. Isolation of antibodies specific to a single conformation-dependant antigenic determinant on the EG95 hydatid vaccine

    PubMed Central

    Read, A.J.; Casey, J.L.; Coley, A.M.; Foley, M.; Gauci, C.G.; Jackson, D.C.; Lightowlers, M.W.

    2009-01-01

    EG95 is a recombinant vaccine protein that elicits protection against hydatid disease in sheep. Previous studies have shown that the host-protective epitopes on EG95 depend on correct conformation and cannot be represented by simple “linear” peptides. By screening random peptide phage display libraries with polyclonal antibodies directed against conformation-dependant epitopes of EG95, we have selected a number of peptides that mimic these epitopes. The selected peptides did not show sequence homology to EG95. Antigen binding assays involving these peptides have provided evidence of at least four conformationally-dependant epitope regions on EG95. One of the selected peptides, E100, has been used to purify antibodies from anti-sera raised in sheep vaccinated with EG95. This yielded monospecific antibodies capable of recognizing recombinant EG95 in ELISA and native EG95 in Western blot assays. This antibody was demonstrated to be effective in antibody-dependant complement-mediated in vitro killing of Echinococcus granulosus oncospheres. Peptide E100 may represent the basis for a quality control assay for EG95 production, and has the potential to become a component of a synthetic peptide-based vaccine against E. granulosus. PMID:19095030

  16. Update of the HLA class I eplet database in the website based registry of antibody-defined HLA epitopes.

    PubMed

    Duquesnoy, R J

    2014-06-01

    Eplets are small configurations of polymorphic amino acid residues on human leukocyte antigen (HLA) molecules and are considered as essential components of HLA epitopes recognized by antibodies. This report describes a new design of the eplet repertoire in HLA-ABC alleles used in Luminex kits for antibody testing. There were three steps (1): identify all combinations of polymorphic residues with HLA molecular modeling within a 3-Å radius, (2) determine polymorphic residue compositions of 3 Å patches from amino acid sequences of HLA alleles in Luminex panels and (3) annotate eplets from one or more patches present on one allele or shared by the same group of alleles. There are now 270 HLA-ABC eplets in the Registry, of which 219 are in antibody-accessible positions on the molecular surface and 51 are defined solely by residue polymorphisms located below the molecular surface. Each eplet has a list of Luminex and non-Luminex alleles for which mismatch acceptability can be determined. PMID:24828055

  17. Ultrastructural localization of beta-amyloid, tau, and ubiquitin epitopes in extracellular neurofibrillary tangles.

    PubMed Central

    Tabaton, M; Cammarata, S; Mancardi, G; Manetto, V; Autilio-Gambetti, L; Perry, G; Gambetti, P

    1991-01-01

    Neurofibrillary tangles (NFTs), a hallmark of Alzheimer disease, are commonly located in perikarya of neurons. In advanced cases of Alzheimer disease, however, NFTs are observed also in the extracellular space. As extracellular NFTs (E-NFTs), and occasionally intracellular NFTs (I-NFTs), are recognized by antibodies to beta-amyloid protein (beta AP), beta AP may be present not only in amyloid deposits but also in paired helical filaments (PHFs), the primary components of NFTs. We compared the antigenic characteristics of I-NFTs and E-NFTs with light- and electron-microscopic immunocytochemistry by using several antibodies to noncontiguous epitopes of the microtubule-associated protein tau and of ubiquitin (Ub) as well as an antiserum to beta AP. At variance with I-NFTs, E-NFTs were made predominantly of straight filaments (SFs), rather than PHFs, that were often separated by astroglial processes and in close association with small beta AP deposits. Occasionally, E-NFTs were made of bundles of amorphous material, which showed no resemblance to SFs, PHFs, or amyloid fibrils. The antigenic changes in E-NFTs suggest that when NFTs become extracellular they lose the N and, possibly, the C termini of tau while maintaining the intermediate region of the molecule; they also lose the N-terminal two-thirds of Ub while the C-terminal conjugation site of Ub is preserved. A small subset of E-NFTs reacted with antibodies to both beta AP and tau. Although in most E-NFTs, the epitopes recognized by tau and Ub antibodies were located in typical PHFs and SFs, the epitopes recognized in this subset of anti-beta AP and anti-tau-positive E-NFTs were located exclusively in the bundles of amorphous material. It is suggested that either beta AP epitopes are present but inaccessible in PHFs and SFs and become exposed after conformational changes occurring in the extracellular space or PHFs and SFs become closely associated with beta AP in the extracellular space. Images PMID:1706517

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

    PubMed Central

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

    2016-01-01

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

  19. Identification and Characterization of an Immunogenic Hybrid Epitope Formed by both HIV gp120 and Human CD4 Proteins ▿

    PubMed Central

    Lewis, George K.; Fouts, Timothy R.; Ibrahim, Sani; Taylor, Brian M.; Salkar, Rachita; Guan, Yongjun; Kamin-Lewis, Roberta; DeVico, Anthony L.

    2011-01-01

    Certain antibodies from HIV-infected humans bind conserved transition state (CD4 induced [CD4i]) domains on the HIV envelope glycoprotein, gp120, and demonstrate extreme dependence on the formation of a gp120-human CD4 receptor complex. The epitopes recognized by these antibodies remain undefined although recent crystallographic studies of the anti-CD4i monoclonal antibody (MAb) 21c suggest that contacts with CD4 as well as gp120 might occur. Here, we explore the possibility of hybrid epitopes that demand the collaboration of both gp120 and CD4 residues to enable antibody reactivity. Analyses with a panel of human anti-CD4i MAbs and gp120-CD4 antigens with specific mutations in predicted binding domains revealed one putative hybrid epitope, defined by the human anti-CD4i MAb 19e. In virological and immunological tests, MAb 19e did not bind native or constrained gp120 except in the presence of CD4. This contrasted with other anti-CD4i MAbs, including MAb 21c, which bound unliganded, full-length gp120 held in a constrained conformation. Conversely, MAb 19e exhibited no specific reactivity with free human CD4. Computational modeling of MAb 19e interactions with gp120-CD4 complexes suggested a distinct binding profile involving antibody heavy chain interactions with CD4 and light chain interactions with gp120. In accordance, targeted mutations in CD4 based on this model specifically reduced MAb 19e interactions with stable gp120-CD4 complexes that retained reactivity with other anti-CD4i MAbs. These data represent a rare instance of an antibody response that is specific to a pathogen-host cell protein interaction and underscore the diversity of immunogenic CD4i epitope structures that exist during natural infection. PMID:21994452

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

    PubMed Central

    Alderete, JF; Neace, Calvin J

    2013-01-01

    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.

  1. Human plasma contains cross-reactive Abeta conformer-specific IgG antibodies.

    PubMed

    O'Nuallain, Brian; Acero, Luis; Williams, Angela D; Koeppen, Helen P McWilliams; Weber, Alfred; Schwarz, Hans P; Wall, Jonathan S; Weiss, Deborah T; Solomon, Alan

    2008-11-25

    Two conformers of aggregated Abeta, i.e., fibrils and oligomers, have been deemed important in the pathogenesis of Alzheimer's disease. We now report that intravenous immune globulin (IVIG) derived from pools of human plasma contains IgGs that recognize conformational epitopes present on fibrils and oligomers, but not their soluble monomeric precursor. We have used affinity chromatography to isolate these antibodies and have shown that they cross-reacted with comparable nanomolar avidity with both types of Abeta aggregates; notably, binding was not inhibited by soluble Abeta monomers. Our studies provide further support for investigating the therapeutic use of IVIG in Alzheimer's disease. PMID:18956886

  2. CD8 T-cell responses against the immunodominant Theileria parva peptide Tp249-59 are composed of two distinct populations specific for overlapping 11-mer and 10-mer epitopes.

    PubMed

    Connelley, Timothy K; Li, Xiaoying; MacHugh, Niall; Colau, Didier; Graham, Simon P; van der Bruggen, Pierre; Taracha, Evans L; Gill, Andy; Morrison, William Ivan

    2016-10-01

    Immunity against Theileria parva is associated with CD8 T-cell responses that exhibit immunodominance, focusing the response against limited numbers of epitopes. As candidates for inclusion in vaccines, characterization of responses against immunodominant epitopes is a key component in novel vaccine development. We have previously demonstrated that the Tp249-59 and Tp1214-224 epitopes dominate CD8 T-cell responses in BoLA-A10 and BoLA-18 MHC I homozygous animals, respectively. In this study, peptide-MHC I tetramers for these epitopes, and a subdominant BoLA-A10-restricted epitope (Tp298-106 ), were generated to facilitate accurate and rapid enumeration of epitope-specific CD8 T cells. During validation of these tetramers a substantial proportion of Tp249-59 -reactive T cells failed to bind the tetramer, suggesting that this population was heterogeneous with respect to the recognized epitope. We demonstrate that Tp250-59 represents a distinct epitope and that tetramers produced with Tp50-59 and Tp49-59 show no cross-reactivity. The Tp249-59 and Tp250-59 epitopes use different serine residues as the N-terminal anchor for binding to the presenting MHC I molecule. Molecular dynamic modelling predicts that the two peptide-MHC I complexes adopt structurally different conformations and Tcell receptor β sequence analysis showed that Tp249-59 and Tp250-59 are recognized by non-overlapping T-cell receptor repertoires. Together these data demonstrate that although differing by only a single residue, Tp249-59 and Tp250-59 epitopes form distinct ligands for T-cell receptor recognition. Tetramer analysis of T. parva-specific CD8 T-cell lines confirmed the immunodominance of Tp1214-224 in BoLA-A18 animals and showed in BoLA-A10 animals that the Tp249-59 epitope response was generally more dominant than the Tp250-59 response and confirmed that the Tp298-106 response was subdominant. PMID:27317384

  3. A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses.

    PubMed

    Boonsathorn, Naphatsawan; Panthong, Sumolrat; Koksunan, Sarawut; Chittaganpitch, Malinee; Phuygun, Siripaporn; Waicharoen, Sunthareeya; Prachasupap, Apichai; Sasaki, Tadahiro; Kubota-Koketsu, Ritsuko; Yasugi, Mayo; Ono, Ken-Ichiro; Arai, Yasuha; Kurosu, Takeshi; Sawanpanyalert, Pathom; Ikuta, Kazuyoshi; Watanabe, Yohei

    2014-09-26

    Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses. PMID:25204499

  4. A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses.

    PubMed

    Hu, Hongxing; Voss, Jarrod; Zhang, Guoliang; Buchy, Philippi; Zuo, Teng; Wang, Lulan; Wang, Feng; Zhou, Fan; Wang, Guiqing; Tsai, Cheguo; Calder, Lesley; Gamblin, Steve J; Zhang, Linqi; Deubel, Vincent; Zhou, Boping; Skehel, John J; Zhou, Paul

    2012-03-01

    Influenza A virus infection is a persistent threat to public health worldwide due to its ability to evade immune surveillance through rapid genetic drift and shift. Current vaccines against influenza A virus provide immunity to viral isolates that are similar to vaccine strains. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. In this study, by using a highly sensitive H5N1 pseudotype-based neutralization assay to screen human monoclonal antibodies produced by memory B cells from an H5N1-infected individual and molecular cloning techniques, we developed three fully human monoclonal antibodies. Among them, antibody 65C6 exhibited potent neutralization activity against all H5 clades and subclades except for subclade 7.2 and prophylactic and therapeutic efficacy against highly pathogenic avian influenza H5N1 viruses in mice. Studies on hemagglutinin (HA)-antibody complexes by electron microscopy and epitope mapping indicate that antibody 65C6 binds to a conformational epitope comprising amino acid residues at positions 118, 121, 161, 164, and 167 (according to mature H5 numbering) on the tip of the membrane-distal globular domain of HA. Thus, we conclude that antibody 65C6 recognizes a neutralization epitope in the globular head of HA that is conserved among almost all divergent H5N1 influenza stains. PMID:22238297

  5. T Cell Epitopes and Post-Translationally Modified Epitopes in Type 1 Diabetes

    PubMed Central

    McGinty, John W.; Marré, Meghan L.; Bajzik, Veronique; Piganelli, Jon D.; James, Eddie A.

    2016-01-01

    Type 1 diabetes (T1D) is an autoimmune disease in which progressive loss of self-tolerance, evidenced by accumulation of auto-antibodies and auto-reactive T cells that recognize diverse self-proteins, leads to immune-mediated destruction of pancreatic beta cells and loss of insulin secretion. In this review, we discuss antigens and epitopes in T1D and the role that post-translational modifications play in circumventing tolerance mechanisms and increasing antigenic diversity. Emerging data suggest that, analogous to other autoimmune diseases such as rheumatoid arthritis and celiac disease, enzymatically modified epitopes are preferentially recognized in T1D. Modifying enzymes such as peptidyl deiminases and tissue transglutaminase are activated in response to beta cell stress, providing a mechanistic link between post-translational modification and interactions with the environment. Although studies of such responses in the at-risk population have been limited, current data suggests that breakdown in tolerance through post-translational modification represents an important checkpoint in the development of T1D. PMID:26370701

  6. Identification of epitopes within a highly immunogenic region of acetylcholine receptor by a phage epitope library.

    PubMed

    Barchan, D; Balass, M; Souroujon, M C; Katchalski-Katzir, E; Fuchs, S

    1995-11-01

    We have employed a hexapeptide phage-epitope library to identify epitopes for a mAb (mAb 5.14), which is directed to a determinant within a highly immunogenic, cytoplasmic region of the alpha-subunit of acetylcholine receptor (AChR). We have selected two different peptide-presenting phages (SWDDIR-phage and LWILTR-phage) which interact specifically with mAb 5.14. This interaction is specifically inhibited by AChR and by synthetic peptides corresponding to the hexapeptides presented by the selected phages. Although mAb 5.14 binds to AChR in its native as well as its denatured form, the selected hexapeptides do not exist as such in the AChR molecule. However, three amino acid sequence homologies with these hexapeptides were shown to be present in the cytoplasmic region of Torpedo AChR. By extending the selected hexapeptides, at one or both ends, with amino acid residues flanking the hexapeptides in the phage, we obtained mimotopes with an up to two order of magnitude higher affinity to the Ab. These extended peptides were able to efficiently block the binding of mAb 5.14 to both peptide-presenting phages, and to AChR. PMID:7594584

  7. [Conformers of carnosine].

    PubMed

    Kliuev, S A

    2006-01-01

    The geometric and energetic parameters of most stable conformations of carnosine were calculated by the semiempirical guantum-chemical method PM3. The carnosine-water-zinc (II) clusters were simulated. PMID:16909845

  8. Hepatitis C virus mutation affects proteasomal epitope processing

    PubMed Central

    Seifert, Ulrike; Liermann, Heike; Racanelli, Vito; Halenius, Anne; Wiese, Manfred; Wedemeyer, Heiner; Ruppert, Thomas; Rispeter, Kay; Henklein, Peter; Sijts, Alice; Hengel, Hartmut; Kloetzel, Peter-M.; Rehermann, Barbara

    2004-01-01

    The high incidence of hepatitis C virus (HCV) persistence raises the question of how HCV interferes with host immune responses. Studying a single-source HCV outbreak, we identified an HCV mutation that impaired correct carboxyterminal cleavage of an immunodominant HLA-A2–restricted CD8 cell epitope that is frequently recognized by recovered patients. The mutation, a conservative HCV nonstructural protein 3 (NS3) tyrosine to phenylalanine substitution, was absent in 54 clones of the infectious source, but present in 15/21 (71%) HLA-A2–positive and in 11/24 (46%) HLA-A2–negative patients with chronic hepatitis C. In order to analyze whether the mutation affected the processing of the HLA-A2–restricted CD8 cell epitope, mutant and wild-type NS3 polypeptides were digested in vitro with 20S constitutive proteasomes and with immunoproteasomes. The presence of the mutation resulted in impaired carboxyterminal cleavage of the epitope. In order to analyze whether impaired epitope processing affected T cell priming in vivo, HLA-A2–transgenic mice were infected with vaccinia viruses encoding either wild-type or mutant HCV NS3. The mutant induced fewer epitope-specific, IFN-γ;–producing and fewer tetramer+ cells than the wild type. These data demonstrate how a conservative mutation in the flanking region of an HCV epitope impairs the induction of epitope-specific CD8+ T cells and reveal a mechanism that may contribute to viral sequence evolution in infected patients. PMID:15254592

  9. Assemblies of Conformal Tanks

    NASA Technical Reports Server (NTRS)

    DeLay, Tom

    2009-01-01

    Assemblies of tanks having shapes that conform to each other and/or conform to other proximate objects have been investigated for use in storing fuels and oxidizers in small available spaces in upper stages of spacecraft. Such assemblies might also prove useful in aircraft, automobiles, boats, and other terrestrial vehicles in which space available for tanks is limited. The basic concept of using conformal tanks to maximize the utilization of limited space is not new in itself: for example, conformal tanks are used in some automobiles to store windshield -washer liquid and coolant that overflows from radiators. The novelty of the present development lies in the concept of an assembly of smaller conformal tanks, as distinguished from a single larger conformal tank. In an assembly of smaller tanks, it would be possible to store different liquids in different tanks. Even if the same liquid were stored in all the tanks, the assembly would offer an advantage by reducing the mechanical disturbance caused by sloshing of fuel in a single larger tank: indeed, the requirement to reduce sloshing is critical in some applications. The figure shows a prototype assembly of conformal tanks. Each tank was fabricated by (1) copper plating a wax tank mandrel to form a liner and (2) wrapping and curing layers of graphite/epoxy composite to form a shell supporting the liner. In this case, the conformal tank surfaces are flat ones where they come in contact with the adjacent tanks. A band of fibers around the outside binds the tanks together tightly in the assembly, which has a quasi-toroidal shape. For proper functioning, it would be necessary to maintain equal pressure in all the tanks.

  10. Quantum massive conformal gravity

    NASA Astrophysics Data System (ADS)

    Faria, F. F.

    2016-04-01

    We first find the linear approximation of the second plus fourth order derivative massive conformal gravity action. Then we reduce the linearized action to separated second order derivative terms, which allows us to quantize the theory by using the standard first order canonical quantization method. It is shown that quantum massive conformal gravity is renormalizable but has ghost states. A possible decoupling of these ghost states at high energies is discussed.

  11. Low frequency of broadly neutralizing HIV antibodies during chronic infection even in quaternary epitope targeting antibodies containing large numbers of somatic mutations.

    PubMed

    Hicar, Mark D; Chen, Xuemin; Kalams, Spyros A; Sojar, Hakimuddin; Landucci, Gary; Forthal, Donald N; Spearman, Paul; Crowe, James E

    2016-02-01

    Neutralizing antibodies (Abs) are thought to be a critical component of an appropriate HIV vaccine response. It has been proposed that Abs recognizing conformationally dependent quaternary epitopes on the HIV envelope (Env) trimer may be necessary to neutralize diverse HIV strains. A number of recently described broadly neutralizing monoclonal Abs (mAbs) recognize complex and quaternary epitopes. Generally, many such Abs exhibit extensive numbers of somatic mutations and unique structural characteristics. We sought to characterize the native antibody (Ab) response against circulating HIV focusing on such conformational responses, without a prior selection based on neutralization. Using a capture system based on VLPs incorporating cleaved envelope protein, we identified a selection of B cells that produce quaternary epitope targeting Abs (QtAbs). Similar to a number of broadly neutralizing Abs, the Ab genes encoding these QtAbs showed extensive numbers of somatic mutations. However, when expressed as recombinant molecules, these Abs failed to neutralize virus or mediate ADCVI activity. Molecular analysis showed unusually high numbers of mutations in the Ab heavy chain framework 3 region of the variable genes. The analysis suggests that large numbers of somatic mutations occur in Ab genes encoding HIV Abs in chronically infected individuals in a non-directed, stochastic, manner. PMID:26748387

  12. Conformational sampling techniques.

    PubMed

    Hatfield, Marcus P D; Lovas, Sándor

    2014-01-01

    The potential energy hyper-surface of a protein relates the potential energy of the protein to its conformational space. This surface is useful in determining the native conformation of a protein or in examining a statistical-mechanical ensemble of structures (canonical ensemble). In determining the potential energy hyper-surface of a protein three aspects must be considered; reducing the degrees of freedom, a method to determine the energy of each conformation and a method to sample the conformational space. For reducing the degrees of freedom the choice of solvent, coarse graining, constraining degrees of freedom and periodic boundary conditions are discussed. The use of quantum mechanics versus molecular mechanics and the choice of force fields are also discussed, as well as the sampling of the conformational space through deterministic and heuristic approaches. Deterministic methods include knowledge-based statistical methods, rotamer libraries, homology modeling, the build-up method, self-consistent electrostatic field, deformation methods, tree-based elimination and eigenvector following routines. The heuristic methods include Monte Carlo chain growing, energy minimizations, metropolis monte carlo and molecular dynamics. In addition, various methods to enhance the conformational search including the deformation or smoothing of the surface, scaling of system parameters, and multi copy searching are also discussed. PMID:23947647

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

    PubMed

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

    2012-05-01

    Bioinformatics analysis was used to predict potential epitopes of Lyssavirus nucleoprotein and highlighted some distinct differences in the quantity and localization of the epitopes disclosed by epitope analysis of monoclonal antibodies against Lyssavirus nucleoprotein. Bioinformatics analysis showed that the domain containing residues 152-164 of Lyssavirus nucleoprotein was a conserved linear epitope that had not been reported previously. Immunization of two rabbits with the corresponding synthetic peptide conjugated to the Keyhole Limpe hemocyanin (KLH) macromolecule resulted in a titer of anti-peptide antibody above 1:200,000 in rabbit sera as detected by indirect enzyme-linked immunosorbent assay (ELISA). Western blot analysis demonstrated that the anti-peptide antibody recognized denatured Lyssavirus nucleoprotein in sodium dodecylsulfonate-polyacrylate gel electrophoresis (SDS-PAGE). Affinity chromatography purification and FITC-labeling of the anti-peptide antibody in rabbit sera was performed. FITC-labeled anti-peptide antibody could recognize Lyssavirus nucleoprotein in BSR cells and canine brain tissues even at a 1:200 dilution. Residues 152-164 of Lyssavirus nucleoprotein were verified as a conserved linear epitope in Lyssavirus. PMID:22405880

  14. Ab and T cell epitopes of influenza A virus, knowledge and opportunities

    PubMed Central

    Bui, Huynh-Hoa; Peters, Bjoern; Assarsson, Erika; Mbawuike, Innocent; Sette, Alessandro

    2007-01-01

    The Immune Epitope Database and Analysis Resources (IEDB) (www.immuneepitope.org) was recently developed to capture epitope related data. IEDB also hosts various bioinformatics tools that can be used to identify novel epitopes as well as to analyze and visualize existing epitope data. Herein, a comprehensive analysis was undertaken (i) to compile and inventory existing knowledge regarding influenza A epitopes and (ii) to determine possible cross-reactivities of identified epitopes among avian H5N1 and human influenza strains. At present, IEDB contains >600 different epitopes derived from 58 different strains and 10 influenza A proteins. By using the IEDB analysis resources, conservancy analyses were performed, and several conserved and possibly cross-reactive epitopes were identified. Significant gaps in the current knowledge were also revealed, including paucity of Ab epitopes in comparison with T cell epitopes, limited number of epitopes reported for avian influenza strains/subtypes, and limited number of epitopes reported from proteins other than hemagglutinin and nucleoprotein. This analysis provides a resource for researchers to access existing influenza epitope data. At the same time, the analysis illustrates gaps in our collective knowledge that should inspire directions for further study of immunity against the influenza A virus. PMID:17200302

  15. Crystal Structure of West Nile Virus Envelope Glycoprotein Reveals Viral Surface Epitopes

    SciTech Connect

    Kanai,R.; Kar, K.; Anthony, K.; Gould, L.; Ledizet, M.; Fikrig, E.; Marasco, W.; Koski, R.; Modis, Y.

    2006-01-01

    West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.

  16. The gp41659 - 671 HIV-1 antibody epitope: a structurally challenging small peptide

    NASA Astrophysics Data System (ADS)

    Zhang, Yuan; Sagui, Celeste

    2014-03-01

    We present the results of extensive Molecular Dynamics (MD) simulations of the tridecapeptide corresponding to residues 659-671 of the envelope glycoprotein gp41 of HIV-1, which spans the 2F5 monoclonal antibody epitope ELDKWA. The most recent AMBER force fields ff99SB and ff12SB in both implicit and explicit solvents have been used for a cumulative time longer than 7.2 μs . We have analyzed the conformational ensembles of the peptide both with and without applied tensile restraints, and found that: (1) The amount of helical populations is important in aqueous solution, but this structure forms part of a flexible conformational ensemble with a rugged free energy landscape with shallow minima, which agrees well with the bulk of the experimental observations; (2) our results are more consistent with the experimental results than those from previous simulations; (3) under uniaxial tension, the disordered peptide first becomes fully helical before melting into turns, loops and 310-helices.

  17. Differential basal-to-apical accessibility of lamin A/C epitopes in the nuclear lamina regulated by changes in cytoskeletal tension

    PubMed Central

    Herzog, Florian A.; Schwartlander, Ruth; Moeller, Jens; Vogel, Viola

    2015-01-01

    Nuclear lamins play central roles at the intersection between cytoplasmic signalling and nuclear events. Here, we show that at least two N- and C-terminal lamin epitopes are not accessible at the basal side of the nuclear envelope under environmental conditions known to upregulate cell contractility. The conformational epitope on the Ig-domain of A-type lamins is more buried in the basal than apical nuclear envelope of human mesenchymal stem cells undergoing osteogenesis (but not adipogenesis), and in fibroblasts adhering to rigid (but not soft) polyacrylamide hydrogels. This structural polarization of the lamina is promoted by compressive forces, emerges during cell spreading, and requires lamin A/C multimerization, intact nucleoskeleton-cytoskeleton linkages (LINC), and apical-actin stress-fibre assembly. Notably, the identified Igepitope overlaps with emerin, DNA and histone binding sites, and comprises various laminopathy mutation sites. Our findings should help deciphering how the physical properties of cellular microenvironments regulate nuclear events. PMID:26301768

  18. Epitope mapping of 7S cashew antigen in complex with antibody by solution-phase H/D exchange monitored by FT-ICR mass spectrometry.

    PubMed

    Guan, Xiaoyan; Noble, Kyle A; Tao, Yeqing; Roux, Kenneth H; Sathe, Shridhar K; Young, Nicolas L; Marshall, Alan G

    2015-06-01

    The potential epitope of a recombinant food allergen protein, cashew Ana o 1, reactive to monoclonal antibody, mAb 2G4, has been mapped by solution-phase amide backbone H/D exchange (HDX) monitored by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Purified mAb 2G4 was incubated with recombinant Ana o 1 (rAna o 1) to form antigen:monoclonal antibody (Ag:mAb) complexes. Complexed and uncomplexed (free) rAna o 1 were then subjected to HDX-MS analysis. Five regions protected from H/D exchange upon mAb binding are identified as potential conformational epitope-contributing segments. PMID:26169135

  19. Conformal Collineations in String Cosmology

    NASA Astrophysics Data System (ADS)

    Baysal, Hüsnü; Camci, U.ğur; Tarhan, İsmail; Yilmaz, İhsan; Yavuz, İlhami; Dolgov, A.

    In this paper, we study the consequences of the existence of conformal collineations (CC) for string cloud in the context of general relativity. Especially, we interest in special conformal collineation (SCC), generated by a special affine conformal collineation (SACC) in the string cloud. Some results on the restrictions imposed by a conformal collineation symmetry in the string cloud are obtained.

  20. Conformational kinetics reveals affinities of protein conformational states

    PubMed Central

    Daniels, Kyle G.; Suo, Yang; Oas, Terrence G.

    2015-01-01

    Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein’s affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states. PMID:26162682

  1. Conformational flexibility of mephenesin.

    PubMed

    Écija, Patricia; Evangelisti, Luca; Vallejo, Montserrat; Basterretxea, Francisco J; Lesarri, Alberto; Castaño, Fernando; Caminati, Walther; Cocinero, Emilio J

    2014-05-22

    The mephenesin molecule (3-(2-methylphenoxy)propane-1,2-diol) serves as a test bank to explore several structural and dynamical issues, such as conformational flexibility, the orientation of the carbon linear chain relative to the benzene plane, or the effect of substituent position on the rotational barrier of a methyl group. The molecule has been studied by rotational spectroscopy in the 4-18 GHz frequency range by Fourier-transform methods in a supersonic expansion. The experiment has been backed by a previous conformational search plus optimization of the lowest energy structures by ab initio and density functional quantum calculations. The three lowest-lying conformers that can interconvert to each other by simple bond rotations have been detected in the jet. Rotational parameters for all structures have been obtained, and methyl torsional barriers have been determined for the two lowest-lying rotamers. The lowest-lying structure of mephenesin is highly planar, with all carbon atoms lying nearly in the benzene ring plane, and is stabilized by the formation of cooperative intramolecular hydrogen bonding. An estimation of the relative abundance of the detected conformers indicates that the energetically most stable conformer will have an abundance near 80% at temperatures relevant for biological activity. PMID:24754523

  2. Conformers of Gaseous Serine.

    PubMed

    He, Kedan; Allen, Wesley D

    2016-08-01

    The myriad conformers of the neutral form of natural amino acid serine (Ser) have been investigated by systematic computations with reliable electronic wave function methods. A total of 85 unique conformers were located using the MP2/cc-pVTZ level of theory. The 12 lowest-energy conformers of serine fall within a 8 kJ mol(-1) window, and for these species, geometric structures, precise relative energies, equilibrium and vibrationally averaged rotational constants, anharmonic vibrational frequencies, infrared intensities, quartic and sextic centrifugal distortion constants, dipole moments, and (14)N nuclear quadrupole coupling constants were computed. The relative energies were refined through composite focal-point analyses employing basis sets as large as aug-cc-pV5Z and correlation treatments through CCSD(T). The rotational constants for seven conformers measured by Fourier-transform microwave spectroscopy are in good agreement with the vibrationally averaged rotational constants computed in this study. Our anharmonic vibrational frequencies are compared to the large number of experimental vibrational absorptions attributable to at least six conformers. PMID:27294314

  3. A sequence pattern common to T cell epitopes.

    PubMed Central

    Rothbard, J B; Taylor, W R

    1988-01-01

    An analysis of the known cytotoxic and helper T cell epitopes has revealed similarity within their primary sequences. These similar motifs, characteristic of the known determinants, have been incorporated into predictive templates that have been used successfully to define eight helper and three cytotoxic epitopes in four different proteins. When the defined epitopes are segregated by restriction element, allele specific subpatterns emerge centering around the general pattern. The presence of similarities argues that the binding of peptide antigens to class I and class II is similar in nature. In addition, these motifs can be used to predict accurately areas within proteins capable of being recognized by individual MHC class I and class II molecules. PMID:2452085

  4. Branched peptide amphiphiles, related epitope compounds and self assembled structures thereof

    DOEpatents

    Stupp, Samuel I.; Guler, Mustafa O.

    2008-11-18

    Branched peptide amphiphilic compounds incorporating one or residues providing a pendant amino group for coupling one or more epitope sequences thereto, such compounds and related compositions for enhanced epitope presentation.

  5. Cholesterol-dependent conformational changes of P-glycoprotein are detected by the 15D3 monoclonal antibody.

    PubMed

    Gutay-Tóth, Zsuzsanna; Fenyvesi, Ferenc; Bársony, Orsolya; Szente, Lajos; Goda, Katalin; Szabó, Gábor; Bacsó, Zsolt

    2016-03-01

    The 15D3 mouse monoclonal antibody (mAb) binds an uncharacterized extracellular epitope of the ATP Binding Cassette (ABC) transporter human P-glycoprotein (Pgp). Depletion of cell plasma membrane cholesterol by using methyl-β-cyclodextrin or other chemically modified β-cyclodextrins decreased the Pgp binding affinity of 15D3 mAb. UIC2 mAb, which is known to distinguish two conformers of this ABC transporter, binds only a fraction of cell surface Pgps. UIC2 mAb non-reactive pools of Pgp can be identified with other extracellular mAbs such as 15D3. Cyclosporin A (CsA) can shift non-reactive Pgps into their UIC2-reactive conformation: a phenomenon called the "UIC2 shift". Competition studies proposed these two mAbs share overlapping epitopes and can reveal conformational changes of Pgp that correlate (r=0.97) with the cholesterol content of cells. An apparent increase in competition of these mAbs suggested a conformational change similar to those found in the presence of CsA. However, the reason turned out not to be the UIC2-shift because cholesterol removal from the plasma membrane (PM) reduced the amount of detectable Pgps by 15D3 mAb. This study showed that 15D3 mAb bound to a conformation sensitive epitope of Pgp that was responsive to PM cholesterol levels. These conformational changes were gradual and not as great as the changes observed between the two conformers recognized by the UIC2 mAb. PMID:26704667

  6. Carcinoembryonic antigen continuous epitopes determined by the spot method.

    PubMed

    Solassol, I; Granier, C; Pèlegrin, A

    2001-01-01

    Carcinoembryonic antigen (CEA) is a heavily glycosylated tumor-associated protein with an N-A1-B1-A2-B2-A3-B3 domain structure. Circulating CEA immunoassays are used for monitoring digestive cancer patients, and radiolabeled anti-CEA monoclonal antibodies (MAb) are used for the diagnosis and therapy of CEA-positive tumors. The five major nonoverlapping epitopes (Gold 1-5) have been broadly correlated with the domain organization, but there is no precise localization of the epitopes at the sequence level. In an attempt to identify the peptide sequences corresponding to the five Gold epitopes on the CEA molecule, we prepared a set of 227 overlapping fifteen-mer peptides corresponding to the complete CEA sequence with the SPOT method. Using five high affinity MAbs directed against the five CEA Gold epitopes, we demonstrated that none of these epitopes could be mimicked by a fifteen-mer peptide sequence. However, using rabbit and goat anti-CEA sera, we identified six major continuous antigenic regions. All are included in the Ig-like domains of the CEA: two in the A1 domain (residues 120-134 and 153-164), one each in the A2 (329-337) and A3 domains (508-513), one at the junction between the A3 and B3 domains (553-561) and one in the B3 domain (565-573). A very homologous sequence (common residues VSPRL) was mapped in each of the three A domains. Thus, in terms of occurrence of continuous epitopes, the Ig-like domains A1, A2, A3 and B3 seem to be the most antigenic parts of CEA. These peptide sequences should be good candidates for the future development of site-specific anti-CEA MAbs. PMID:11275797

  7. Functional, structural and epitopic prediction of hypothetical proteins of Mycobacterium tuberculosis H37Rv: An in silico approach for prioritizing the targets.

    PubMed

    Gazi, Md Amran; Kibria, Mohammad Golam; Mahfuz, Mustafa; Islam, Md Rezaul; Ghosh, Prakash; Afsar, Md Nure Alam; Khan, Md Arif; Ahmed, Tahmeed

    2016-10-15

    The global control of tuberculosis (TB) remains a great challenge from the standpoint of diagnosis, detection of drug resistance, and treatment. Major serodiagnostic limitations include low sensitivity and high cost in detecting TB. On the other hand, treatment measures are often hindered by low efficacies of commonly used drugs and resistance developed by the bacteria. Hence, there is a need to look into newer diagnostic and therapeutic targets. The proteome information available suggests that among the 3906 proteins in Mycobacterium tuberculosis H37Rv, about quarter remain classified as hypothetical uncharacterized set. This study involves a combination of a number of bioinformatics tools to analyze those hypothetical proteins (HPs). An entire set of 999 proteins was primarily screened for protein sequences having conserved domains with high confidence using a combination of the latest versions of protein family databases. Subsequently, 98 of such potential target proteins were extensively analyzed by means of physicochemical characteristics, protein-protein interaction, sub-cellular localization, structural similarity and functional classification. Next, we predicted antigenic proteins from the entire set and identified B and T cell epitopes of these proteins in M. tuberculosis H37Rv. We predicted the function of these HPs belong to various classes of proteins such as enzymes, transporters, receptors, structural proteins, transcription regulators and other proteins. However, the structural similarity prediction of the annotated proteins substantiated the functional classification of those proteins. Consequently, based on higher antigenicity score and sub-cellular localization, we choose two (NP_216420.1, NP_216903.1) of the antigenic proteins to exemplify B and T cell epitope prediction approach. Finally we found 15 epitopes those located partially or fully in the linear epitope region. We found 21 conformational epitopes by using Ellipro server as well. In

  8. Conserved hypothetical protein Rv1977 in Mycobacterium tuberculosis strains contains sequence polymorphisms and might be involved in ongoing immune evasion

    PubMed Central

    Jiang, Yi; Liu, Haican; Wang, Xuezhi; Li, Guilian; Qiu, Yan; Dou, Xiangfeng; Wan, Kanglin

    2015-01-01

    Host immune pressure and associated parasite immune evasion are key features of host-pathogen co-evolution. A previous study showed that human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. Here, we selected 151 clinical Mycobacterium tuberculosis isolates from China, amplified gene encoding Rv1977 and compared the sequences. The results showed that Rv1977, a conserved hypothetical protein, is not conserved in M. tuberculosis strains and there are polymorphisms existed in the protein. Some mutations, especially one frameshift mutation, occurred in the antigen Rv1977, which is uncommon in M.tb strains and may lead to the protein function altering. Mutations and deletion in the gene all affect one of three T cell epitopes and the changed T cell epitope contained more than one variable position, which may suggest ongoing immune evasion. PMID:26261576

  9. Data on the epitope mapping of soybean A2 and A3 glycinin.

    PubMed

    Saeed, Hanaa; Gagnon, Christine; Cober, Elroy; Gleddie, Steve

    2016-09-01

    The data information provided in this article relate to our research article "Using patient serum to epitope map soybean glycinins reveals common epitopes shared with many legumes and tree nuts" (Saeed et al., 2016) [1]. Here we provide western blot detection of glycinin subunits by soy-sensitive human sera, ELISA screens with overlapping synthetic peptides (epitope mapping), and various database/server epitope searches. PMID:27294180

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

    PubMed Central

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

    2010-01-01

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

  11. Polymorphous computing fabric

    DOEpatents

    Wolinski, Christophe Czeslaw; Gokhale, Maya B.; McCabe, Kevin Peter

    2011-01-18

    Fabric-based computing systems and methods are disclosed. A fabric-based computing system can include a polymorphous computing fabric that can be customized on a per application basis and a host processor in communication with said polymorphous computing fabric. The polymorphous computing fabric includes a cellular architecture that can be highly parameterized to enable a customized synthesis of fabric instances for a variety of enhanced application performances thereof. A global memory concept can also be included that provides the host processor random access to all variables and instructions associated with the polymorphous computing fabric.

  12. Extended conformal algebras

    NASA Astrophysics Data System (ADS)

    Bouwknegt, Peter

    1988-06-01

    We investigate extensions of the Virasoro algebra by a single primary field of integer or halfinteger conformal dimension Δ. We argue that for vanishing structure constant CΔΔΔ, the extended conformal algebra can only be associative for a generic c-value if Δ=1/2, 1, 3/2, 2 or 3. For the other Δ<=5 we compute the finite set of allowed c-values and identify the rational solutions. The case CΔΔΔ≠0 is also briefly discussed. I would like to thank Kareljan Schoutens for discussions and Sander Bais for a careful reading of the manuscript.

  13. Charged conformal Killing spinors

    SciTech Connect

    Lischewski, Andree

    2015-01-15

    We study the twistor equation on pseudo-Riemannian Spin{sup c}-manifolds whose solutions we call charged conformal Killing spinors (CCKSs). We derive several integrability conditions for the existence of CCKS and study their relations to spinor bilinears. A construction principle for Lorentzian manifolds admitting CCKS with nontrivial charge starting from CR-geometry is presented. We obtain a partial classification result in the Lorentzian case under the additional assumption that the associated Dirac current is normal conformal and complete the classification of manifolds admitting CCKS in all dimensions and signatures ≤5 which has recently been initiated in the study of supersymmetric field theories on curved space.

  14. Epitope mapping by epitope excision, hydrogen/deuterium exchange, and peptide-panning techniques combined with in silico analysis.

    PubMed

    Clementi, Nicola; Mancini, Nicasio; Criscuolo, Elena; Cappelletti, Francesca; Clementi, Massimo; Burioni, Roberto

    2014-01-01

    The fine characterization of protective B cell epitopes plays a pivotal role in the development of novel vaccines. The development of epitope-based vaccines, in fact, cannot be possible without a clear definition of the antigenic regions involved in the binding between the protective antibody (Ab) and its molecular target. To achieve this result, different epitope-mapping approaches have been widely described (Clementi et al. Drug Discov Today 18(9-10):464-471, 2013). Nowadays, the best way to characterize an Ab bound region is still the resolution of Ab-antigen (Ag) co-crystal structure. Unfortunately, the crystallization approaches are not always feasible. However, different experimental strategies aimed to predict Ab-Ag interaction and followed by in silico analysis of the results may be good surrogate approaches to achieve this result. Here, we review few experimental techniques followed by the use of "basic" informatics tools for the analysis of the results. PMID:24515481

  15. Cross-reactive, linear B cell epitopes of the influenza virus matrix protein 1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: Evaluate antibody responses to the conserved influenza matrix protein. Background: Little is known about the B cell epitopes in conserved internal influenza proteins or their role in viral immunity and immunopathogenesis. Based on epitope information present in the Immune Epitope Database...

  16. Pathological conformations involving the amino terminus of tau occur early in Alzheimer's disease and are differentially detected by monoclonal antibodies.

    PubMed

    Combs, Benjamin; Hamel, Chelsey; Kanaan, Nicholas M

    2016-10-01

    Conformational changes involving the amino terminus of the tau protein are among the earliest alterations associated with tau pathology in Alzheimer's disease and other tauopathies. This region of tau contains a phosphatase-activating domain (PAD) that is aberrantly exposed in pathological forms of the protein, an event that is associated with disruptions in anterograde fast axonal transport. We utilized four antibodies that recognize the amino terminus of tau, TNT1, TNT2 (a novel antibody), Tau12, and Tau13, to further study this important region. Using scanning alanine mutations in recombinant tau proteins, we refined the epitopes of each antibody. We examined the antibodies' relative abilities to specifically label pathological tau in non-denaturing and denaturing assays to gain insight into some of the mechanistic details of PAD exposure. We then determined the pattern of tau pathology labeled by each antibody in human hippocampal sections at various disease stages in order to characterize PAD exposure in the context of disease progression. The characteristics of reactivity for the antibodies fell into two groups. TNT1 and TNT2 recognized epitopes within amino acids 7-12 and specifically identified recombinant tau aggregates and pathological tau from Alzheimer's disease brains in a conformation-dependent manner. These antibodies labeled early pre-tangle pathology from neurons in early Braak stages and colocalized with thiazine red, a marker of fibrillar pathology, in classic neurofibrillary tangles. However, late tangles were negative for TNT1 and TNT2 indicating a loss of the epitope in later stages of tangle evolution. In contrast, Tau12 and Tau13 both identified discontinuous epitopes in the amino terminus and were unable to differentiate between normal and pathological tau in biochemical and tissue immunohistological assays. Despite the close proximity of these epitopes, the antibodies demonstrated remarkably different abilities to identify pathological

  17. New p22-Phox Monoclonal Antibodies: Identification of a Conformational Probe for Cytochrome b558

    PubMed Central

    Campion, Yannick; Jesaitis, Algirdas J.; Nguyen, Minh Vu Chuong; Grichine, Alexei; Herenger, Yvan; Baillet, Athan; Berthier, Sylvie; Morel, Françoise; Paclet, Marie-Hélène

    2009-01-01

    The phagocyte NADPH oxidase, belonging to the NADPH oxidase family (Nox), is dedicated to the production of bactericidal reactive oxygen species. The enzyme catalytic center is the cytochrome b558, formed by 2 subunits, Nox2 (gp91-phox) and p22-phox. Cytochrome b558 activation results from a conformational change induced by cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac). The catalytic subunit is Nox2, while p22-phox is essential for both Nox2 maturation and the membrane anchorage of regulatory proteins. Moreover, it has been shown to be necessary for novel Nox activity. In order to characterize both p22-phox topology and cytochrome b558 conformational change, 6 monoclonal antibodies were produced against purified cytochrome b558. Phage display epitope mapping combined with a truncation analysis of recombinant p22-phox allowed the identification of epitope regions. Some of these antibodies almost completely inhibited in vitro reconstituted NADPH oxidase activity. Data analysis identified antibodies that recognized epitopes involved in either Nox2 maturation or Nox2 activation. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils showed that the monoclonal antibody 12E6 bound preferentially active cytochrome b558. These monoclonal antibodies provided novel and unique probes to investigate maturation, activation and activity, not only of Nox2 but also of novel Nox. PMID:20375611

  18. Conformational instability governed by disulfide bonds partitions the dominant from subdominant helper T-cell responses specific for HIV-1 envelope glycoprotein gp120

    PubMed Central

    Nguyen, Hong-Nam P.; Steede, N. Kalaya; Robinson, James E.; Landry, Samuel J.

    2015-01-01

    Most individuals infected with human immunodeficiency virus type 1 (HIV-1) generate a CD4+ T-cell response that is dominated by a few epitopes. Immunodominance may be counterproductive because a broad CD4+ T-cell response is associated with reduced viral load. Previous studies indicated that antigen three-dimensional structure controls antigen processing and presentation and therefore CD4+ T-cell epitope dominance. Dominant epitopes occur adjacent to the V1-V2, V3, and V4 loops because proteolytic antigen processing in the loops promotes presentation of adjacent sequences. In this study, three gp120 (strain JR-FL) variants were constructed, in which deletions of single outer-domain disulfide bonds were expected to introduce local conformational flexibility and promote presentation of additional CD4+ T-cell epitopes. Following mucosal immunization of C57BL/6 mice with wild-type or variant gp120 lacking the V3-flanking disulfide bond, the typical pattern of dominant epitopes was observed, suggesting that the disulfide bond posed no barrier to antigen presentation. In mice that lacked gamma interferoninducible lysosomal thioreductase (GILT), proliferative responses to the typically dominant epitopes of gp120 were selectively depressed, and the dominance pattern was rearranged. Deletion of the V3-flanking disulfide bond or one of the V4-flanking disulfide bonds partially restored highly proliferative responses to the typically dominant epitopes. These results reveal an acute dependence of dominant CD4+ T-cell responses on the native gp120 conformation. PMID:25944298

  19. Conformational instability governed by disulfide bonds partitions the dominant from subdominant helper T-cell responses specific for HIV-1 envelope glycoprotein gp120.

    PubMed

    Nguyen, Hong-Nam P; Steede, N Kalaya; Robinson, James E; Landry, Samuel J

    2015-06-01

    Most individuals infected with human immunodeficiency virus type 1 (HIV-1) generate a CD4(+) T-cell response that is dominated by a few epitopes. Immunodominance may be counterproductive because a broad CD4(+) T-cell response is associated with reduced viral load. Previous studies indicated that antigen three-dimensional structure controls antigen processing and presentation and therefore CD4(+) T-cell epitope dominance. Dominant epitopes occur adjacent to the V1-V2, V3, and V4 loops because proteolytic antigen processing in the loops promotes presentation of adjacent sequences. In this study, three gp120 (strain JR-FL) variants were constructed, in which deletions of single outer-domain disulfide bonds were expected to introduce local conformational flexibility and promote presentation of additional CD4(+) T-cell epitopes. Following mucosal immunization of C57BL/6 mice with wild-type or variant gp120 lacking the V3-flanking disulfide bond, the typical pattern of dominant epitopes was observed, suggesting that the disulfide bond posed no barrier to antigen presentation. In mice that lacked gamma interferon-inducible lysosomal thioreductase (GILT), proliferative responses to the typically dominant epitopes of gp120 were selectively depressed, and the dominance pattern was rearranged. Deletion of the V3-flanking disulfide bond or one of the V4-flanking disulfide bonds partially restored highly proliferative responses to the typically dominant epitopes. These results reveal an acute dependence of dominant CD4(+) T-cell responses on the native gp120 conformation. PMID:25944298

  20. Conformal cloak for waves

    SciTech Connect

    Chen Huanyang; Leonhardt, Ulf; Tyc, Tomas

    2011-05-15

    Conformal invisibility devices are only supposed to work within the valid range of geometrical optics. Here, we show by numerical simulations and analytical arguments that for certain quantized frequencies, they are nearly perfect even in a regime that clearly violates geometrical optics. The quantization condition follows from the analogy between the Helmholtz equation and the stationary Schroedinger equation.

  1. Extended conformal field theories

    NASA Astrophysics Data System (ADS)

    Taormina, Anne

    1990-08-01

    Some extended conformal field theories are briefly reviewed. They illustrate how non minimal models of the Virasoro algebra (c≥1) can become minimal with respect to a larger algebra. The accent is put on N-extended superconformal algebras, which are relevant in superstring compactification.

  2. PERSONALITY AND CONFORMITY.

    ERIC Educational Resources Information Center

    BAROCAS, RALPH; GORLOW, LEON

    AN INVESTIGATION WAS MADE OF THE RELATIONSHIP BETWEEN PERSONALITY FACTORS AND CONFORMITY. THE SUBJECTS WERE 243 RANDOMLY SELECTED STUDENTS ENROLLED IN COLLEGE PSYCHOLOGY COURSES WHO WERE DIVIDED INTO GROUPS OF 97, 96, AND 50 SUBJECTS. A PERSONALITY FACTOR INVENTORY WAS OBTAINED FROM RESPONSES TO A LARGE LIST OF TRUE-FALSE PERSONALITY ITEM…

  3. Animal culture: chimpanzee conformity?

    PubMed

    van Schaik, Carel P

    2012-05-22

    Culture-like phenomena in wild animals have received much attention, but how good is the evidence and how similar are they to human culture? New data on chimpanzees suggest their culture may even have an element of conformity. PMID:22625856

  4. OptiTope—a web server for the selection of an optimal set of peptides for epitope-based vaccines

    PubMed Central

    Toussaint, Nora C.; Kohlbacher, Oliver

    2009-01-01

    Epitope-based vaccines (EVs) have recently been attracting significant interest. They trigger an immune response by confronting the immune system with immunogenic peptides derived from, e.g. viral- or cancer-related proteins. Binding of these peptides to proteins from the major histocompatibility complex (MHC) is crucial for immune system activation. However, since the MHC is highly polymorphic, different patients typically bind different repertoires of peptides. Furthermore, economical and regulatory issues impose strong limitations on the number of peptides that can be included in an EV. Hence, it is crucial to identify the optimal set of peptides for a vaccine, given constraints such as MHC allele probabilities in the target population, peptide mutation rates and maximum number of selected peptides. OptiTope aims at assisting immunologists in this critical task. With OptiTope, we provide an easy-to-use tool to determine a provably optimal set of epitopes with respect to overall immunogenicity in a specific individual (personalized medicine) or a target population (e.g. a certain ethnic group). OptiTope is available at http://www.epitoolkit.org/optitope. PMID:19420066

  5. Conformational changes in biopolymers

    NASA Astrophysics Data System (ADS)

    Ivanov, Vassili

    2005-12-01

    Biopolymer conformational changes are involved in many biological processes. This thesis summarizes some theoretical and experimental approaches which I have taken at UCLA to explore conformational changes in biopolymers. The reversible thermal denaturation of the DNA double helix is, perhaps, the simplest example of biopolymer conformational change. I have developed a statistical mechanics model of DNA melting with reduced degrees of freedom, which allows base stacking interaction to be taken into account and treat base pairing and stacking separately. Unlike previous models, this model describes both the unpairing and unstacking parts of the experimental melting curves and explains the observed temperature dependence of the effective thermodynamic parameters used in models of the nearest neighbor type. I developed a basic kinetic model for irreversible thermal denaturation of F-actin, which incorporates depolymerization of F-actin from the ends and breaking of F-actin fiber in the middle. The model explains the cooperativity of F-actin thermal denaturation observed by D. Pavlov et al. in differential calorimetry measurements. CG-rich DNA sequences form left-handed Z-DNA at high ionic strength or upon binding of polyvalent ions and some proteins. I studied experimentally the B-to-Z transition of the (CG)6 dodecamer. Improvement of the locally linearized model used to interpret the data gives evidence for an intermediate state in the B-to-Z transition of DNA, contrary to previous research on this subject. In the past 15 years it has become possible to study the conformational changes of biomolecules using single-molecule techniques. In collaboration with other lab members I performed a single-molecule experiment, where we monitored the displacement of a micrometer-size bead tethered to a surface by a DNA probe undergoing the conformational change. This technique allows probing of conformational changes with subnanometer accuracy. We applied the method to detect

  6. Identification of polymorphism in ethylone hydrochloride: synthesis and characterization.

    PubMed

    Maheux, Chad R; Alarcon, Idralyn Q; Copeland, Catherine R; Cameron, T Stanley; Linden, Anthony; Grossert, J Stuart

    2016-08-01

    Ethylone, a synthetic cathinone with psychoactive properties, is a designer drug which has appeared on the recreational drug market in recent years. Since 2012, illicit shipments of ethylone hydrochloride have been intercepted with increasing frequency at the Canadian border. Analysis has revealed that ethylone hydrochloride exists as two distinct polymorphs. In addition, several minor impurities were detected in some seized exhibits. In this study, the two conformational polymorphs of ethylone hydrochloride have been synthesized and fully characterized by FTIR, FT-Raman, powder XRD, GC-MS, ESI-MS/MS and NMR ((13) C CPMAS, (1) H, (13) C). The two polymorphs can be distinguished by vibrational spectroscopy, solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction. The FTIR data are applied to the identification of both polymorphs of ethylone hydrochloride (mixed with methylone hydrochloride) in a laboratory submission labelled as 'Ocean Snow Ultra'. The data presented in this study will assist forensic scientists in the differentiation of the two ethylone hydrochloride polymorphs. This report, alongside our recent article on the single crystal X-ray structure of a second polymorph of this synthetic cathinone, is the first to confirm polymorphism in ethylone hydrochloride. © 2015 Canada Border Services Agency. Drug Testing and Analysis published by John Wiley & Sons, Ltd. © 2015 Canada Border Services Agency. Drug Testing and Analysis published by John Wiley & Sons, Ltd. PMID:26344849

  7. Galilean conformal and superconformal symmetries

    SciTech Connect

    Lukierski, J.

    2012-10-15

    Firstly we discuss briefly three different algebras named as nonrelativistic (NR) conformal: Schroedinger, Galilean conformal, and infinite algebra of local NR conformal isometries. Further we shall consider in some detail Galilean conformal algebra (GCA) obtained in the limit c{yields}{infinity} from relativistic conformal algebraO(d+1, 2) (d-number of space dimensions). Two different contraction limits providing GCA and some recently considered realizations will be briefly discussed. Finally by considering NR contraction of D = 4 superconformal algebra the Galilei conformal superalgebra (GCSA) is obtained, in the formulation using complexWeyl supercharges.

  8. Two highly similar LAEDDTNAQKT and LTDKIGTEI epitopes in G glycoprotein may be useful for effective epitope based vaccine design against pathogenic Henipavirus.

    PubMed

    Parvege, Md Masud; Rahman, Monzilur; Nibir, Yead Morshed; Hossain, Mohammad Shahnoor

    2016-04-01

    Nipah virus and Hendra virus, two members of the genus Henipavirus, are newly emerging zoonotic pathogens which cause acute respiratory illness and severe encephalitis in human. Lack of the effective antiviral therapy endorses the urgency for the development of vaccine against these deadly viruses. In this study, we employed various computational approaches to identify epitopes which has the potential for vaccine development. By analyzing the immune parameters of the conserved sequences of G glycoprotein using various databases and bioinformatics tools, we identified two potential epitopes which may be used as peptide vaccines. Using different B cell epitope prediction servers, four highly similar B cell epitopes were identified. Immunoinformatics analyses revealed that LAEDDTNAQKT is a highly flexible and accessible B-cell epitope to antibody. Highly similar putative CTL epitopes were analyzed for their binding with the HLA-C 12*03 molecule. Docking simulation assay revealed that LTDKIGTEI has significantly lower binding energy, which bolstered its potential as epitope-based vaccine design. Finally, cytotoxicity analysis has also justified their potential as promising epitope-based vaccine candidate. In sum, our computational analysis indicates that either LAEDDTNAQKT or LTDKIGTEI epitope holds a promise for the development of universal vaccine against all kinds of pathogenic Henipavirus. Further in vivo and in vitro studies are necessary to validate the obtained findings. PMID:26970211

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

    PubMed Central

    2010-01-01

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

  10. Exposure of Epitope Residues on the Outer Face of the Chikungunya Virus Envelope Trimer Determines Antibody Neutralizing Efficacy

    PubMed Central

    Fong, Rachel H.; Banik, Soma S. R.; Mattia, Kimberly; Barnes, Trevor; Tucker, David; Liss, Nathan; Lu, Kai; Selvarajah, Suganya; Srinivasan, Surabhi; Mabila, Manu; Miller, Adam; Muench, Marcus O.; Michault, Alain; Rucker, Joseph B.; Paes, Cheryl; Simmons, Graham; Kahle, Kristen M.

    2014-01-01

    ABSTRACT Chikungunya virus (CHIKV) is a reemerging alphavirus that causes a debilitating arthritic disease and infects millions of people and for which no specific treatment is available. Like many alphaviruses, the structural targets on CHIKV that elicit a protective humoral immune response in humans are poorly defined. Here we used phage display against virus-like particles (VLPs) to isolate seven human monoclonal antibodies (MAbs) against the CHIKV envelope glycoproteins E2 and E1. One MAb, IM-CKV063, was highly neutralizing (50% inhibitory concentration, 7.4 ng/ml), demonstrated high-affinity binding (320 pM), and was capable of therapeutic and prophylactic protection in multiple animal models up to 24 h postexposure. Epitope mapping using a comprehensive shotgun mutagenesis library of 910 mutants with E2/E1 alanine mutations demonstrated that IM-CKV063 binds to an intersubunit conformational epitope on domain A, a functionally important region of E2. MAbs against the highly conserved fusion loop have not previously been reported but were also isolated in our studies. Fusion loop MAbs were broadly cross-reactive against diverse alphaviruses but were nonneutralizing. Fusion loop MAb reactivity was affected by temperature and reactivity conditions, suggesting that the fusion loop is hidden in infectious virions. Visualization of the binding sites of 15 different MAbs on the structure of E2/E1 revealed that all epitopes are located at the membrane-distal region of the E2/E1 spike. Interestingly, epitopes on the exposed topmost and outer surfaces of the E2/E1 trimer structure were neutralizing, whereas epitopes facing the interior of the trimer were not, providing a rationale for vaccine design and therapeutic MAb development using the intact CHIKV E2/E1 trimer. IMPORTANCE CHIKV is the most important alphavirus affecting humans, resulting in a chronic arthritic condition that can persist for months or years. In recent years, millions of people have been infected

  11. Polymorphism of the myostatin gene and its association with growth traits in chicken.

    PubMed

    Bhattacharya, T K; Chatterjee, R N

    2013-04-01

    An experiment was carried out on myostatin gene with the objectives of identification of polymorphism in the myostatin gene and estimation of the effect of polymorphism on growth traits in chickens. Single-stranded conformation polymorphism followed by sequencing was performed to reveal polymorphism of the gene. A total of 13 haplotypes were observed across 3 chicken lines (PB-1 and CB as broiler lines and IWI as the layer line). Myostatin haplogroups had a significant effect on BW at 28, 42, and 49 d of age in the PB-1 line. The significant association of haplogroups was observed with BW at d 14 and 49 in the CB line. In the IWI layer line, the myostatin gene was polymorphic but had no significant association with growth traits. It is concluded that the myostatin gene was polymorphic and had a significant effect on growth traits in broiler chickens. PMID:23472013

  12. The Elastic Constants and Related Mechanical Properties of the Monoclinic Polymorph of the Carbamazepine Molecular Crystal

    NASA Astrophysics Data System (ADS)

    Mohapatra, Himansu; Eckhardt, Craig J.

    2007-03-01

    Polymorphism is the property of a compound to crystallize in two or more crystalline phases containing different arrangements and/or conformations of the molecules in the crystal lattice. The Phenomenon of polymorphism is a major issue in the pharmaceutical industry especially in relation to drug uptake in the body, tablet processing and growth. This has led to considerable interest in predicting and understanding properties of drug polymorphs, and more recently the mechanical properties of the polymorphs. In this work, Brillouin scattering is used to probe the acoustic phonons of the monoclinic (P21/c) polymorph of the drug, carbamazepine (CBZ). By sampling a variety of acoustic phonons, the complete elastic constant tensor has been determined for this CBZ polymorph. The observed trend in the elastic constants: C11< C22˜C33 is qualitatively associated with the crystal growth pattern seen in CBZ. Investigation into the anisotropy of the intermolecular interactions has been investigated further by calculation of linear compressibilities.

  13. MUC-1 Tumor Antigen Agonist Epitopes for Enhancing T-cell Responses to Human Tumors | NCI Technology Transfer Center | TTC

    Cancer.gov

    Scientists at NIH have identified 7 new agonist epitopes of the MUC-1 tumor associated antigen. Compared to their native epitope counterparts, peptides reflecting these agonist epitopes have been shown to enhance the generation of human tumor cells, which in turn have a greater ability to kill human tumor cells endogenously expressing the native MUC-1 epitope.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  16. Selection of a peptide mimicking neutralization epitope of hepatitis E virus with phage peptide display technology

    PubMed Central

    Gu, Ying; Zhang, Jun; Wang, Ying-Bing; Li, Shao-Wei; Yang, Hai-Jie; Luo, Wen-Xin; Xia, Ning-Shao

    2004-01-01

    AIM: To select the peptide mimicking the neutralization epitope of hepatitis E virus which bound to non-type-specific and conformational monoclonal antibodies (mAbs) 8C11 and 8H3 fromed 7-peptide phage display library, and expressed the peptide recombinant with HBcAg in E.coli, and to observe whether the recombinant HBcAg could still form virus like particle (VLP) and to test the activation of the recombinant polyprotein and chemo-synthesized peptide that was selected by mAb 8H3. METHODS: 8C11 and 8H3 were used to screen for binding peptides through a 7-peptide phage display library. After 4 rounds of panning, monoclonal phages were selected and sequenced. The obtained dominant peptide coding sequences was then synthesized and inserted into amino acid 78 to 83 of hepatitis B core antigen (HBcAg), and then expressed in E.coli. Activity of the recombinant proteins was detected by Western blotting, VLPs of the recombinant polyproteins were tested by transmission electron microscopy and binding activity of the chemo-synthesized peptide was confirmed by BIAcore biosensor. RESULTS: Twenty-one positive monoclonal phages (10 for 8C11, and 11 for 8H3) were selected and the inserted fragments were sequenced. The DNA sequence coding for the obtained dominant peptides 8C11 (N’-His-Pro-Thr-Leu-Leu-Arg-Ile-C’, named 8C11A) and 8H3 (N’-Ser-Ile-Leu-Pro- Tyr-Pro-Tyr-C’, named 8H3A) were then synthesized and cloned to the HBcAg vector, then expressed in E.coli. The recombinant proteins aggregated into homodimer or polymer on SDS-PAGE, and could bind to mAb 8C11 and 8H3 in Western blotting. At the same time, the recombinant polyprotein could form virus like particles (VLPs), which could be visualized on electron micrograph. The dominant peptide 8H3A selected by mAb 8H3 was further chemo-synthesized, and its binding to mAb 8H3 could be detected by BIAcore biosensor. CONCLUSION: These results implicate that conformational neutralizing epitope can be partially modeled by a short

  17. Multiscale conformal pattern transfer

    PubMed Central

    Lodewijks, Kristof; Miljkovic, Vladimir; Massiot, Inès; Mekonnen, Addis; Verre, Ruggero; Olsson, Eva; Dmitriev, Alexandre

    2016-01-01

    We demonstrate a method for seamless transfer from a parent flat substrate of basically any lithographic top-down or bottom-up pattern onto essentially any kind of surface. The nano- or microscale patterns, spanning macroscopic surface areas, can be transferred with high conformity onto a large variety of surfaces when such patterns are produced on a thin carbon film, grown on top of a sacrificial layer. The latter allows lifting the patterns from the flat parent substrate onto a water-air interface to be picked up by the host surface of choice. We illustrate the power of this technique by functionalizing broad range of materials including glass, plastics, metals, rough semiconductors and polymers, highlighting the potential applications in in situ colorimetry of the chemistry of materials, anti-counterfeit technologies, biomolecular and biomedical studies, light-matter interactions at the nanoscale, conformal photovoltaics and flexible electronics. PMID:27329824

  18. Multiscale conformal pattern transfer

    NASA Astrophysics Data System (ADS)

    Lodewijks, Kristof; Miljkovic, Vladimir; Massiot, Inès; Mekonnen, Addis; Verre, Ruggero; Olsson, Eva; Dmitriev, Alexandre

    2016-06-01

    We demonstrate a method for seamless transfer from a parent flat substrate of basically any lithographic top-down or bottom-up pattern onto essentially any kind of surface. The nano- or microscale patterns, spanning macroscopic surface areas, can be transferred with high conformity onto a large variety of surfaces when such patterns are produced on a thin carbon film, grown on top of a sacrificial layer. The latter allows lifting the patterns from the flat parent substrate onto a water-air interface to be picked up by the host surface of choice. We illustrate the power of this technique by functionalizing broad range of materials including glass, plastics, metals, rough semiconductors and polymers, highlighting the potential applications in in situ colorimetry of the chemistry of materials, anti-counterfeit technologies, biomolecular and biomedical studies, light-matter interactions at the nanoscale, conformal photovoltaics and flexible electronics.

  19. Multiscale conformal pattern transfer.

    PubMed

    Lodewijks, Kristof; Miljkovic, Vladimir; Massiot, Inès; Mekonnen, Addis; Verre, Ruggero; Olsson, Eva; Dmitriev, Alexandre

    2016-01-01

    We demonstrate a method for seamless transfer from a parent flat substrate of basically any lithographic top-down or bottom-up pattern onto essentially any kind of surface. The nano- or microscale patterns, spanning macroscopic surface areas, can be transferred with high conformity onto a large variety of surfaces when such patterns are produced on a thin carbon film, grown on top of a sacrificial layer. The latter allows lifting the patterns from the flat parent substrate onto a water-air interface to be picked up by the host surface of choice. We illustrate the power of this technique by functionalizing broad range of materials including glass, plastics, metals, rough semiconductors and polymers, highlighting the potential applications in in situ colorimetry of the chemistry of materials, anti-counterfeit technologies, biomolecular and biomedical studies, light-matter interactions at the nanoscale, conformal photovoltaics and flexible electronics. PMID:27329824

  20. Conformal gripping device

    NASA Technical Reports Server (NTRS)

    Vranish, John M. (Inventor)

    2009-01-01

    The present invention relates to a conformal gripping device. In an embodiment of the present invention a conformal gripper device may be disclosed comprising a frame that includes an array of movable pins. The device may also include a roller locking and unlocking system within the frame. The system may comprise a pair of locking rollers for each row of gripper pins to facilitate locking and unlocking the array of gripper pins on a column-by-column basis. The system may also include a striker element that may force the locking rollers to roll along an angled roll surface to facilitate unlocking of the array of pins on a column-by-column basis. The system may further include an electromagnetic actuator or solenoid and permanent magnets to facilitate movement of the striker element and the locking rollers.

  1. Conformational flexibility of aspartame.

    PubMed

    Toniolo, Claudio; Temussi, Pierandrea

    2016-05-01

    L-Aspartyl-L-phenylalanine methyl ester, better known as aspartame, is not only one of the most used artificial sweeteners, but also a very interesting molecule with respect to the correlation between molecular structure and taste. The extreme conformational flexibility of this dipeptide posed a huge difficulty when researchers tried to use it as a lead compound to design new sweeteners. In particular, it was difficult to take advantage of its molecular model as a mold to infer the shape of the, then unknown, active site of the sweet taste receptor. Here, we follow the story of the 3D structural aspects of aspartame from early conformational studies to recent docking into homology models of the receptor. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 376-384, 2016. PMID:27038223

  2. The conformal bootstrap

    NASA Astrophysics Data System (ADS)

    Poland, David; Simmons-Duffin, David

    2016-06-01

    The conformal bootstrap was proposed in the 1970s as a strategy for calculating the properties of second-order phase transitions. After spectacular success elucidating two-dimensional systems, little progress was made on systems in higher dimensions until a recent renaissance beginning in 2008. We report on some of the main results and ideas from this renaissance, focusing on new determinations of critical exponents and correlation functions in the three-dimensional Ising and O(N) models.

  3. Conformal scalar field wormholes

    NASA Technical Reports Server (NTRS)

    Halliwell, Jonathan J.; Laflamme, Raymond

    1989-01-01

    The Euclidian Einstein equations with a cosmological constant and a conformally coupled scalar field are solved, taking the metric to be of the Robertson-Walker type. In the case Lambda = 0, solutions are found which represent a wormhole connecting two asymptotically flat Euclidian regions. In the case Lambda greater than 0, the solutions represent tunneling from a small Tolman-like universe to a large Robertson-Walker universe.

  4. Conformations of organophosphine oxides

    DOE PAGESBeta

    De Silva, Nuwan; Zahariev, Federico; Hay, Benjamin P.; Gordon, Mark S.; Windus, Theresa L.

    2015-07-17

    The conformations of a series of organophosphine oxides, OP(CH3)2R, where R = methyl, ethyl, isopropyl, tert-butyl, vinyl, and phenyl, are predicted using the MP2/cc-pVTZ level of theory. Comparison of potential energy surfaces for rotation about P–C bonds with crystal structure data reveals a strong correlation between predicted location and energetics of minima and histograms of dihedral angle distributions observed in the solid state. In addition, the most stable conformers are those that minimize the extent of steric repulsion between adjacent rotor substituents, and the torsional barriers tend to increase with the steric bulk of the rotating alkyl group. MM3 forcemore » field parameters were adjusted to fit the MP2 results, providing a fast and accurate model for predicting organophosphine oxides shapes—an essential part of understanding the chemistry of these compounds. As a result, the predictive power of the modified MM3 model was tested against MP2/cc-pVTZ conformations for triethylphosphine oxide, OP(CH2CH3)3, and triphenylphosphine oxide, OP(Ph)3.« less

  5. Conformations of organophosphine oxides

    SciTech Connect

    De Silva, Nuwan; Zahariev, Federico; Hay, Benjamin P.; Gordon, Mark S.; Windus, Theresa L.

    2015-07-17

    The conformations of a series of organophosphine oxides, OP(CH3)2R, where R = methyl, ethyl, isopropyl, tert-butyl, vinyl, and phenyl, are predicted using the MP2/cc-pVTZ level of theory. Comparison of potential energy surfaces for rotation about P–C bonds with crystal structure data reveals a strong correlation between predicted location and energetics of minima and histograms of dihedral angle distributions observed in the solid state. In addition, the most stable conformers are those that minimize the extent of steric repulsion between adjacent rotor substituents, and the torsional barriers tend to increase with the steric bulk of the rotating alkyl group. MM3 force field parameters were adjusted to fit the MP2 results, providing a fast and accurate model for predicting organophosphine oxides shapes—an essential part of understanding the chemistry of these compounds. As a result, the predictive power of the modified MM3 model was tested against MP2/cc-pVTZ conformations for triethylphosphine oxide, OP(CH2CH3)3, and triphenylphosphine oxide, OP(Ph)3.

  6. Hot conformal gauge theories

    NASA Astrophysics Data System (ADS)

    Mojaza, Matin; Pica, Claudio; Sannino, Francesco

    2010-12-01

    We compute the nonzero temperature free energy up to the order g6ln⁡(1/g) in the coupling constant for vectorlike SU(N) gauge theories featuring matter transforming according to different representations of the underlying gauge group. The number of matter fields, i.e. flavors, is arranged in such a way that the theory develops a perturbative stable infrared fixed point at zero temperature. Because of large distance conformality we trade the coupling constant with its fixed point value and define a reduced free energy which depends only on the number of flavors, colors, and matter representation. We show that the reduced free energy changes sign, at the second, fifth, and sixth order in the coupling, when decreasing the number of flavors from the upper end of the conformal window. If the change in sign is interpreted as a signal of an instability of the system then we infer a critical number of flavors. Surprisingly this number, if computed to the order g2, agrees with previous predictions for the lower boundary of the conformal window for nonsupersymmetric gauge theories. The higher order results tend to predict a higher number of critical flavors. These are universal properties, i.e. they are independent of the specific matter representation.

  7. Structural analysis of Anopheles midgut aminopeptidase N reveals a novel malaria transmission-blocking vaccine B-cell epitope

    PubMed Central

    Atkinson, Sarah C.; Armistead, Jennifer S.; Mathias, Derrick K.; Sandeu, Maurice M.; Tao, Dingyin; Borhani-Dizaji, Nahid; Tarimo, Brian B.; Morlais, Isabelle; Dinglasan, Rhoel R.; Borg, Natalie A.

    2015-01-01

    Mosquito-based malaria transmission-blocking vaccines (mTBVs) target midgut-surface antigens of the Plasmodium parasite's obligate vector, the Anopheles mosquito. The alanyl aminopeptidase N (AnAPN1) is the leading mTBV immunogen; however AnAPN1's role in Plasmodium infection of the mosquito and how anti-AnAPN1 antibodies functionally block parasite transmission remains elusive. Here we present the 2.65 Å crystal structure of AnAPN1 and the immunoreactivity and transmission-blocking profile of three AnAPN1 monoclonal antibodies (mAb), including mAb 4H5B7, which effectively block transmission of natural strains of Plasmodium falciparum. Utilizing the AnAPN1 structure we map the conformation-dependent 4H5B7 neo-epitope to a previously uncharacterized region on domain 1, and further demonstrate that non-human primate neo-epitope-specific IgG also block parasite transmission. We discuss the prospect of a novel biological function of AnAPN1 as a receptor for Plasmodium in the mosquito midgut and the implications for redesigning the AnAPN1 mTBV. PMID:26075520

  8. Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses.

    PubMed

    Grandea, Andres G; Olsen, Ole A; Cox, Thomas C; Renshaw, Mark; Hammond, Philip W; Chan-Hui, Po-Ying; Mitcham, Jennifer L; Cieplak, Witold; Stewart, Shaun M; Grantham, Michael L; Pekosz, Andrew; Kiso, Maki; Shinya, Kyoko; Hatta, Masato; Kawaoka, Yoshihiro; Moyle, Matthew

    2010-07-13

    Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome, which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG(+) memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains. PMID:20615945

  9. Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses

    PubMed Central

    Grandea, Andres G.; Olsen, Ole A.; Cox, Thomas C.; Renshaw, Mark; Hammond, Philip W.; Chan-Hui, Po-Ying; Mitcham, Jennifer L.; Cieplak, Witold; Stewart, Shaun M.; Grantham, Michael L.; Pekosz, Andrew; Kiso, Maki; Shinya, Kyoko; Hatta, Masato; Kawaoka, Yoshihiro; Moyle, Matthew

    2010-01-01

    Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome, which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG+ memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains. PMID:20615945

  10. Structure and Immunogenicity of a Peptide Vaccine, Including the Complete HIV-1 gp41 2F5 Epitope

    PubMed Central

    Serrano, Soraya; Araujo, Aitziber; Apellániz, Beatriz; Bryson, Steve; Carravilla, Pablo; de la Arada, Igor; Huarte, Nerea; Rujas, Edurne; Pai, Emil F.; Arrondo, José L. R.; Domene, Carmen; Jiménez, María Angeles; Nieva, José L.

    2014-01-01

    The membrane-proximal external region (MPER) of gp41 harbors the epitope recognized by the broadly neutralizing anti-HIV 2F5 antibody, a research focus in HIV-1 vaccine development. In this work, we analyze the structure and immunogenic properties of MPERp, a peptide vaccine that includes the following: (i) the complete sequence protected from proteolysis by the 2F5 paratope; (ii) downstream residues postulated to establish weak contacts with the CDR-H3 loop of the antibody, which are believed to be crucial for neutralization; and (iii) an aromatic rich anchor to the membrane interface. MPERp structures solved in dodecylphosphocholine micelles and 25% 1,1,1,3,3,3-hexafluoro-2-propanol (v/v) confirmed folding of the complete 2F5 epitope within continuous kinked helices. Infrared spectroscopy (IR) measurements demonstrated the retention of main helical conformations in immunogenic formulations based on alum, Freund's adjuvant, or two different types of liposomes. Binding to membrane-inserted MPERp, IR, molecular dynamics simulations, and characterization of the immune responses further suggested that packed helical bundles partially inserted into the lipid bilayer, rather than monomeric helices adsorbed to the membrane interface, could encompass effective MPER peptide vaccines. Together, our data constitute a proof-of-concept to support MPER-based peptides in combination with liposomes as stand-alone immunogens and suggest new approaches for structure-aided MPER vaccine development. PMID:24429284

  11. Are there generic mechanisms governing interactions between nanoparticles and cells? Epitope mapping the outer layer of the protein material interface

    NASA Astrophysics Data System (ADS)

    Lynch, Iseult

    2007-01-01

    In this paper we discuss the possibility of a general paradigm for cell-biomaterial and cell-nanoparticle interactions. The basis of the paradigm is that the nature of the biomaterial or nanoparticle surface is not the important parameter, but rather the nature of the outermost layer of adsorbed proteins as well as long-lived misfolded proteins shed from the surfaces. If the adsorbed protein is irreversibly adsorbed onto the surface it may be sufficiently disrupted so that a variety of peptide units (here termed “cryptic epitopes”) not usually expressed in nature at the surface of the protein become exposed. Similarly, where there is a slow exchange time with the surface, surface-induced perturbations may lead to long-lived misfolded proteins being shed from the surface and continuing to express altered surface peptide sequences. In cases where the proteins have lost most of their tertiary structure, anomalous peptide sequences and geometries that are not displayed at the surface by the native protein may in fact be presented after surface adsorption of a protein. Such anomalous surface expressions could contain novel epitopes that trigger various signalling pathways or even diseases. Thus, future approaches to understanding cell-biomaterial and cell-nanoparticle interactions should focus on characterising the outer layer of the adsorbed proteins, or “epitope mapping” as well as examining the possibility of formation of essentially “new” proteins as a result of desorption of conformationally or geometrically altered proteins.

  12. Crystal Structure of Insulin-Regulated Aminopeptidase with Bound Substrate Analogue Provides Insight on Antigenic Epitope Precursor Recognition and Processing.

    PubMed

    Mpakali, Anastasia; Saridakis, Emmanuel; Harlos, Karl; Zhao, Yuguang; Papakyriakou, Athanasios; Kokkala, Paraskevi; Georgiadis, Dimitris; Stratikos, Efstratios

    2015-09-15

    Aminopeptidases that generate antigenic peptides influence immunodominance and adaptive cytotoxic immune responses. The mechanisms that allow these enzymes to efficiently process a vast number of different long peptide substrates are poorly understood. In this work, we report the structure of insulin-regulated aminopeptidase, an enzyme that prepares antigenic epitopes for cross-presentation in dendritic cells, in complex with an antigenic peptide precursor analog. Insulin-regulated aminopeptidase is found in a semiclosed conformation with an extended internal cavity with limited access to the solvent. The N-terminal moiety of the peptide is located at the active site, positioned optimally for catalysis, whereas the C-terminal moiety of the peptide is stabilized along the extended internal cavity lodged between domains II and IV. Hydrophobic interactions and shape complementarity enhance peptide affinity beyond the catalytic site and support a limited selectivity model for antigenic peptide selection that may underlie the generation of complex immunopeptidomes. PMID:26259583

  13. A paradigm shift: Cancer therapy with peptide-based B-cell epitopes and peptide immunotherapeutics targeting multiple solid tumor types: Emerging concepts and validation of combination immunotherapy

    PubMed Central

    Kaumaya, Pravin TP

    2015-01-01

    There is a recognizable and urgent need to speed the development and application of novel, more efficacious anti-cancer vaccine therapies that inhibit tumor progression and prevent acquisition of tumor resistance. We have created and established a portfolio of validated peptide epitopes against multiple receptor tyrosine kinases and we have identified the most biologically effective combinations of EGFR (HER-1), HER-2, HER-3, VEGF and IGF-1R peptide vaccines/mimics to selectively inhibit multiple receptors and signaling pathways. The strategy is based on the use of chimeric conformational B-cell epitope peptides incorporating “promiscuous” T-cell epitopes that afford the possibility of generating an enduring immune response, eliciting protein-reactive high-affinity anti-peptide antibodies as potential vaccines and peptide mimics that act as antagonists to receptor signaling that drive cancer metastasis. In this review we will summarize our ongoing studies based on the development of combinatorial immunotherapeutic strategies that act synergistically to enhance immune-mediated tumor killing aimed at addressing mechanisms of tumor resistance for several tumor types. PMID:25874884

  14. Conformal superalgebras via tractor calculus

    NASA Astrophysics Data System (ADS)

    Lischewski, Andree

    2015-01-01

    We use the manifestly conformally invariant description of a Lorentzian conformal structure in terms of a parabolic Cartan geometry in order to introduce a superalgebra structure on the space of twistor spinors and normal conformal vector fields formulated in purely algebraic terms on parallel sections in tractor bundles. Via a fixed metric in the conformal class, one reproduces a conformal superalgebra structure that has been considered in the literature before. The tractor approach, however, makes clear that the failure of this object to be a Lie superalgebra in certain cases is due to purely algebraic identities on the spinor module and to special properties of the conformal holonomy representation. Moreover, it naturally generalizes to higher signatures. This yields new formulas for constructing new twistor spinors and higher order normal conformal Killing forms out of existing ones, generalizing the well-known spinorial Lie derivative. Moreover, we derive restrictions on the possible dimension of the space of twistor spinors in any metric signature.

  15. Polymorphism of triphenyl phosphite

    NASA Astrophysics Data System (ADS)

    Baran, J.; Davydova, N. A.; Drozd, M.

    2014-03-01

    The glass-forming liquid triphenyl phosphite (TPP) has recently attracted much attention due to the possible existence of a polyamorphism, i.e., the existence of two or more amorphous phases. In the present work we provide experimental evidence of the existence of a polymorphism in TPP. In addition to the already known conventional crystalline phase, which melts at 299.1 K, it has been found that TPP can crystallize in another polymorphic phase. The new polymorph can be obtained from the liquid phase due to direct cooling from the room temperature up to 245 K where it is held for 15 min and then heated up to 270 K. At 270 K crystallization of the new polymorph occurs, which melts at 291.6 K.

  16. Variable expression of epitopes on the surface of Mycoplasma gallisepticum demonstrated with monoclonal antibodies.

    PubMed

    Bencina, D; Kleven, S H; Elfaki, M G; Snoj, A; Dovc, P; Dorrer, D; Russ, I

    1994-03-01

    Twelve monoclonal antibodies (Mabs) against Mycoplasma gallisepticum (Mg) strains F, R, S6(208) and PET2 were used for analysis of epitopes of 22 Mg strains. Six Mabs recognized surface epitopes in the majority of strains, but did not react with variant strains like K 503 and K 703. Two Mabs reacted with epitopes on about 56 kilodalton (kDa) proteins and showing consistent expression on Mg colonies. Three Mabs recognized three different variable surface epitopes associated with about 67 kDa proteins and one Mab variable epitope on about 33 and 80 kDa proteins. Two-dimensional immunoblotting showed considerable differences in the charge of proteins bearing variable surface epitopes in different Mg strains. Subcloning of four low passage Mg strains using Mabs for screening populations that derived from a single colony with defined surface epitopes showed that some colonies may switch surface epitopes associated with 67 and 80 kDa proteins. This switching was reversible and generated subpopulations of Mg expressing different combinations of surface epitopes. Phenotypic switching of epitopes probably occurs also in vivo and may be the mechanism enabling Mg to evade the host immune response. PMID:18671069

  17. Novel conformation-specific monoclonal antibodies against amyloidogenic forms of transthyretin

    PubMed Central

    Higaki, Jeffrey N.; Chakrabartty, Avi; Galant, Natalie J.; Hadley, Kevin C.; Hammerson, Bradley; Nijjar, Tarlochan; Torres, Ronald; Tapia, Jose R.; Salmans, Joshua; Barbour, Robin; Tam, Stephen J.; Flanagan, Ken; Zago, Wagner; Kinney, Gene G.

    2016-01-01

    Abstract Introduction: Transthyretin amyloidosis (ATTR amyloidosis) is caused by the misfolding and deposition of the transthyretin (TTR) protein and results in progressive multi-organ dysfunction. TTR epitopes exposed by dissociation and misfolding are targets for immunotherapeutic antibodies. We developed and characterized antibodies that selectively bound to misfolded, non-native conformations of TTR. Methods: Antibody clones were generated by immunizing mice with an antigenic peptide comprising a cryptotope within the TTR sequence and screened for specific binding to non-native TTR conformations, suppression of in vitro TTR fibrillogenesis, promotion of antibody-dependent phagocytic uptake of mis-folded TTR and specific immunolabeling of ATTR amyloidosis patient-derived tissue. Results: Four identified monoclonal antibodies were characterized. These antibodies selectively bound the target epitope on monomeric and non-native misfolded forms of TTR and strongly suppressed TTR fibril formation in vitro. These antibodies bound fluorescently tagged aggregated TTR, targeting it for phagocytic uptake by macrophage THP-1 cells, and amyloid-positive TTR deposits in heart tissue from patients with ATTR amyloidosis, but did not bind to other types of amyloid deposits or normal tissue. Conclusions: Conformation-specific anti-TTR antibodies selectively bind amyloidogenic but not native TTR. These novel antibodies may be therapeutically useful in preventing deposition and promoting clearance of TTR amyloid and in diagnosing TTR amyloidosis. PMID:26981744

  18. Shear, heat and pH induced conformational changes of wheat gluten - Impact on antigenicity.

    PubMed

    Rahaman, Toheder; Vasiljevic, Todor; Ramchandran, Lata

    2016-04-01

    Processing can induce conformational changes of food proteins depending on the conditions used that may affect their antigenicity. This study investigated the effect of pH (3,5,7) temperature (80,90,100 °C) and shear (500,1000,1500 s(-1)) on the conformational changes (surface hydrophobicity, FTIR, SDS-PAGE and thiol content) of gluten in relation to its antigenicity (determined by Enzyme-linked Immunosorbent Assay). Overall, at pH 3, up to 90 °C, conformational changes and possible burial of some antigenic hydrophobic residues resulted in reduction of antigenicity to one-third that of control. Further heating to 100 °C caused increase in antigenicity due to exposure of some hidden epitopes. However, at pH 5 and 7, the antigenicity declined only at 100 °C due to modification in thiol content and related structural changes causing destruction and/or masking of some epitopes. Shear alone had no effect on antigenicity of gluten but could have a synergistic influence at pH 7 and 100 °C. PMID:26593481

  19. Epitope Predictions Indicate the Presence of Two Distinct Types of Epitope-Antibody-Reactivities Determined by Epitope Profiling of Intravenous Immunoglobulins

    PubMed Central

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

    2013-01-01

    Epitope-antibody-reactivities (EAR) of intravenous immunoglobulins (IVIGs) determined for 75,534 peptides by microarray analysis demonstrate that roughly 9% of peptides derived from 870 different human protein sequences react with antibodies present in IVIG. Computational prediction of linear B cell epitopes was conducted using machine learning with an ensemble of classifiers in combination with position weight matrix (PWM) analysis. Machine learning slightly outperformed PWM with area under the curve (AUC) of 0.884 vs. 0.849. Two different types of epitope-antibody recognition-modes (Type I EAR and Type II EAR) were found. Peptides of Type I EAR are high in tyrosine, tryptophan and phenylalanine, and low in asparagine, glutamine and glutamic acid residues, whereas for peptides of Type II EAR it is the other way around. Representative crystal structures present in the Protein Data Bank (PDB) of Type I EAR are PDB 1TZI and PDB 2DD8, while PDB 2FD6 and 2J4W are typical for Type II EAR. Type I EAR peptides share predicted propensities for being presented by MHC class I and class II complexes. The latter interaction possibly favors T cell-dependent antibody responses including IgG class switching. Peptides of Type II EAR are predicted not to be preferentially presented by MHC complexes, thus implying the involvement of T cell-independent IgG class switch mechanisms. The high extent of IgG immunoglobulin reactivity with human peptides implies that circulating IgG molecules are prone to bind to human protein/peptide structures under non-pathological, non-inflammatory conditions. A webserver for predicting EAR of peptide sequences is available at www.sysmed-immun.eu/EAR. PMID:24244326

  20. Polymorphism in liver-stage malaria vaccine candidate proteins: immune evasion and implications for vaccine design.

    PubMed

    Flanagan, Katie L; Wilson, Kirsty L; Plebanski, Magdalena

    2016-03-01

    The pre-erythrocytic stage of infection by malaria parasites represents a key target for vaccines that aim to eradicate malaria. Two important broad immune evasion strategies that can interfere with vaccine efficacy include the induction of dendritic cell (DC) dysfunction and regulatory T cells (Tregs) by blood-stage malaria parasites, leading to inefficient priming of T cells targeting liver-stage infections. The parasite also uses 'surgical strike' strategies, whereby polymorphism in pre-erythrocytic antigens can interfere with host immunity. Specifically, we review how even single amino acid changes in T cell epitopes can lead to loss of binding to major histocompatibility complex (MHC), lack of cross-reactivity, or antagonism and immune interference, where simultaneous or sequential stimulation with related variants of the same T cell epitope can cause T cell anergy or the conversion of effector to immunosuppressive T cell phenotypes. PMID:26610026

  1. Engineering Recombinant Reoviruses To Display gp41 Membrane-Proximal External-Region Epitopes from HIV-1.

    PubMed

    Boehme, Karl W; Ikizler, Mine'; Iskarpatyoti, Jason A; Wetzel, J Denise; Willis, Jordan; Crowe, James E; LaBranche, Celia C; Montefiori, David C; Wilson, Gregory J; Dermody, Terence S

    2016-01-01

    The gp41 membrane-proximal external region (MPER) is a target for broadly neutralizing antibody responses against human immunodeficiency virus type 1 (HIV-1). However, replication-defective virus vaccines currently under evaluation in clinical trials do not efficiently elicit MPER-specific antibodies. Structural modeling suggests that the MPER forms an α-helical coiled coil that is required for function and immunogenicity. To maintain the native MPER conformation, we used reverse genetics to engineer replication-competent reovirus vectors that displayed MPER sequences in the α-helical coiled-coil tail domain of viral attachment protein σ1. Sequences in reovirus strain type 1 Lang (T1L) σ1 were exchanged with sequences encoding HIV-1 strain Ba-L MPER epitope 2F5 or the entire MPER. Individual 2F5 or MPER substitutions were introduced at virion-proximal or virion-distal sites in the σ1 tail. Recombinant reoviruses containing heterologous HIV-1 sequences were viable and produced progeny yields comparable to those with wild-type virus. HIV-1 sequences were retained following 10 serial passages in cell culture, indicating that the substitutions were genetically stable. Recombinant viruses engineered to display the 2F5 epitope or full-length MPER in σ1 were recognized by purified 2F5 antibody. Inoculation of mice with 2F5-containing vectors or rabbits with 2F5- or MPER-containing vectors elicited anti-reovirus antibodies, but HIV-1-specific antibodies were not detected. Together, these findings indicate that heterologous sequences that form α-helices can functionally replace native sequences in the α-helical tail domain of reovirus attachment protein σ1. However, although these vectors retain native antigenicity, they were not immunogenic, illustrating the difficulty of experimentally inducing immune responses to this essential region of HIV-1. IMPORTANCE Vaccines to protect against HIV-1, the causative agent of AIDS, are not approved for use. Antibodies that

  2. Engineering Recombinant Reoviruses To Display gp41 Membrane-Proximal External-Region Epitopes from HIV-1

    PubMed Central

    Boehme, Karl W.; Ikizler, Mine'; Iskarpatyoti, Jason A.; Wetzel, J. Denise; Willis, Jordan; Crowe, James E.; LaBranche, Celia C.; Montefiori, David C.

    2016-01-01

    ABSTRACT The gp41 membrane-proximal external region (MPER) is a target for broadly neutralizing antibody responses against human immunodeficiency virus type 1 (HIV-1). However, replication-defective virus vaccines currently under evaluation in clinical trials do not efficiently elicit MPER-specific antibodies. Structural modeling suggests that the MPER forms an α-helical coiled coil that is required for function and immunogenicity. To maintain the native MPER conformation, we used reverse genetics to engineer replication-competent reovirus vectors that displayed MPER sequences in the α-helical coiled-coil tail domain of viral attachment protein σ1. Sequences in reovirus strain type 1 Lang (T1L) σ1 were exchanged with sequences encoding HIV-1 strain Ba-L MPER epitope 2F5 or the entire MPER. Individual 2F5 or MPER substitutions were introduced at virion-proximal or virion-distal sites in the σ1 tail. Recombinant reoviruses containing heterologous HIV-1 sequences were viable and produced progeny yields comparable to those with wild-type virus. HIV-1 sequences were retained following 10 serial passages in cell culture, indicating that the substitutions were genetically stable. Recombinant viruses engineered to display the 2F5 epitope or full-length MPER in σ1 were recognized by purified 2F5 antibody. Inoculation of mice with 2F5-containing vectors or rabbits with 2F5- or MPER-containing vectors elicited anti-reovirus antibodies, but HIV-1-specific antibodies were not detected. Together, these findings indicate that heterologous sequences that form α-helices can functionally replace native sequences in the α-helical tail domain of reovirus attachment protein σ1. However, although these vectors retain native antigenicity, they were not immunogenic, illustrating the difficulty of experimentally inducing immune responses to this essential region of HIV-1. IMPORTANCE Vaccines to protect against HIV-1, the causative agent of AIDS, are not approved for use

  3. Identification of a major continuous epitope of human alpha crystallin

    NASA Technical Reports Server (NTRS)

    Takemoto, L.; Emmons, T.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Human lens proteins were digested with trypsin or V8 protease, and the resulting peptides resolved on a C18 reverse phase column. Fractions from this column were probed with polyclonal antiserum made against the whole alpha crystallin molecule. Peptides in the seropositive fraction were purified to homogeneity, then characterized by mass spectral analysis and partial Edman degradation. The tryptic and V8 digests contained only one seropositive peptide that was derived from the C-terminal region of the alpha-A molecule. To determine the exact boundaries of the epitope, various size analogues of this region were synthesized and probed with anti-alpha serum. Together, these studies demonstrate that the major continuous epitope of the alpha-A chain includes the sequence KPTSAPS, corresponding to residues 166-172 of the human alpha-A crystallin chain.

  4. OSI Conformance Testing for Bibliographic Applications.

    ERIC Educational Resources Information Center

    Arbez, Gilbert; Swain, Leigh

    1990-01-01

    Describes the development of Open Systems Interconnection (OSI) conformance testing sites, conformance testing tools, and conformance testing services. Discusses related topics such as interoperability testing, arbitration testing, and international harmonization of conformance testing. A glossary is included. (24 references) (SD)

  5. Efficient production of chimeric Human papillomavirus 16 L1 protein bearing the M2e influenza epitope in Nicotiana benthamiana plants

    PubMed Central

    2011-01-01

    Background Human papillomavirus 16 (HPV-16) L1 protein has the capacity to self-assemble into capsomers or virus-like particles (VLPs) that are highly immunogenic, allowing their use in vaccine production. Successful expression of HPV-16 L1 protein has been reported in plants, and plant-produced VLPs have been shown to be immunogenic after administration to animals. Results We investigated the potential of HPV-16 L1 to act as a carrier of two foreign epitopes from Influenza A virus: (i) M2e2-24, ectodomain of the M2 protein (M2e), that is highly conserved among all influenza A isolates, or (ii) M2e2-9, a shorter version of M2e containing the N-terminal highly conserved epitope, that is common for both M1 and M2 influenza proteins. A synthetic HPV-16 L1 gene optimized with human codon usage was used as a backbone gene to design four chimeric sequences containing either the M2e2-24 or the M2e2-9 epitope in two predicted surface-exposed L1 positions. All chimeric constructs were transiently expressed in plants using the Cowpea mosaic virus-derived expression vector, pEAQ-HT. Chimeras were recognized by a panel of linear and conformation-specific anti HPV-16 L1 MAbs, and two of them also reacted with the anti-influenza MAb. Electron microscopy showed that chimeric proteins made in plants spontaneously assembled in higher order structures, such as VLPs of T = 1 or T = 7 symmetry, or capsomers. Conclusions In this study, we report for the first time the transient expression and the self-assembly of a chimeric HPV-16 L1 bearing the M2e influenza epitope in plants, representing also the first record of a successful expression of chimeric HPV-16 L1 carrying an epitope of a heterologous virus in plants. This study further confirms the usefulness of human papillomavirus particles as carriers of exogenous epitopes and their potential relevance for the production in plants of monovalent or multivalent vaccines. PMID:22085463

  6. Leaf growth is conformal.

    PubMed

    Alim, Karen; Armon, Shahaf; Shraiman, Boris I; Boudaoud, Arezki

    2016-01-01

    Growth pattern dynamics lie at the heart of morphogenesis. Here, we investigate the growth of plant leaves. We compute the conformal transformation that maps the contour of a leaf at a given stage onto the contour of the same leaf at a later stage. Based on the mapping we predict the local displacement field in the leaf blade and find it to agree with the experimentally measured displacement field to 92%. This approach is applicable to any two-dimensional system with locally isotropic growth, enabling the deduction of the whole growth field just from observation of the tissue contour. PMID:27597439

  7. Metamaterials with conformational nonlinearity

    NASA Astrophysics Data System (ADS)

    Lapine, Mikhail; Shadrivov, Ilya V.; Powell, David A.; Kivshar, Yuri S.

    2011-11-01

    Within a decade of fruitful development, metamaterials became a prominent area of research, bridging theoretical and applied electrodynamics, electrical engineering and material science. Being man-made structures, metamaterials offer a particularly useful playground to develop interdisciplinary concepts. Here we demonstrate a novel principle in metamaterial assembly which integrates electromagnetic, mechanical, and thermal responses within their elements. Through these mechanisms, the conformation of the meta-molecules changes, providing a dual mechanism for nonlinearity and offering nonlinear chirality. Our proposal opens a wide road towards further developments of nonlinear metamaterials and photonic structures, adding extra flexibility to their design and control.

  8. The developmental regulation of the L2/HNK-1 and L3 carbohydrate epitopes in mouse brain. Evidence for separate control of lipid- and protein-bound epitopes.

    PubMed

    Breen, K C

    1989-04-10

    The carbohydrate epitopes L2/HNK-1 and L3 have previously been identified on various neural cell adhesion molecules and have been suggested to play a role in the mediation of cell-cell adhesion. In this study, the developmental expression of the two epitopes in soluble, membrane-bound and chloroform/methanol-extracted fractions of the constituent mouse brain regions was examined by enzyme-linked immunosorbent assay (ELISA). The protein-bound epitopes were shown to be uniformly developmentally regulated, with levels peaking at postnatal day 20 (P20). The epitopes in a crude chloroform/methanol fraction, however, demonstrated a different pattern, with L2 peaking earlier at postnatal day zero (P0). These results suggest a possible interaction between the control of the two pools of the epitope. PMID:2468531

  9. A General Method to Discover Epitopes from Sera

    PubMed Central

    Whittemore, Kurt; Johnston, Stephen Albert; Sykes, Kathryn; Shen, Luhui

    2016-01-01

    Antigen-antibody complexes are central players in an effective immune response. However, finding those interactions relevant to a particular disease state can be arduous. Nonetheless many paths to discovery have been explored since deciphering these interactions can greatly facilitate the development of new diagnostics, therapeutics, and vaccines. In silico B cell epitope mapping approaches have been widely pursued, though success has not been consistent. Antibody mixtures in immune sera have been used as handles for biologically relevant antigens, but these and other experimental approaches have proven resource intensive and time consuming. In addition, these methods are often tailored to individual diseases or a specific proteome, rather than providing a universal platform. Most of these methods are not able to identify the specific antibody’s epitopes from unknown antigens, such as un-annotated neo antigens in cancer. Alternatively, a peptide library comprised of sequences unrestricted by naturally-found protein space provides for a universal search for mimotopes of an antibody’s epitope. Here we present the utility of such a non-natural random sequence library of 10,000 peptides physically addressed on a microarray for mimotope discovery without sequence information of the specific antigen. The peptide arrays were probed with serum from an antigen-immunized rabbit, or alternatively probed with serum pre-absorbed with the same immunizing antigen. With this positive and negative screening scheme, we identified the library-peptides as the mimotopes of the antigen. The unique library peptides were successfully used to isolate antigen-specific antibodies from complete immune serum. Sequence analysis of these peptides revealed the epitopes in the immunized antigen. We present this method as an inexpensive, efficient method for identifying mimotopes of any antibody’s targets. These mimotopes should be useful in defining both components of the antigen

  10. Genotyping for cytochrome P450 polymorphisms.

    PubMed

    Daly, Ann K; King, Barry P; Leathart, Julian B S

    2006-01-01

    Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) analysis are described. Rapid high-throughput techniques are also available for analyses of this type, but they require access to specialized equipment and are not considered here. General guidelines for performing amplification using PCR are described together with electrophoresis protocols for analysis of restriction digests of PCR products with agarose and polyacrylamide gels including the use of polyacrylamide-based gels for SSCP analysis. Protocols for the following specific isoforms and alleles are also provided: CYP1A1 (*2B and *4 alleles), CYP2C8 (*3 and *4 alleles), CYP2C9 (*2, *3, and *11 alleles), CYP2C19 (*2 and *3 alleles), CYP2D6 (*3, *4, *5, and *6 alleles), CYP2E1 (*5A, *5B, and *6 alleles), and CYP3A5 (*3 allele). PMID:16719392

  11. The amyloid fold of Gad m 1 epitopes governs IgE binding.

    PubMed

    Sánchez, Rosa; Martínez, Javier; Castro, Ana; Pedrosa, María; Quirce, Santiago; Rodríguez-Pérez, Rosa; Gasset, María

    2016-01-01

    Amyloids are polymeric structural states formed from locally or totally unfolded protein chains that permit surface reorganizations, stability enhancements and interaction properties that are absent in the precursor monomers. β-Parvalbumin, the major allergen in fish allergy, forms amyloids that are recognized by IgE in the patient sera, suggesting a yet unknown pathological role for these assemblies. We used Gad m 1 as the fish β-parvalbumin model and a combination of approaches, including peptide arrays, recombinant wt and mutant chains, biophysical characterizations, protease digestions, mass spectrometry, dot-blot and ELISA assays to gain insights into the role of amyloids in the IgE interaction. We found that Gad m 1 immunoreactive regions behave as sequence-dependent conformational epitopes that provide a 1000-fold increase in affinity and the structural repetitiveness required for optimal IgE binding and cross-linking upon folding into amyloids. These findings support the amyloid state as a key entity in type I food allergy. PMID:27597317

  12. The amyloid fold of Gad m 1 epitopes governs IgE binding

    PubMed Central

    Sánchez, Rosa; Martínez, Javier; Castro, Ana; Pedrosa, María; Quirce, Santiago; Rodríguez-Pérez, Rosa; Gasset, María

    2016-01-01

    Amyloids are polymeric structural states formed from locally or totally unfolded protein chains that permit surface reorganizations, stability enhancements and interaction properties that are absent in the precursor monomers. β-Parvalbumin, the major allergen in fish allergy, forms amyloids that are recognized by IgE in the patient sera, suggesting a yet unknown pathological role for these assemblies. We used Gad m 1 as the fish β-parvalbumin model and a combination of approaches, including peptide arrays, recombinant wt and mutant chains, biophysical characterizations, protease digestions, mass spectrometry, dot-blot and ELISA assays to gain insights into the role of amyloids in the IgE interaction. We found that Gad m 1 immunoreactive regions behave as sequence-dependent conformational epitopes that provide a 1000-fold increase in affinity and the structural repetitiveness required for optimal IgE binding and cross-linking upon folding into amyloids. These findings support the amyloid state as a key entity in type I food allergy. PMID:27597317

  13. Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody

    PubMed Central

    Malito, Enrico; Biancucci, Marco; Faleri, Agnese; Ferlenghi, Ilaria; Scarselli, Maria; Maruggi, Giulietta; Lo Surdo, Paola; Veggi, Daniele; Liguori, Alessia; Santini, Laura; Bertoldi, Isabella; Petracca, Roberto; Marchi, Sara; Romagnoli, Giacomo; Cartocci, Elena; Vercellino, Irene; Savino, Silvana; Spraggon, Glen; Norais, Nathalie; Pizza, Mariagrazia; Rappuoli, Rino; Masignani, Vega; Bottomley, Matthew James

    2014-01-01

    Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5–15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA. PMID:25404323

  14. Production of Influenza Virus HA1 Harboring Native-Like Epitopes by Pichia pastoris.

    PubMed

    Lin, Qingshan; Yang, Kunyu; He, Fangping; Jiang, Jie; Li, Tingting; Chen, Zhenqin; Li, Rui; Chen, Yixin; Li, Shaowei; Zhao, Qinjian; Xia, Ningshao

    2016-08-01

    The outbreak of the H5N1 highly pathogenic avian influenza which exhibits high variation had brought a serious threat to the safety of humanity. To overcome this high variation, hemagglutinin-based recombinant subunit vaccine with rational design has been considered as a substitute for traditional virion-based vaccine development. Here, we expressed HA1 part of the hemagglutinin protein using the Pichia pastoris expression system and attained a high yield of about 120 mg/L through the use of fed-batch scalable fermentation. HA1 protein in the culture supernatant was purified using two-step ion-exchange chromatography. The resultant HA1 protein was homogeneous in solution in a glycosylated form, as confirmed by endoglycosidase H treatment. Sedimentation velocity tests, silver staining of protein gels, and immunoblotting were used for verification. The native HA1 reacted well with conformational, cross-genotype, neutralizing monoclonal antibodies, whereas a loss of binding activity was noted with the denatured HA1 form. Moreover, the murine anti-HA1 serum exhibited a virus-capture capability in the hemagglutination inhibition assay, which suggests that HA1 harbors native-like epitopes. In conclusion, soluble HA1 was efficiently expressed and purified in this study. The functional glycosylated protein will be an alternative for the development of recombinant protein-based influenza vaccine. PMID:27040529

  15. Common food allergens and their IgE-binding epitopes.

    PubMed

    Matsuo, Hiroaki; Yokooji, Tomoharu; Taogoshi, Takanori

    2015-10-01

    Food allergy is an adverse immune response to certain kinds of food. Although any food can cause allergic reactions, chicken egg, cow's milk, wheat, shellfish, fruit, and buckwheat account for 75% of food allergies in Japan. Allergen-specific immunoglobulin E (IgE) antibodies play a pivotal role in the development of food allergy. Recent advances in molecular biological techniques have enabled the efficient analysis of food allergens. As a result, many food allergens have been identified, and their molecular structure and IgE-binding epitopes have also been identified. Studies of allergens have demonstrated that IgE antibodies specific to allergen components and/or the peptide epitopes are good indicators for the identification of patients with food allergy, prediction of clinical severity and development of tolerance. In this review, we summarize our current knowledge regarding the allergens and IgE epitopes in the well-researched allergies to chicken egg, cow's milk, wheat, shrimp, and peanut. PMID:26433529

  16. Versatile Virus-Like Particle Carrier for Epitope Based Vaccines

    PubMed Central

    Tissot, Alain C.; Renhofa, Regina; Schmitz, Nicole; Cielens, Indulis; Meijerink, Edwin; Ose, Velta; Jennings, Gary T.; Saudan, Philippe; Pumpens, Paul; Bachmann, Martin F.

    2010-01-01

    Background Recombinant proteins and in particular single domains or peptides are often poorly immunogenic unless conjugated to a carrier protein. Virus-like-particles are a very efficient means to confer high immunogenicity to antigens. We report here the development of virus-like-particles (VLPs) derived from the RNA bacteriophage AP205 for epitope-based vaccines. Methodology/Principal Findings Peptides of angiotensin II, S.typhi outer membrane protein (D2), CXCR4 receptor, HIV1 Nef, gonadotropin releasing hormone (GnRH), Influenza A M2-protein were fused to either N- or C-terminus of AP205 coat protein. The A205-peptide fusions assembled into VLPs, and peptides displayed on the VLP were highly immunogenic in mice. GnRH fused to the C-terminus of AP205 induced a strong antibody response that inhibited GnRH function in vivo. Exposure of the M2-protein peptide at the N-terminus of AP205 resulted in a strong M2-specific antibody response upon immunization, protecting 100% of mice from a lethal influenza infection. Conclusions/Significance AP205 VLPs are therefore a very efficient and new vaccine system, suitable for complex and long epitopes, of up to at least 55 amino acid residues in length. AP205 VLPs confer a high immunogenicity to displayed epitopes, as shown by inhibition of endogenous GnRH and protective immunity against influenza infection. PMID:20352110

  17. An immunogen containing four tandem 10E8 epitope repeats with exposed key residues induces antibodies that neutralize HIV-1 and activates an ADCC reporter gene.

    PubMed

    Sun, Zhiwu; Zhu, Yun; Wang, Qian; Ye, Ling; Dai, Yanyan; Su, Shan; Yu, Fei; Ying, Tianlei; Yang, Chinglai; Jiang, Shibo; Lu, Lu

    2016-01-01

    After three decades of intensive research efforts, an effective vaccine against HIV-1 remains to be developed. Several broadly neutralizing antibodies to HIV-1, such as 10E8, recognize the membrane proximal external region (MPER) of the HIV-1 gp41 protein. Thus, the MPER is considered to be a very important target for vaccine design. However, the MPER segment has very weak immunogenicity and tends to insert its epitope residues into the cell membrane, thereby avoiding antibody binding. To address this complication in vaccine development, we herein designed a peptide, designated 10E8-4P, containing four copies of the 10E8 epitope as an immunogen. As predicted by structural simulation, 10E8-4P exhibits a well-arranged tandem helical conformation, with the key residues in the 10E8 epitope oriented at different angles, thus suggesting that some of these key residues may be exposed outside of the lipid membrane. Compared with a peptide containing a single 10E8 epitope (10E8-1P), 10E8-4P not only exhibited better antigenicity but also elicited neutralizing antibody response against HIV-1 pseudoviruses, whereas 10E8-1P could not induce detectable neutralizing antibody response. Importantly, antibodies elicited by 10E8-4P also possessed a strong ability to activate an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter gene, thus suggesting that they may have ADCC activity. Therefore, this strategy shows promise for further optimization and application in future HIV-1 vaccine design. PMID:27329850

  18. An immunogen containing four tandem 10E8 epitope repeats with exposed key residues induces antibodies that neutralize HIV-1 and activates an ADCC reporter gene

    PubMed Central

    Sun, Zhiwu; Zhu, Yun; Wang, Qian; Ye, Ling; Dai, Yanyan; Su, Shan; Yu, Fei; Ying, Tianlei; Yang, Chinglai; Jiang, Shibo; Lu, Lu

    2016-01-01

    After three decades of intensive research efforts, an effective vaccine against HIV-1 remains to be developed. Several broadly neutralizing antibodies to HIV-1, such as 10E8, recognize the membrane proximal external region (MPER) of the HIV-1 gp41 protein. Thus, the MPER is considered to be a very important target for vaccine design. However, the MPER segment has very weak immunogenicity and tends to insert its epitope residues into the cell membrane, thereby avoiding antibody binding. To address this complication in vaccine development, we herein designed a peptide, designated 10E8-4P, containing four copies of the 10E8 epitope as an immunogen. As predicted by structural simulation, 10E8-4P exhibits a well-arranged tandem helical conformation, with the key residues in the 10E8 epitope oriented at different angles, thus suggesting that some of these key residues may be exposed outside of the lipid membrane. Compared with a peptide containing a single 10E8 epitope (10E8-1P), 10E8-4P not only exhibited better antigenicity but also elicited neutralizing antibody response against HIV-1 pseudoviruses, whereas 10E8-1P could not induce detectable neutralizing antibody response. Importantly, antibodies elicited by 10E8-4P also possessed a strong ability to activate an antibody-dependent cell-mediated cytotoxicity (ADCC) reporter gene, thus suggesting that they may have ADCC activity. Therefore, this strategy shows promise for further optimization and application in future HIV-1 vaccine design. PMID:27329850

  19. GPI-anchored single chain Fv - an effective way to capture transiently-exposed neutralization epitopes on HIV-1 envelope spike

    PubMed Central

    2010-01-01

    Background Identification of broad neutralization epitopes in HIV-1 envelope spikes is paramount for HIV-1 vaccine development. A few broad neutralization epitopes identified so far are present on the surface of native HIV-1 envelope spikes whose recognition by antibodies does not depend on conformational changes of the envelope spikes. However, HIV-1 envelope spikes also contain transiently-exposed neutralization epitopes, which are more difficult to identify. Results In this study, we constructed single chain Fvs (scFvs) derived from seven human monoclonal antibodies and genetically linked them with or without a glycosyl-phosphatidylinositol (GPI) attachment signal. We show that with a GPI attachment signal the scFvs are targeted to lipid rafts of plasma membranes. In addition, we demonstrate that four of the GPI-anchored scFvs, but not their secreted counterparts, neutralize HIV-1 with various degrees of breadth and potency. Among them, GPI-anchored scFv (X5) exhibits extremely potent and broad neutralization activity against multiple clades of HIV-1 strains tested. Moreover, we show that GPI-anchored scFv (4E10) also exhibited more potent neutralization activity than its secretory counterpart. Finally, we demonstrate that expression of GPI-anchored scFv (X5) in the lipid raft of plasma membrane of human CD4+ T cells confers long-term resistance to HIV-1 infection, HIV-1 envelope-mediated cell-cell fusion, and the infection of HIV-1 captured and transferred by human DCs. Conclusions Thus GPI-anchored scFv could be used as a general and effective way to identify antibodies that react with transiently-exposed neutralization epitopes in envelope proteins of HIV-1 and other enveloped viruses. The GPI-anchored scFv (X5), because of its breadth and potency, should have a great potential to be developed into anti-viral agent for HIV-1 prevention and therapy. PMID:20923574

  20. Analysis of epitope-specific immune responses induced by vaccination with structurally folded and unfolded recombinant Bet v 1 allergen derivatives in man.

    PubMed

    Pree, Ines; Reisinger, Jürgen; Focke, Margit; Vrtala, Susanne; Pauli, Gabrielle; van Hage, Marianne; Cromwell, Oliver; Gadermaier, Elisabeth; Egger, Cornelia; Reider, Norbert; Horak, Friedrich; Valenta, Rudolf; Niederberger, Verena

    2007-10-15

    Previously, we have constructed recombinant derivatives of the major birch pollen allergen, Bet v 1, with a more than 100-fold reduced ability to induce IgE-mediated allergic reactions. These derivatives differed from each other because the two recombinant Bet v 1 fragments represented unfolded molecules whereas the recombinant trimer resembled most of the structural fold of the Bet v 1 allergen. In this study, we analyzed the Ab (IgE, IgG subclass, IgA, IgM) response to Bet v 1, recombinant and synthetic Bet v 1-derived peptides in birch pollen allergic patients who had been vaccinated with the derivatives or adjuvant alone. Furthermore, we studied the induction of IgE-mediated skin responses in these patients using Bet v 1 and Bet v 1 fragments. Both types of vaccines induced a comparable IgG1 and IgG4 response against new sequential epitopes which overlap with the conformational IgE epitopes of Bet v 1. This response was 4- to 5-fold higher than that induced by immunotherapy with birch pollen extract. Trimer more than fragments induced also IgE responses against new epitopes and a transient increase in skin sensitivity to the fragments at the beginning of therapy. However, skin reactions to Bet v 1 tended to decrease one year after treatment in both actively treated groups. We demonstrate that vaccination with folded and unfolded recombinant allergen derivatives induces IgG Abs against new epitopes. These data may be important for the development of therapeutic as well as prophylactic vaccines based on recombinant allergens. PMID:17911617

  1. Conformally symmetric traversable wormholes

    SciTech Connect

    Boehmer, Christian G.; Harko, Tiberiu; Lobo, Francisco S. N.

    2007-10-15

    Exact solutions of traversable wormholes are found under the assumption of spherical symmetry and the existence of a nonstatic conformal symmetry, which presents a more systematic approach in searching for exact wormhole solutions. In this work, a wide variety of solutions are deduced by considering choices for the form function, a specific linear equation of state relating the energy density and the pressure anisotropy, and various phantom wormhole geometries are explored. A large class of solutions impose that the spatial distribution of the exotic matter is restricted to the throat neighborhood, with a cutoff of the stress-energy tensor at a finite junction interface, although asymptotically flat exact solutions are also found. Using the 'volume integral quantifier', it is found that the conformally symmetric phantom wormhole geometries may, in principle, be constructed by infinitesimally small amounts of averaged null energy condition violating matter. Considering the tidal acceleration traversability conditions for the phantom wormhole geometry, specific wormhole dimensions and the traversal velocity are also deduced.

  2. Eikonalization of conformal blocks

    SciTech Connect

    Fitzpatrick, A. Liam; Kaplan, Jared; Walters, Matthew T.; Wang, Junpu

    2015-09-03

    Classical field configurations such as the Coulomb potential and Schwarzschild solution are built from the t-channel exchange of many light degrees of freedom. We study the CFT analog of this phenomenon, which we term the 'eikonalization' of conformal blocks. We show that when an operator T appears in the OPE Ο(x)Ο(0), then the large spin Fock space states [TT···T] also appear in this OPE with a computable coefficient. The sum over the exchange of these Fock space states in an correlator build the classical 'T field' in the dual AdS description. In some limits the sum of all Fock space exchanges can be represented as the exponential of a single T exchange in the 4-pt correlator of O. Our results should be useful for systematizing 1/ℓ perturbation theory in general CFTs and simplifying the computation of large spin OPE coefficients. As examples we obtain the leading log ℓ dependence of Fock space conformal block coefficients, and we directly compute the OPE coefficients of the simplest ‘triple-trace’ operators.

  3. Eikonalization of conformal blocks

    DOE PAGESBeta

    Fitzpatrick, A. Liam; Kaplan, Jared; Walters, Matthew T.; Wang, Junpu

    2015-09-03

    Classical field configurations such as the Coulomb potential and Schwarzschild solution are built from the t-channel exchange of many light degrees of freedom. We study the CFT analog of this phenomenon, which we term the 'eikonalization' of conformal blocks. We show that when an operator T appears in the OPE Ο(x)Ο(0), then the large spin Fock space states [TT···T]ℓ also appear in this OPE with a computable coefficient. The sum over the exchange of these Fock space states in an correlator build the classical 'T field' in the dual AdS description. In some limits the sum of all Fock spacemore » exchanges can be represented as the exponential of a single T exchange in the 4-pt correlator of O. Our results should be useful for systematizing 1/ℓ perturbation theory in general CFTs and simplifying the computation of large spin OPE coefficients. As examples we obtain the leading log ℓ dependence of Fock space conformal block coefficients, and we directly compute the OPE coefficients of the simplest ‘triple-trace’ operators.« less

  4. Immunization with synthetic peptides containing epitopes of the class 1 outer-membrane protein of Neisseria meningitidis: production of bactericidal antibodies on immunization with a cyclic peptide.

    PubMed

    Christodoulides, M; McGuinness, B T; Heckels, J E

    1993-08-01

    The class 1 outer-membrane protein of Neisseria meningitidis is the target for subtype-specific, bactericidal monoclonal antibodies (mAbs). The epitopes recognized by these antibodies have been mapped previously to linear peptides corresponding to the sequences thought to be exposed at the apices of surface-exposed loops of the protein. In this work several synthetic peptides containing the subtype Pl.16b epitope have been synthetized with the aim of inducing a polyclonal immune response resembling the reactivity of the mAbs. Initially, peptides of 9 and 15 amino acid residues were synthesized and used for immunization after coupling to a carrier protein. The reactivity of the resulting antisera, with synthetic linear decapeptides, resembled that seen in previous epitope mapping experiments with the protective mAbs. However, despite the induction of antibodies having the desired specificity, the antisera reacted poorly with the native protein in outer membranes, and were non-bactericidal. A 36mer peptide, consisting of the entire surface-exposed loop 4 of the class 1 protein was then synthesized and used for immunization as (i) free peptide, (ii) peptide coupled to carrier and (iii) peptide subjected to cyclization, in an attempt to restrict it to conformations that might more closely resemble the native loop structure. In contrast to antisera raised against linear peptides, antibodies raised by immunization with the 36mer cyclic peptide, did not react with linear peptides recognized by the mAbs, but instead appeared to recognize conformational determinants. This antiserum promoted complement-mediated bactericidal killing of the homologous meningococcal strain, demonstrating the potential of synthetic peptide immunogens for inducing a protective immune response against group B meningococci. PMID:7691983

  5. The value of HIV protective epitope research for informed vaccine design against diverse viral pathogens

    PubMed Central

    Kramer, Victor G; Byrareddy, Siddappa N

    2014-01-01

    The success of vaccine regimens against viral pathogens hinges on the elicitation of protective responses. Hypervariable pathogens such as HIV avoid neutralization by masking protective epitopes with more immunogenic decoys. The identification of protective, conserved epitopes is crucial for future vaccine candidate design. The strategies employed for identification of HIV protective epitopes will also aid towards rational vaccine design for other viral pathogens. PMID:24964950

  6. Recent Advances in Conformal Gravity

    NASA Astrophysics Data System (ADS)

    O'Brien, James; Chaykov, Spasen

    2016-03-01

    In recent years, significant advances have been made in alternative gravitational theories. Although MOND remains the leading candidate among the alternative models, Conformal Gravity has been studied by Mannheim and O'Brien to solve the rotation curve problem without the need for dark matter. Recently, Mannheim, O'Brien and Chaykov have begun solving other gravitational questions in Conformal Gravity. In this presentation, we highlight the new work of Conformal Gravity's application to random motions of clusters (the original Zwicky problem), gravitational bending of light, gravitational lensing and a very recent survey of dwarf galaxy rotation curves. We will show in each case that Conformal Gravity can provide an accurate explanation and prediction of the data without the need for dark matter. Coupled with the fact that Conformal Gravity is a fully re-normalizable metric theory of gravity, these results help to push Conformal Gravity onto a competitive stage against other alternative models.

  7. Presentation of native epitopes in the V1/V2 and V3 regions of human immunodeficiency virus type 1 gp120 by fusion glycoproteins containing isolated gp120 domains.

    PubMed Central

    Kayman, S C; Wu, Z; Revesz, K; Chen, H; Kopelman, R; Pinter, A

    1994-01-01

    The immune response to viral glycoproteins is often directed against conformation- and/or glycosylation-dependent structures; synthetic peptides and bacterially expressed proteins are inadequate probes for the mapping of such epitopes. This report describes a retroviral vector system that presents such native epitopes on chimeric glycoproteins in which protein fragments of interest are fused to the C terminus of the N-terminal domain of the murine leukemia virus surface protein, gp70. The system was used to express two disulfide-bonded domains from gp120, the surface protein of human immunodeficiency virus type 1 (HIV-1), that include potent neutralization epitopes. The resulting fusion glycoproteins were synthesized at high levels and were efficiently transported and secreted. A fusion protein containing the HXB2 V1/V2 domain was recognized by an HIVIIIB-infected patient serum as well as by 17 of 36 HIV-1 seropositive hemophiliac, homosexual male and intravenous drug user patient sera. Many of these HIV+ human sera reacted with V1/V2 domains from several HIV-1 clones expressed in fusion glycoproteins, indicating the presence of cross-reactive antibodies against epitopes in the V1/V2 domain. Recognition of gp(1-263):V1/V2HXB2 by the HIVIIIB-infected human patient serum was largely blocked by synthetic peptides matching V1 but not V2 sequences, while recognition of this construct by a broadly cross-reactive hemophiliac patient serum was not blocked by individual V1 or V2 peptides or by mixtures of these peptides. A construct containing the V3 domain of the IIIB strain of HIV-1, gp(1-263):V3HXB2, was recognized by sera from a human and a chimpanzee that had been infected by HIVIIIB but not by sera from hemophiliac patients who had been infected with HIV-1 of MN-like V3 serotype. The reactive sera had significantly higher titers when assayed against gp(1-263):V3HXB2 than when assayed against matching V3 peptides. Immunoprecipitation of this fusion glycoprotein by the

  8. Conformal Gravity and Gravitational Waves

    NASA Astrophysics Data System (ADS)

    Fabbri, Luca; Paranjape, M. B.

    We consider monochromatic, plane gravitational waves in a conformally invariant theory of general relativity. We show that the simple, standard ansatz for the metric, usually that which is taken for the linearized theory of these waves, is reducible to the metric of Minkowski spacetime via a sequence of conformal and coordinate transformations. This implies that we have in fact, exact plane wave solutions. However they are simply coordinate/conformal artifacts. As a consequence, they carry no energy.

  9. Killing and conformal Killing tensors

    NASA Astrophysics Data System (ADS)

    Heil, Konstantin; Moroianu, Andrei; Semmelmann, Uwe

    2016-08-01

    We introduce an appropriate formalism in order to study conformal Killing (symmetric) tensors on Riemannian manifolds. We reprove in a simple way some known results in the field and obtain several new results, like the classification of conformal Killing 2-tensors on Riemannian products of compact manifolds, Weitzenböck formulas leading to non-existence results, and construct various examples of manifolds with conformal Killing tensors.

  10. A tumour necrosis factor alpha polymorphism is not associated with rheumatoid arthritis.

    PubMed Central

    Wilson, A G; de Vries, N; van de Putte, L B; Duff, G W

    1995-01-01

    OBJECTIVE--To determine whether a polymorphism within the tumour necrosis factor alpha (TNF alpha) gene is associated with susceptibility to, or severity of, rheumatoid arthritis (RA). METHODS--Consecutive patients with recent onset RA were enrolled in a prospective trial. DNA was collected, disease activity was measured at presentation, and radiographic progression at three years was assessed. Typing of TNF alpha was by polymerase chain reaction and single stranded conformation polymorphism analysis. RESULTS--No association of TNF alpha alleles and susceptibility to, or severity of, RA was demonstrated. CONCLUSIONS--These results indicate that this TNF alpha polymorphism does not play a part in the genetic background of RA. PMID:7668906

  11. Identification of dominant epitopes of synthetic immunocontraceptive vaccines that induce antibodies in dogs.

    PubMed

    Yu, Meng; Zeng, Weiguang; Pagnon, Joanne; Walker, John; Ghosh, Souravi; Wang, Lin-Fa; Jackson, David C

    2005-08-31

    The specificities of immunoglobulin G antibodies obtained from the sera of dogs inoculated with totally synthetic immunocontraceptive vaccine candidates based on luteinising hormone releasing hormone (LHRH: amino acid sequence HWSYGLRPG) were examined using peptides expressed in a phage display library. The three vaccine candidates each contained a different T helper-cell epitope chemically linked with the same LHRH amino acid sequence HWSYGLRPG and all of them elicited high antibody titres against the hormone. Delineation of epitopes recognised by sera from vaccinated dogs using a phage display library indicated that two of the three vaccine candidates induced antibody directed to the consensus sequence xHWSxxLxxx whereas the third vaccine candidate induced antibody against the consensus sequence xxxxxxxRPx. Two of the three vaccine candidates elicited antibodies against B cell epitopes present within the helper T-cell epitope component of the vaccine whereas the third vaccine did not. The occurrence of anti-T helper cell epitope antibodies appeared to have little or no effect on the generation of the anti-LHRH responses indicating that carrier-induced epitope suppression was not operating here. Our results also demonstrated that with animal sera of high quality, it is possible to delineate immunodominant epitopes recognised by polyclonal antibodies with high efficiency using phage display library. The approach has utility in the definition of immunodominant epitopes, which may "decoy" antibody responses away from other epitopes, which may be more useful in prophylaxis or therapy. PMID:15927323

  12. A versatile PCR-based tandem epitope tagging system for Streptomyces coelicolor genome.

    PubMed

    Kim, Ji-Nu; Yi, Jeong Sang; Lee, Bo-Rahm; Kim, Eun-Jung; Kim, Min Woo; Song, Yoseb; Cho, Byung-Kwan; Kim, Byung-Gee

    2012-07-20

    Epitope tagging approaches have been widely used for the analysis of functions, interactions and subcellular distributions of proteins. However, incorporating epitope sequence into protein loci in Streptomyces is time-consuming procedure due to the absence of the versatile tagging methods. Here, we developed a versatile PCR-based tandem epitope tagging tool for the Streptomyces genome engineering. We constructed a series of template plasmids that carry repeated sequence of c-myc epitope, Flp recombinase target (FRT) sites, and apramycin resistance marker to insert epitope tags into any desired spot of the chromosomal loci. A DNA module which includes the tandem epitope-encoding sequence and a selectable marker was amplified by PCR with primers that carry homologous extensions to the last portion and downstream region of the targeted gene. We fused the epitope tags at the 3' region of global transcription factors of Streptomyces coelicolor to test the validity of this system. The proper insertion of the epitope tag was confirmed by PCR and western blot analysis. The recombinants showed the identical phenotype to the wild-type that proved the conservation of in vivo function of the tagged proteins. Finally, the direct binding targets were successfully detected by chromatin immunoprecipitation with the increase in the signal-to-noise ratio. The epitope tagging system describes here would provide wide applications to study the protein functions in S. coelicolor. PMID:22704935

  13. Comparative Analysis of Human B Cell Epitopes Based on BCG Genomes

    PubMed Central

    Liu, Haican; Zhao, Xiuqin; Wan, Kanglin

    2016-01-01

    Background. Tuberculosis is a huge global health problem. BCG is the only vaccine used for about 100 years against TB, but the reasons for protection variability in populations remain unclear. To improve BCG efficacy and develop a strategy for new vaccines, the underlying genetic differences among BCG subtypes should be understood urgently. Methods and Findings. Human B cell epitope data were collected from the Immune Epitope Database. Epitope sequences were mapped with those of 15 genomes, including 13 BCGs, M. bovis AF2122/97, and M. tuberculosis H37Rv, to identify epitopes distribution. Among 398 experimentally verified B cell epitopes, 321 (80.7%) were conserved, while the remaining 77 (19.3%) were lost to varying degrees in BCGs. The variable protective efficacy of BCGs may result from the degree of B cell epitopes deficiency. Conclusions. Here we firstly analyzed the genetic characteristics of BCGs based on B cell epitopes and found that B cell epitopes distribution may contribute to vaccine efficacy. Restoration of important antigens or effective B cell epitopes in BCG could be a useful strategy for vaccine development. PMID:27382565

  14. Comparative Analysis of Human B Cell Epitopes Based on BCG Genomes.

    PubMed

    Li, Machao; Liu, Haican; Zhao, Xiuqin; Wan, Kanglin

    2016-01-01

    Background. Tuberculosis is a huge global health problem. BCG is the only vaccine used for about 100 years against TB, but the reasons for protection variability in populations remain unclear. To improve BCG efficacy and develop a strategy for new vaccines, the underlying genetic differences among BCG subtypes should be understood urgently. Methods and Findings. Human B cell epitope data were collected from the Immune Epitope Database. Epitope sequences were mapped with those of 15 genomes, including 13 BCGs, M. bovis AF2122/97, and M. tuberculosis H37Rv, to identify epitopes distribution. Among 398 experimentally verified B cell epitopes, 321 (80.7%) were conserved, while the remaining 77 (19.3%) were lost to varying degrees in BCGs. The variable protective efficacy of BCGs may result from the degree of B cell epitopes deficiency. Conclusions. Here we firstly analyzed the genetic characteristics of BCGs based on B cell epitopes and found that B cell epitopes distribution may contribute to vaccine efficacy. Restoration of important antigens or effective B cell epitopes in BCG could be a useful strategy for vaccine development. PMID:27382565

  15. Loop Virasoro Lie conformal algebra

    SciTech Connect

    Wu, Henan Chen, Qiufan; Yue, Xiaoqing

    2014-01-15

    The Lie conformal algebra of loop Virasoro algebra, denoted by CW, is introduced in this paper. Explicitly, CW is a Lie conformal algebra with C[∂]-basis (L{sub i} | i∈Z) and λ-brackets [L{sub i} {sub λ} L{sub j}] = (−∂−2λ)L{sub i+j}. Then conformal derivations of CW are determined. Finally, rank one conformal modules and Z-graded free intermediate series modules over CW are classified.

  16. Reflections on conformal spectra

    NASA Astrophysics Data System (ADS)

    Kim, Hyungrok; Kravchuk, Petr; Ooguri, Hirosi

    2016-04-01

    We use modular invariance and crossing symmetry of conformal field theory to reveal approximate reflection symmetries in the spectral decompositions of the partition function in two dimensions in the limit of large central charge and of the four-point function in any dimension in the limit of large scaling dimensions Δ0 of external operators. We use these symmetries to motivate universal upper bounds on the spectrum and the operator product expansion coefficients, which we then derive by independent techniques. Some of the bounds for four-point functions are valid for finite Δ0 as well as for large Δ0. We discuss a similar symmetry in a large spacetime dimension limit. Finally, we comment on the analogue of the Cardy formula and sparse light spectrum condition for the four-point function.

  17. Capturing Chromosome Conformation

    NASA Astrophysics Data System (ADS)

    Dekker, Job; Rippe, Karsten; Dekker, Martijn; Kleckner, Nancy

    2002-02-01

    We describe an approach to detect the frequency of interaction between any two genomic loci. Generation of a matrix of interaction frequencies between sites on the same or different chromosomes reveals their relative spatial disposition and provides information about the physical properties of the chromatin fiber. This methodology can be applied to the spatial organization of entire genomes in organisms from bacteria to human. Using the yeast Saccharomyces cerevisiae, we could confirm known qualitative features of chromosome organization within the nucleus and dynamic changes in that organization during meiosis. We also analyzed yeast chromosome III at the G1 stage of the cell cycle. We found that chromatin is highly flexible throughout. Furthermore, functionally distinct AT- and GC-rich domains were found to exhibit different conformations, and a population-average 3D model of chromosome III could be determined. Chromosome III emerges as a contorted ring.

  18. Chenopodium album pollen profilin (Che a 2): homology modeling and evaluation of cross-reactivity with allergenic profilins based on predicted potential IgE epitopes and IgE reactivity analysis.

    PubMed

    Amini, Akram; sankian, Mojtaba; Assarehzadegan, Mohammad Ali; Vahedi, Fatemeh; Varasteh, AbdolReza

    2011-04-01

    The inhalation of Chenopodium album (C. album) pollen has been reported as an important cause of allergic respiratory symptoms. The aim of this study was to produce the recombinant profilin of C. album (rChe a 2) pollen and to investigate its cross-reactivity with other plant-derived profilins based on potential conformational epitopes and IgE reactivity analysis. Che a 2-coding sequence was cloned, expressed, and purified using one step metal affinity chromatography to recover high-purity target protein. We assessed cross-reactivity and predicted IgE potential epitopes among rChe a 2 and other plant-derived profilins. Immunodetection and inhibition assays using sixteen individual sera from C. album allergic patients demonstrated that purified rChe a 2 could be the same as that in the crude extract. The results of inhibition assays among rChe a 2 and other plant-derived profilins were in accordance with those of the homology of predicted conserved conformational regions. In this study, amino acid sequence homology analysis showed that a high degree of IgE cross-reactivity among plant-derived profilins may depend on predicted potential IgE epitopes. PMID:21086179

  19. HIV-1 epitope-specific CD8+ T cell responses strongly associated with delayed disease progression cross-recognize epitope variants efficiently.

    PubMed

    Turnbull, Emma L; Lopes, A Ross; Jones, Nicola A; Cornforth, David; Newton, Phillipa; Aldam, Diana; Pellegrino, Pierre; Turner, Jo; Williams, Ian; Wilson, Craig M; Goepfert, Paul A; Maini, Mala K; Borrow, Persephone

    2006-05-15

    The ability of HIV-1-specific CD8(+) T cell responses to recognize epitope variants resulting from viral sequence variation in vivo may affect the ease with which HIV-1 can escape T cell control and impact on the rate of disease progression in HIV-1-infected humans. Here, we studied the functional cross-reactivity of CD8 responses to HIV-1 epitopes restricted by HLA class I alleles associated with differential prognosis of infection. We show that the epitope-specific responses exhibiting the most efficient cross-recognition of amino acid-substituted variants were those strongly associated with delayed progression to disease. Not all epitopes restricted by the same HLA class I allele showed similar variant cross-recognition efficiency, consistent with the hypothesis that the reported associations between particular HLA class I alleles and rate of disease progression may be due to the quality of responses to certain "critical" epitopes. Irrespective of their efficiency of functional cross-recognition, CD8(+) T cells of all HIV-1 epitope specificities examined showed focused TCR usage. Furthermore, interpatient variability in variant cross-reactivity correlated well with use of different dominant TCR Vbeta families, suggesting that flexibility is not conferred by the overall clonal breadth of the response but instead by properties of the dominant TCR(s) used for epitope recognition. A better understanding of the features of T cell responses associated with long-term control of viral replication should facilitate rational vaccine design. PMID:16670322

  20. Mapping B-cell epitopes for the peroxidoxin of Leishmania (Viannia) braziliensis and its potential for the clinical diagnosis of tegumentary and visceral leishmaniasis.

    PubMed

    Menezes-Souza, Daniel; Mendes, Tiago Antônio de Oliveira; Nagem, Ronaldo Alves Pinto; Santos, Thaís Teodoro de Oliveira; Silva, Ana Luíza Teixeira; Santoro, Marcelo Matos; de Carvalho, Silvio Fernando Guimarães; Coelho, Eduardo Antônio Ferraz; Bartholomeu, Daniella Castanheira; Fujiwara, Ricardo Toshio

    2014-01-01

    The search toward the establishment of novel serological tests for the diagnosis of leishmaniasis and proper differential diagnosis may represent one alternative to the invasive parasitological methods currently used to identify infected individuals. In the present work, we investigated the potential use of recombinant peroxidoxin (rPeroxidoxin) of Leishmania (Viannia) braziliensis as a potential antigen for the immunodiagnosis of human tegumentary (TL) and visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). Linear B-cell epitope mapping was performed to identify polymorphic epitopes when comparing orthologous sequences present in Trypanosoma cruzi, the agent for Chagas disease (CD), and the Homo sapiens and Canis familiaris hosts. The serological assay (ELISA) demonstrated that TL, VL and CVL individuals showed high levels of antibodies against rPeroxidoxin, allowing identification of infected ones with considerable sensitivity and great ability to discriminate (specificity) between non-infected and CD individuals (98.46% and 100%; 98.18% and 95.71%; 95.79% and 100%, respectively). An rPeroxidoxin ELISA also showed a greater ability to discriminate between vaccinated and infected animals, which is an important requirement for the public campaign control of CVL. A depletion ELISA assay using soluble peptides of this B-cell epitope confirmed the recognition of these sites only by Leishmania-infected individuals. Moreover, this work identifies two antigenic polymorphic linear B-cell epitopes of L. braziliensis. Specific recognition of TL and VL patients was confirmed by significantly decreased IgG reactivity against rPeroxidoxin after depletion of peptide-1- and peptide-2-specific antibodies (peptide 1: reduced by 32%, 42% and 5% for CL, ML and VL, respectively; peptide-2: reduced by 24%, 22% and 13% for CL, ML and VL, respectively) and only peptide-2 for CVL (reduced 9%). Overall, rPeroxidoxin may be a potential antigen for the immunodiagnosis of TL

  1. Biogenesis of influenza a virus hemagglutinin cross-protective stem epitopes.

    PubMed

    Magadán, Javier G; Altman, Meghan O; Ince, William L; Hickman, Heather D; Stevens, James; Chevalier, Aaron; Baker, David; Wilson, Patrick C; Ahmed, Rafi; Bennink, Jack R; Yewdell, Jonathan W

    2014-06-01

    Antigenic variation in the globular domain of influenza A virus (IAV) hemagglutinin (HA) precludes effective immunity to this major human pathogen. Although the HA stem is highly conserved between influenza virus strains, HA stem-reactive antibodies (StRAbs) were long considered biologically inert. It is now clear, however, that StRAbs reduce viral replication in animal models and protect against pathogenicity and death, supporting the potential of HA stem-based immunogens as drift-resistant vaccines. Optimally designing StRAb-inducing immunogens and understanding StRAb effector functions require thorough comprehension of HA stem structure and antigenicity. Here, we study the biogenesis of HA stem epitopes recognized in cells infected with various drifted IAV H1N1 strains using mouse and human StRAbs. Using a novel immunofluorescence (IF)-based assay, we find that human StRAbs bind monomeric HA in the endoplasmic reticulum (ER) and trimerized HA in the Golgi complex (GC) with similar high avidity, potentially good news for producing effective monomeric HA stem immunogens. Though HA stem epitopes are nestled among several N-linked oligosaccharides, glycosylation is not required for full antigenicity. Rather, as N-linked glycans increase in size during intracellular transport of HA through the GC, StRAb binding becomes temperature-sensitive, binding poorly to HA at 4°C and well at 37°C. A de novo designed, 65-residue protein binds the mature HA stem independently of temperature, consistent with a lack of N-linked oligosaccharide steric hindrance due to its small size. Likewise, StRAbs bind recombinant HA carrying simple N-linked glycans in a temperature-independent manner. Chemical cross-linking experiments show that N-linked oligosaccharides likely influence StRAb binding by direct local effects rather than by globally modifying the conformational flexibility of HA. Our findings indicate that StRAb binding to HA is precarious, raising the possibility that

  2. Thrombophilic polymorphisms in Israel.

    PubMed

    Zoossmann-Diskin, Avshalom; Gazit, Ephraim; Peleg, Leah; Shohat, Mordechai; Turner, David

    2008-01-01

    Three thrombophilic polymorphisms, FV G1691A, FII G20210A and MTHFR C677T were investigated in Israeli populations by FRET, (fluorescence resonance energy transfer) real-time PCR. We observe extensive variability in the frequencies of each of the polymorphisms, as has been observed in the study of other polymorphisms in these populations. Very high allele frequencies for FV G1691A (the highest 0.087 in Turkish and Greek Jews) and FII G20210A (the highest 0.061 in Georgian Jews) in some of the Israeli populations justify a clinical investigation to assess their risk for venous thrombosis. Principal Coordinates Analysis demonstrates that the Jewish populations are interspersed among the non-Jewish populations. The resemblance of some Jewish populations to certain non-Jewish populations coincides with findings based on classical markers. PMID:18583164

  3. Replacement between conformity and counter-conformity in consumption decisions.

    PubMed

    Chou, Ting-Jui; Chang, En-Chung; Dai, Qi; Wong, Veronica

    2013-02-01

    This study assessed, in a Chinese context, how self-esteem interacts with perceived similarity and uniqueness to yield cognitive dissonance, and whether the dissonance leads to self-reported conformity or counter-conformity behavior. Participants were 408 respondents from 4 major Chinese cities (M age = 33.0 yr., SD = 4.3; 48% men). Self-perceptions of uniqueness, similarity, cognitive dissonance, self-esteem and need to behave in conformity or counter-conformity were measured. A theoretical model was assessed in four situations, relating the ratings of self-esteem and perceived similarity/uniqueness to the way other people at a wedding were dressed, and the resultant cognitive dissonance and conformity/ counter-conformity behavior. Regardless of high or low self-esteem, all participants reported cognitive dissonance when they were told that they were dressed extremely similarly to or extremely differently from the other people attending the wedding. However, the conforming/counter-conforming strategies used by participants to resolve the cognitive dissonance differed. When encountering dissonance induced by the perceived extreme uniqueness of dress, participants with low self-esteem tended to say they would dress next time so as to conform with the way others were dressed, while those with high self-esteem indicated they would continue their counter-conformity in attire. When encountering dissonance induced by the perceived extreme similarity to others, both those with high and low self-esteem tended to say they would dress in an unorthodox manner to surprise other people in the future. PMID:23654033

  4. Investigating the impact of hepatitis B virus surface gene polymorphism on antigenicity using ex vivo phenotyping.

    PubMed

    Ijaz, Samreen; Szypulska, Renata; Andrews, Nick; Tedder, Richard S

    2012-11-01

    The hepatitis B virus (HBV) surface antigen (HBsAg) is a complex protein, and understanding accurately the impact of amino acid changes on the antigenicity of the immunodominant a determinant must take this complexity into consideration. Epitope mapping with four mAbs was used to phenotype HBsAg directly from patients' sera to investigate the effect of mutations in their native genetic backbone. The expected mAb reactivity was established initially for samples harbouring 'wild-type' HBsAg sequences across genotypes A-E. The alteration of HBsAg antigenicity, defined by mAb epitope loss, was demonstrated in a number of samples with sequence-inferred amino acid changes. Individual mutations within the mapped epitopes to which the mAbs were directed usually affected their binding. However, the loss of more than one epitope was observed as the number of mutations within a sequence increased. Conversely, not all mutations occurring in the a determinant altered the HBsAg conformation. The genotype backbone, the specific amino acid substitution and amino acid changes occurring outside the major antigenic region appeared to be important in determining expression of the predicted epitope loss. These data clearly demonstrate that sequence-based methods alone may not accurately define HBsAg phenotype. This phenotyping methodology allows for the rapid and accurate identification of antigenically altered viruses and will greatly enhance current HBV surveillance, research and diagnostic activities. The data generated can be used to inform on public health issues relating to prevalence, transmission and impact of HBsAg mutants in HBV-infected populations. PMID:22855781

  5. Reconstitution of CD8 T Cells Protective against Cytomegalovirus in a Mouse Model of Hematopoietic Cell Transplantation: Dynamics and Inessentiality of Epitope Immunodominance.

    PubMed

    Holtappels, Rafaela; Lemmermann, Niels A W; Podlech, Jürgen; Ebert, Stefan; Reddehase, Matthias J

    2016-01-01

    Successful reconstitution of cytomegalovirus (CMV)-specific CD8(+) T cells by hematopoietic cell transplantation (HCT) gives a favorable prognosis for the control of CMV reactivation and prevention of CMV disease after hematoablative therapy of hematopoietic malignancies. In the transient immunocompromised state after HCT, pre-emptive cytoimmunotherapy with viral epitope-specific effector or memory CD8(+) T cells is a promising option to speed up antiviral control. Despite high-coding capacity of CMVs and a broad CD8(+) T-cell response on the population level, which reflects polymorphism in major histocompatibility complex class-I (MHC-I) glycoproteins, the response in terms of quantity of CD8(+) T cells in any individual is directed against a limited set of CMV-encoded epitopes selected for presentation by the private repertoire of MHC-I molecules. Such epitopes are known as "immunodominant" epitopes (IDEs). Besides host immunogenetics, genetic variance in CMV strains harbored as latent viruses by an individual HCT recipient can also determine the set of IDEs, which complicates a "personalized immunotherapy." It is, therefore, an important question if IDE-specific CD8(+) T-cell reconstitution after HCT is critical or dispensable for antiviral control. As viruses with targeted mutations of IDEs cannot be experimentally tested in HCT patients, we employed the well-established mouse model of HCT. Notably, control of murine CMV (mCMV) after HCT was comparably efficient for IDE-deletion mutant mCMV-Δ4IDE and the corresponding IDE-expressing revertant virus mCMV-Δ4IDE-rev. Thus, antigenicity-loss mutations in IDEs do not result in loss-of-function of a polyclonal CD8(+) T-cell population. Although IDE deletion was not associated with global changes in the response to non-IDE epitopes, the collective of non-IDE-specific CD8(+) T-cells infiltrates infected tissue and confines infection within nodular inflammatory foci. We conclude from the model, and predict also for

  6. Reconstitution of CD8 T Cells Protective against Cytomegalovirus in a Mouse Model of Hematopoietic Cell Transplantation: Dynamics and Inessentiality of Epitope Immunodominance

    PubMed Central

    Holtappels, Rafaela; Lemmermann, Niels A. W.; Podlech, Jürgen; Ebert, Stefan; Reddehase, Matthias J.

    2016-01-01

    Successful reconstitution of cytomegalovirus (CMV)-specific CD8+ T cells by hematopoietic cell transplantation (HCT) gives a favorable prognosis for the control of CMV reactivation and prevention of CMV disease after hematoablative therapy of hematopoietic malignancies. In the transient immunocompromised state after HCT, pre-emptive cytoimmunotherapy with viral epitope-specific effector or memory CD8+ T cells is a promising option to speed up antiviral control. Despite high-coding capacity of CMVs and a broad CD8+ T-cell response on the population level, which reflects polymorphism in major histocompatibility complex class-I (MHC-I) glycoproteins, the response in terms of quantity of CD8+ T cells in any individual is directed against a limited set of CMV-encoded epitopes selected for presentation by the private repertoire of MHC-I molecules. Such epitopes are known as “immunodominant” epitopes (IDEs). Besides host immunogenetics, genetic variance in CMV strains harbored as latent viruses by an individual HCT recipient can also determine the set of IDEs, which complicates a “personalized immunotherapy.” It is, therefore, an important question if IDE-specific CD8+ T-cell reconstitution after HCT is critical or dispensable for antiviral control. As viruses with targeted mutations of IDEs cannot be experimentally tested in HCT patients, we employed the well-established mouse model of HCT. Notably, control of murine CMV (mCMV) after HCT was comparably efficient for IDE-deletion mutant mCMV-Δ4IDE and the corresponding IDE-expressing revertant virus mCMV-Δ4IDE-rev. Thus, antigenicity-loss mutations in IDEs do not result in loss-of-function of a polyclonal CD8+ T-cell population. Although IDE deletion was not associated with global changes in the response to non-IDE epitopes, the collective of non-IDE-specific CD8+ T-cells infiltrates infected tissue and confines infection within nodular inflammatory foci. We conclude from the model, and predict also for human

  7. Disappearing Polymorphs Revisited

    PubMed Central

    Bučar, Dejan-Krešimir; Lancaster, Robert W; Bernstein, Joel

    2015-01-01

    Nearly twenty years ago, Dunitz and Bernstein described a selection of intriguing cases of polymorphs that disappear. The inability to obtain a crystal form that has previously been prepared is indeed a frustrating and potentially serious problem for solid-state scientists. This Review discusses recent occurrences and examples of disappearing polymorphs (as well as the emergence of elusive crystal forms) to demonstrate the enduring relevance of this troublesome, but always captivating, phenomenon in solid-state research. A number of these instances have been central issues in patent litigations. This Review, therefore, also highlights the complex relationship between crystal chemistry and the law. PMID:26031248

  8. Vaccine Design for H5N1 Based on B- and T-cell Epitope Predictions

    PubMed Central

    Tambunan, Usman Sumo Friend; Sipahutar, Feimmy Ruth Pratiwi; Parikesit, Arli Aditya; Kerami, Djati

    2016-01-01

    From 2003 to 2013, Indonesia had the highest number of avian influenza A cases in humans, with 192 cases and 160 fatalities. Avian influenza is caused by influenza virus type A, such as subtype H5N1. This virus has two glycoproteins: hemagglutinin and neuraminidase, which will become the primary target to be neutralized by vaccine. Vaccine is the most effective immunologic intervention. In this study, we use the epitope-based vaccine design from hemagglutinin and neuraminidase of H5N1 Indonesian strain virus by using immunoinformatics approach in order to predict the binding of B-cell and T-cell epitopes (class I and class II human leukocyte antigen [HLA]). BCPREDS was used to predict the B-cell epitope. Propred, Propred I, netMHCpan, and netMHCIIpan were used to predict the T-cell epitope. Two B-cell epitopes of hemagglutinin candidates and one B-cell epitope of neuraminidase candidates were obtained to bind T-cell CD4+ (class II HLA), and also five T-cell epitope hemagglutinin and four T-cell epitope neuraminidase were obtained to bind T-cell CD8+ (class I HLA). The visualization of epitopes was done using MOE 2008.10. It shows that the binding affinity of epitope–HLA was based on minimum binding free energy (ΔGbinding). Based on this result, visualization, and dynamic simulation, four hemagglutinin epitopes (MEKIVLLLA, CPYLGSPSF, KCQTPMGAI, and IGTSTLNQR) and two neuraminidase epitopes (NPNQKIITI and CYPDAGEIT) were computed as having the best binding affinity from HLA ligand. The results mentioned above are from in silico experiments and need to be validated using wet experiment. PMID:27147821

  9. Plasmodium vivax Promiscuous T-Helper Epitopes Defined and Evaluated as Linear Peptide Chimera Immunogens

    PubMed Central

    Caro-Aguilar, Ivette; Rodríguez, Alexandra; Calvo-Calle, J. Mauricio; Guzmán, Fanny; De la Vega, Patricia; Elkin Patarroyo, Manuel; Galinski, Mary R.; Moreno, Alberto

    2002-01-01

    Clinical trials of malaria vaccines have confirmed that parasite-derived T-cell epitopes are required to elicit consistent and long-lasting immune responses. We report here the identification and functional characterization of six T-cell epitopes that are present in the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) and bind promiscuously to four different HLA-DRB1∗ alleles. Each of these peptides induced lymphoproliferative responses in cells from individuals with previous P. vivax infections. Furthermore, linear-peptide chimeras containing the promiscuous PvMSP-1 T-cell epitopes, synthesized in tandem with the Plasmodium falciparum immunodominant circumsporozoite protein (CSP) B-cell epitope, induced high specific antibody titers, cytokine production, long-lasting immune responses, and immunoglobulin G isotype class switching in BALB/c mice. A linear-peptide chimera containing an allele-restricted P. falciparum T-cell epitope with the CSP B-cell epitope was not effective. Two out of the six promiscuous T-cell epitopes exhibiting the highest anti-peptide response also contain B-cell epitopes. Antisera generated against these B-cell epitopes recognize P. vivax merozoites in immunofluorescence assays. Importantly, the anti-peptide antibodies generated to the CSP B-cell epitope inhibited the invasion of P. falciparum sporozoites into human hepatocytes. These data and the simplicity of design of the chimeric constructs highlight the potential of multimeric, multistage, and multispecies linear-peptide chimeras containing parasite promiscuous T-cell epitopes for malaria vaccine development. PMID:12065487

  10. Classical Virasoro irregular conformal block

    NASA Astrophysics Data System (ADS)

    Rim, Chaiho; Zhang, Hong

    2015-07-01

    Virasoro irregular conformal block with arbitrary rank is obtained for the classical limit or equivalently Nekrasov-Shatashvili limit using the beta-deformed irregular matrix model (Penner-type matrix model for the irregular conformal block). The same result is derived using the generalized Mathieu equation which is equivalent to the loop equation of the irregular matrix model.

  11. Conformal gravity and time

    NASA Astrophysics Data System (ADS)

    Hazboun, Jeffrey Shafiq

    2014-10-01

    Cartan geometry provides a rich formalism from which to look at various geometrically motivated extensions to general relativity. In this manuscript, we start by motivating reasons to extend the theory of general relativity. We then introduce the reader to our technique, called the quotient manifold method, for extending the geometry of spacetime. We will specifically look at the class of theories formed from the various quotients of the conformal group. Starting with the conformal symmetries of Euclidean space, we construct a manifold where time manifests as a part of the geometry. Though there is no matter present in the geome- try studied here, geometric terms analogous to dark energy and dark matter appear when we write down the Einstein tensor. Specifically, the quotient of the conformal group of Euclidean four-space by its Weyl subgroup results in a geometry possessing many of the properties of relativistic phase space, including both a natural symplectic form and nondegenerate Killing metric. We show the general solution possesses orthogonal Lagrangian submanifolds, with the induced metric and the spin connection on the submanifolds necessarily Lorentzian, despite the Euclidean starting point. By examining the structure equations of the biconformal space in an orthonormal frame adapted to its phase space properties, we also find two new tensor fields exist in this geometry, not present in Riemannian geometry. The first is a combination of the Weyl vector with the scale factor on the metric, and determines the time-like directions on the submanifolds. The second comes from the components of the spin connection, symmetric with respect to the new metric. Though this field comes from the spin connection, it transforms ho- mogeneously. Finally, we show in the absence of Cartan curvature or sources, the configuration space has geometric terms equivalent to a perfect fluid and a cosmological constant. We complete the analysis of this homogeneous space by

  12. Characterizing the Conformational Landscape of Flavivirus Fusion Peptides via Simulation and Experiment

    NASA Astrophysics Data System (ADS)

    Marzinek, Jan K.; Lakshminarayanan, Rajamani; Goh, Eunice; Huber, Roland G.; Panzade, Sadhana; Verma, Chandra; Bond, Peter J.

    2016-01-01

    Conformational changes in the envelope proteins of flaviviruses help to expose the highly conserved fusion peptide (FP), a region which is critical to membrane fusion and host cell infection, and which represents a significant target for antiviral drugs and antibodies. In principle, extended timescale atomic-resolution simulations may be used to characterize the dynamics of such peptides. However, the resultant accuracy is critically dependent upon both the underlying force field and sufficient conformational sampling. In the present study, we report a comprehensive comparison of three simulation methods and four force fields comprising a total of more than 40 μs of sampling. Additionally, we describe the conformational landscape of the FP fold across all flavivirus family members. All investigated methods sampled conformations close to available X-ray structures, but exhibited differently populated ensembles. The best force field / sampling combination was sufficiently accurate to predict that the solvated peptide fold is less ordered than in the crystallographic state, which was subsequently confirmed via circular dichroism and spectrofluorometric measurements. Finally, the conformational landscape of a mutant incapable of membrane fusion was significantly shallower than wild-type variants, suggesting that dynamics should be considered when therapeutically targeting FP epitopes.

  13. Characterizing the Conformational Landscape of Flavivirus Fusion Peptides via Simulation and Experiment

    PubMed Central

    Marzinek, Jan K.; Lakshminarayanan, Rajamani; Goh, Eunice; Huber, Roland G.; Panzade, Sadhana; Verma, Chandra; Bond, Peter J.

    2016-01-01

    Conformational changes in the envelope proteins of flaviviruses help to expose the highly conserved fusion peptide (FP), a region which is critical to membrane fusion and host cell infection, and which represents a significant target for antiviral drugs and antibodies. In principle, extended timescale atomic-resolution simulations may be used to characterize the dynamics of such peptides. However, the resultant accuracy is critically dependent upon both the underlying force field and sufficient conformational sampling. In the present study, we report a comprehensive comparison of three simulation methods and four force fields comprising a total of more than 40 μs of sampling. Additionally, we describe the conformational landscape of the FP fold across all flavivirus family members. All investigated methods sampled conformations close to available X-ray structures, but exhibited differently populated ensembles. The best force field / sampling combination was sufficiently accurate to predict that the solvated peptide fold is less ordered than in the crystallographic state, which was subsequently confirmed via circular dichroism and spectrofluorometric measurements. Finally, the conformational landscape of a mutant incapable of membrane fusion was significantly shallower than wild-type variants, suggesting that dynamics should be considered when therapeutically targeting FP epitopes. PMID:26785994

  14. Plasmodium falciparum Rosetting Epitopes Converge in the SD3-Loop of PfEMP1-DBL1α

    PubMed Central

    Angeletti, Davide; Albrecht, Letusa; Blomqvist, Karin; Quintana, María del Pilar; Akhter, Tahmina; Bächle, Susanna M.; Sawyer, Alan; Sandalova, Tatyana; Achour, Adnane; Wahlgren, Mats; Moll, Kirsten

    2012-01-01

    The ability of Plasmodium falciparum parasitized RBC (pRBC) to form rosettes with normal RBC is linked to the virulence of the parasite and RBC polymorphisms that weaken rosetting confer protection against severe malaria. The adhesin PfEMP1 mediates the binding and specific antibodies prevent sequestration in the micro-vasculature, as seen in animal models. Here we demonstrate that epitopes targeted by rosette disrupting antibodies converge in the loop of subdomain 3 (SD3) which connects the h6 and h7 α-helices of PfEMP1-DBL1α. Both monoclonal antibodies and polyclonal IgG, that bound to epitopes in the SD3-loop, stained the surface of pRBC, disrupted rosettes and blocked direct binding of recombinant NTS-DBL1α to RBC. Depletion of polyclonal IgG raised to NTS-DBL1α on a SD3 loop-peptide removed the anti-rosetting activity. Immunizations with recombinant subdomain 1 (SD1), subdomain 2 (SD2) or SD3 all generated antibodies reacting with the pRBC-surface but only the sera of animals immunized with SD3 disrupted rosettes. SD3-sequences were found to segregate phylogenetically into two groups (A/B). Group A included rosetting sequences that were associated with two cysteine-residues present in the SD2-domain while group B included those with three or more cysteines. Our results suggest that the SD3 loop of PfEMP1-DBL1α is an important target of anti-rosetting activity, clarifying the molecular basis of the development of variant-specific rosette disrupting antibodies. PMID:23227205

  15. Different Vaccine Vectors Delivering the Same Antigen Elicit CD8+ T Cell Responses with Distinct Clonotype and Epitope Specificity

    SciTech Connect

    Honda, M.; Robinson, H.; Wang, R.; Kong, W.-P.; Kanekiyo, M.; Akahata, W.; Xu, L.; Matsuo, K.; Natarajan, K.; Asher, T. E.; Price, D. A.; Douek, D. C.; Margulies, D. H.; Nabel, G. J.

    2009-08-15

    Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternative vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.

  16. Different Vaccine Vectors Delivering the Same Antigen Elicit CD8plus T Cell Responses with Distinct Clonotype and Epitope Specificity

    SciTech Connect

    M Honda; R Wang; W Kong; M Kanekiyo; Q Akahata; L Xu; K Matsuo; K Natarajan; H Robinson; et al.

    2011-12-31

    Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternative vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.

  17. Specific antibody response to oligomannosidic epitopes in Crohn's disease.

    PubMed Central

    Sendid, B; Colombel, J F; Jacquinot, P M; Faille, C; Fruit, J; Cortot, A; Lucidarme, D; Camus, D; Poulain, D

    1996-01-01

    Elevated antibody levels against the yeast Saccharomyces cerevisiae have been reported in sera from patients with Crohn's disease and not with ulcerative colitis. The aim of the study was to identify the nature of the epitopes supporting this antibody response. Whole cells from different S. cerevisiae strains were selected in immunofluorescence assay for their ability to differentiate the antibody responses of patients with Crohn's disease and ulcerative colitis. Their cell wall phosphopeptidomannans were then tested as antigen in enzyme-linked immunosorbent assay (ELISA) against sera from 42 patients with Crohn's disease, 20 patients with ulcerative colitis, and 34 healthy controls. Graded chemical degradations were performed on the most reactive strain phosphopeptidomannan. The discriminating epitope was determined through gas-liquid chromatography-mass spectrometry. The greatest discrimination among patients with Crohn's disease, ulcerative colitis, and controls was obtained with Su1, a S. cerevisiae strain used in brewing of beer. ELISA directed against phosphopeptidomannan of this strain was 64% sensitive and 77% specific for discriminating Crohn's disease versus ulcerative colitis and 71% sensitive and 89% specific for Crohn's disease versus controls. Periodate oxidation and selective degradation demonstrated that the most important polysaccharide epitope was shared by both the acid-stable and the alkali-labile domains of the phosphopeptidomannan. The determination of oligomannose sequences of S. cerevisiae Su1 phosphopeptidomannans suggested that a mannotetraose, Man (1 --> 3)Man(1 --> 2)Man(1 --> 2)Man, supported the serological response seen in Crohn's disease. Further identification of the immunogen eliciting this antibody response as a marker of the disease may help to understand its etiology. PMID:8991640

  18. Epitope spreading induced by immunization with synthetic SSB peptides

    PubMed Central

    DING, MIN; ZHANG, JIANZHONG

    2016-01-01

    Sjogren's syndrome type B (SSB)/La antibody is an autoantibody generally observed in connective tissue diseases whereas double-stranded deoxyribonucleic acid (dsDNA) antibodies are the most characteristic autoantibodies found in systemic lupus erythematosus (SLE) patients. The relationship of these autoantibodies remains unclear. The aim of the study was to determine the profile of antibody production in rabbits immunized with synthetic SSB peptides alone or with dsDNA. For this purpose, 214–225aa peptide of SSB antigen was synthesized based on the organic chemistry solid-phase peptide synthesis. Rabbits were immunized with the following antigens: i) synthetic SSB peptides linked with keyhole limpet hemocyanin (KLH); ii) dsDNA; iii) SSB plus dsDNA; iv) KLH; and v) phosphate-buffered saline. SSB peptide antibody was measured using the enzyme-linked immunosorbent assay while extractable nuclear antigens (ENA) antibody and dsDNA antibody were measured by immunoblotting and immunofluorescence, respectively. The results showed that a specific anti-SSB peptide antibody was produced following immunization with SSB epitope alone or with dsDNA. The SSB peptide antibody titer in the coimmunization group was higher than that of the SSB alone group. In addition, antibodies against ribonucleoprotein (RNP), Smith and/or dsDNA were detected in rabbits of the coimmunization group. The presence of anti-dsDNA antibodies in the rabbits immunized with SSB peptide suggested the induction of epitope spreading. In conclusions, SSB antibodies were produced in rabbits immunized with SSB peptide or SSB+dsDNA, whereas SSB antibody titers were higher in the coimmunization group. Furthermore, coimmunization was associated with epitope spreading. PMID:27347030

  19. Projectors, shadows, and conformal blocks

    NASA Astrophysics Data System (ADS)

    Simmons-Duffin, David

    2014-04-01

    We introduce a method for computing conformal blocks of operators in arbitrary Lorentz representations in any spacetime dimension, making it possible to apply bootstrap techniques to operators with spin. The key idea is to implement the "shadow formalism" of Ferrara, Gatto, Grillo, and Parisi in a setting where conformal invariance is manifest. Conformal blocks in d-dimensions can be expressed as integrals over the projective null-cone in the "embedding space" d+1,1. Taking care with their analytic structure, these integrals can be evaluated in great generality, reducing the computation of conformal blocks to a bookkeeping exercise. To facilitate calculations in four-dimensional CFTs, we introduce techniques for writing down conformally-invariant correlators using auxiliary twistor variables, and demonstrate their use in some simple examples.

  20. Constraints on the conformation of the cytoplasmic face of dark-adapted and light-excited rhodopsin inferred from antirhodopsin antibody imprints

    PubMed Central

    Bailey, Brian W.; Mumey, Brendan; Hargrave, Paul A.; Arendt, Anatol; Ernst, Oliver P.; Hofmann, Klaus Peter; Callis, Patrik R.; Burritt, James B.; Jesaitis, Algirdas J.; Dratz, Edward A.

    2003-01-01

    Rhodopsin is the best-understood member of the large G protein–coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the "antibody imprint" method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets. PMID:14573859

  1. Effects of Conformism on the Cultural Evolution of Social Behaviour

    PubMed Central

    Molleman, Lucas; Pen, Ido; Weissing, Franz J.

    2013-01-01

    Models of cultural evolution study how the distribution of cultural traits changes over time. The dynamics of cultural evolution strongly depends on the way these traits are transmitted between individuals by social learning. Two prominent forms of social learning are payoff-based learning (imitating others that have higher payoffs) and conformist learning (imitating locally common behaviours). How payoff-based and conformist learning affect the cultural evolution of cooperation is currently a matter of lively debate, but few studies systematically analyse the interplay of these forms of social learning. Here we perform such a study by investigating how the interaction of payoff-based and conformist learning affects the outcome of cultural evolution in three social contexts. First, we develop a simple argument that provides insights into how the outcome of cultural evolution will change when more and more conformist learning is added to payoff-based learning. In a social dilemma (e.g. a Prisoner’s Dilemma), conformism can turn cooperation into a stable equilibrium; in an evasion game (e.g. a Hawk-Dove game or a Snowdrift game) conformism tends to destabilize the polymorphic equilibrium; and in a coordination game (e.g. a Stag Hunt game), conformism changes the basin of attraction of the two equilibria. Second, we analyse a stochastic event-based model, revealing that conformism increases the speed of cultural evolution towards pure equilibria. Individual-based simulations as well as the analysis of the diffusion approximation of the stochastic model by and large confirm our findings. Third, we investigate the effect of an increasing degree of conformism on cultural group selection in a group-structured population. We conclude that, in contrast to statements in the literature, conformism hinders rather than promotes the evolution of cooperation. PMID:23874528

  2. Monoclonal Antibodies Bind A SNP-Sensitive Epitope that is Present Uniquely in Mycobacterium avium Subspecies Paratuberculosis

    PubMed Central

    Bannantine, John P.; Stabel, Judith R.; Lamont, Elise A.; Briggs, Robert E.; Sreevatsan, Srinand

    2011-01-01

    Due to a close genetic relatedness, there is no known antibody that detects Mycobacterium avium subspecies paratuberculosis (MAP), which causes Johne’s disease in cattle and sheep, and does not cross-react with other M. avium subspecies. In the present study, a monoclonal antibody (MAb; 17A12) was identified from mice immunized with a cell membrane fraction of MAP strain K-10. This antibody is 100% specific as it detected a 25-kDa protein in all 29 MAP whole cell lysates, but did not bind to any of the 29 non-paratuberculosis strains tested in immunoblot assays. However, the antibody revealed variable reactivity levels in MAP strains as it detected higher levels in bovine isolates but comparably lower levels in ovine isolates of MAP. In order to identify the target binding protein for 17A12, a lambda phage expression library of MAP genomic fragments was screened with the MAb. Four reactive clones were identified, sequenced and all shown to be overlapping. Further analysis revealed all four clones expressed an unknown protein encoded by a sequence that is not annotated in the K-10 genome and overlapped with MAP3422c on the opposing DNA strand. The epitope of 17A12 was precisely defined to seven amino acids and was used to query the K-10 genome. Similarity searches revealed another protein, encoded by MAP1025, possessed a similar epitope (one-amino acid mismatch) that also reacted strongly to the antibody. A single nucleotide polymorphism (SNP) in MAP1025 was then identified by comparative sequence analysis, which results in a Pro28His change at residue 28, the first amino acid within the 17A12 epitope. This SNP is present in all MAP strains but absent in all non-MAP strains and accounts for the specificity of the 17A12 antibody. This new antibody is the first ever isolated that binds only to the paratuberculosis subspecies of M. avium and opens new possibilities for the specific detection of this significant ruminant pathogen. PMID:21845186

  3. Structural insight into epitopes in the pregnancy-associated malaria protein VAR2CSA.

    PubMed

    Andersen, Pernille; Nielsen, Morten A; Resende, Mafalda; Rask, Thomas S; Dahlbäck, Madeleine; Theander, Thor; Lund, Ole; Salanti, Ali

    2008-02-01

    Pregnancy-associated malaria is caused by Plasmodium falciparum malaria parasites binding specifically to chondroitin sulfate A in the placenta. This sequestration of parasites is a major cause of low birth weight in infants and anemia in the mothers. VAR2CSA, a polymorphic multi-domain protein of the PfEMP1 family, is the main parasite ligand for CSA binding, and identification of protective antibody epitopes is essential for VAR2CSA vaccine development. Attempts to determine the crystallographic structures of VAR2CSA or its domains have not been successful yet. In this study, we propose 3D models for each of the VAR2CSA DBL domains and we show that regions in the fold of VAR2CSA inter-domain 2 and a PfEMP1 CIDR domain seem to be homologous to the EBA-175 and Pk alpha-DBL fold. This suggests that ID2 could be a functional domain. We also identify regions of VAR2CSA present on the surface of native VAR2CSA by comparing reactivity of plasma containing anti-VAR2CSA antibodies in peptide array experiments before and after incubation with native VAR2CSA. By this method we identify conserved VAR2CSA regions targeted by antibodies that react with the native molecule expressed on infected erythrocytes. By mapping the data onto the DBL models we present evidence suggesting that the S1+S2 DBL sub-domains are generally surface-exposed in most domains, whereas the S3 sub-domains are less exposed in native VAR2CSA. These results comprise an important step towards understanding the structure of VAR2CSA on the surface of CSA-binding infected erythrocytes. PMID:18282103

  4. Warped conformal field theory

    NASA Astrophysics Data System (ADS)

    Detournay, Stéphane; Hartman, Thomas; Hofman, Diego M.

    2012-12-01

    We study field theories in two spacetime dimensions invariant under a chiral scaling symmetry that acts only on right-movers. The local symmetries include one copy of the Virasoro algebra and a U(1) current algebra. This differs from the two-dimensional conformal group but in some respects is equally powerful in constraining the theory. In particular, the symmetries on a torus lead to modular covariance of the partition function, which is used to derive a universal formula for the asymptotic density of states. For an application we turn to the holographic description of black holes in quantum gravity, motivated by the fact that the symmetries in the near-horizon geometry of any extremal black hole are identical to those of a two-dimensional field theory with chiral scaling. We consider two examples: black holes in warped AdS3 in topologically massive gravity and in string theory. In both cases, the density of states in the two-dimensional field theory reproduces the Bekenstein-Hawking entropy of black holes in the gravity theory.

  5. Dynamics of protein conformations

    NASA Astrophysics Data System (ADS)

    Stepanova, Maria

    2010-10-01

    A novel theoretical methodology is introduced to identify dynamic structural domains and analyze local flexibility in proteins. The methodology employs a multiscale approach combining identification of essential collective coordinates based on the covariance analysis of molecular dynamics trajectories, construction of the Mori projection operator with these essential coordinates, and analysis of the corresponding generalized Langevin equations [M.Stepanova, Phys.Rev.E 76(2007)051918]. Because the approach employs a rigorous theory, the outcomes are physically transparent: the dynamic domains are associated with regions of relative rigidity in the protein, whereas off-domain regions are relatively soft. This also allows scoring the flexibility in the macromolecule with atomic-level resolution [N.Blinov, M.Berjanskii, D.S.Wishart, and M.Stepanova, Biochemistry, 48(2009)1488]. The applications include the domain coarse-graining and characterization of conformational stability in protein G and prion proteins. The results are compared with published NMR experiments. Potential applications for structural biology, bioinformatics, and drug design are discussed.

  6. Highly conserved influenza A virus epitope sequences as candidates of H3N2 flu vaccine targets.

    PubMed

    Wu, Ko-Wen; Chien, Chih-Yi; Li, Shiao-Wen; King, Chwan-Chuen; Chang, Chuan-Hsiung

    2012-08-01

    This study focused on identifying the conserved epitopes in a single subtype A (H3N2)-as candidates for vaccine targets. We identified a total of 32 conserved epitopes in four viral proteins [22 HA, 4PB1, 3 NA, 3 NP]. Evaluation of conserved epitopes in coverage during 1968-2010 revealed that (1) 12 HA conserved epitopes were highly present in the circulating viruses; (2) the remaining 10 HA conserved epitopes appeared with lower percentage but a significantly increasing trend after 1989 [p<0.001]; and (3) the conserved epitopes in NA, NP and PB1 are also highly frequent in wild-type viruses. These conserved epitopes also covered an extremely high percentage of the 16 vaccine strains during the 42 year period. The identification of highly conserved epitopes using our approach can also be applied to develop broad-spectrum vaccines. PMID:22698979

  7. Enzyme polymorphisms in Canarium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fifty-two accessions of Canarium involving seven species, C. ovatum, C. album, C. megalanthum, C. harveyi, C. indicum, C. mehenbethene, and C. odontophyllum were studied for isozyme polymorphisms. Starch gel electrophoresis with a histidine-citrate buffer system (pH 6.5) was employed to assay six en...

  8. Polymorphous Perversity in Texts

    ERIC Educational Resources Information Center

    Johnson-Eilola, Johndan

    2012-01-01

    Here's the tricky part: If we teach ourselves and our students that texts are made to be broken apart, remixed, remade, do we lose the polymorphous perversity that brought us pleasure in the first place? Does the pleasure of transgression evaporate when the borders are opened?

  9. Investigation of Uranium Polymorphs

    SciTech Connect

    Sweet, Lucas E.; Henager, Charles H.; Hu, Shenyang Y.; Johnson, Timothy J.; Meier, David E.; Peper, Shane M.; Schwantes, Jon M.

    2011-08-01

    The UO3-water system is complex and has not been fully characterized, even though these species are common throughout the nuclear fuel cycle. As an example, most production schemes for UO3 result in a mixture of up to six or more different polymorphic phases, and small differences in these conditions will affect phase genesis that ultimately result in measureable changes to the end product. As a result, this feature of the UO3-water system may be useful as a means for determining process history. This research effort attempts to better characterize the UO3-water system with a variety of optical techniques for the purpose of developing some predictive capability for estimating process history in polymorphic phases of unknown origin. Three commercially relevant preparation methods for the production of UO3 were explored. Previously unreported low temperature routes to β- and γ-UO3 were discovered. Raman and fluorescence spectroscopic libraries were established for pure and mixed polymorphic forms of UO3 in addition to the common hydrolysis products of UO3. An advantage of the sensitivity of optical fluorescence microscopy over XRD has been demonstrated. Preliminary aging studies of the α and γ forms of UO3 have been conducted. In addition, development of a 3-D phase field model used to predict phase genesis of the system was initiated. Thermodynamic and structural constants that will feed the model have been gathered from the literature for most of the UO3 polymorphic phases.

  10. Association between HRH4 polymorphisms and ankylosing spondylitis susceptibility

    PubMed Central

    Ran, Bo; Wang, Yongcheng; Zhang, Yonggang; Mao, Keya; Wang, Yan

    2015-01-01

    Target: The purpose of the study was to investigate the association between the histamine H4 receptor (HRH4) polymorphisms and the susceptibility to ankylosing spondylitis (AS). Methods: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to analyze the HRH4 rs8088140 and rs657132 polymorphisms. Linkage disequilibrium and haplotype analyses were conducted with Haploview software. The genotypes distributions of HRH4 polymorphisms in the control group were tested by Hardy-Weinberg equilibrium (HWE), allele, genotype and haplotype frequencies between the cases and control groups were compared by χ2 test. The controls were matched with cases by age and gender. The relative risk of AS with HRH4 polymorphisms was represented by odds ratio (OR) with 95% confidence interval (CI) calculated by χ2 test. Results: The genotypes distributions of HRH4 rs8088140, rs657132 polymorphisms in controls conformed to HWE. The frequency of rs657132 AA genotype in the case group was obviously higher than that in the control group (P=0.040), and so was the A allele (OR=2.572, 95% CI=1.475-4.486, P=0.022). The frequency differences of A-A haplotype between two groups had statistical significance (P=0.011, OR=2.071, 95% CI=1.172-3.660) through haplotype analysis, indicating A-A might be the susceptible haplotype to AS. Conclusion: The AA genotypes of HRH4 rs657132 polymorphism may be the susceptible factors for AS, and rs657132 plays a role in generation of AS. In addition, A-A haplotype in rs8088140-rs657132 is also increased the risk of AS. PMID:26823878

  11. Immunochemical characterization of two thyroid-stimulating hormone beta-subunit epitopes.

    PubMed Central

    Fairlie, W D; Stanton, P G; Hearn, M T

    1995-01-01

    The epitopes of human thyroid-stimulating hormone (hTSH) recognized by two murine monoclonal antibodies (MAbs), designated MAb 279 and MAb 299, have been characterized. These MAbs are highly specific for the beta-subunit of TSH. The epitope recognized by MAb 279 appears to be completely conserved between bovine and human TSH and partially conserved in the porcine species. The TSH beta-subunit epitope recognized by MAb 299 is only partially conserved between the human, bovine and porcine species. Both MAbs are capable of inhibiting the binding of TSH to its receptor in a TSH radioreceptor assay, indicating that the epitopes either coincide or are located close to the TSH beta-subunit receptor-binding sites. The carbohydrate moieties of the TSH beta-subunit appear to play little or no role in the epitope recognition by MAb 279 or MAb 299 while the integrity of the disulphide bonds are essential. The epitopic recognition may also involve lysine residues, as determined by the immunoreactivity with both MAbs following citraconylation of TSH. In addition, the amino acid sequence region between residues bTSH beta 34-44 could be excised by trypsin digestion of bovine TSH beta (bTSH beta) without eliminating epitopic recognition by either MAb. These results provide further insight into the relationship between the structure of the TSH beta-subunit epitopes and location of the receptor-binding sites. Images Figure 2 PMID:7538754

  12. Computer-aided design of T-cell epitope-based vaccines: addressing population coverage.

    PubMed

    Oyarzun, P; Kobe, B

    2015-10-01

    Epitope-based vaccines (EVs) make use of short antigen-derived peptides corresponding to immune epitopes, which are administered to trigger a protective humoral and/or cellular immune response. EVs potentially allow for precise control over the immune response activation by focusing on the most relevant - immunogenic and conserved - antigen regions. Experimental screening of large sets of peptides is time-consuming and costly; therefore, in silico methods that facilitate T-cell epitope mapping of protein antigens are paramount for EV development. The prediction of T-cell epitopes focuses on the peptide presentation process by proteins encoded by the major histocompatibility complex (MHC). Because different MHCs have different specificities and T-cell epitope repertoires, individuals are likely to respond to a different set of peptides from a given pathogen in genetically heterogeneous human populations. In addition, protective immune responses are only expected if T-cell epitopes are restricted by MHC proteins expressed at high frequencies in the target population. Therefore, without careful consideration of the specificity and prevalence of the MHC proteins, EVs could fail to adequately cover the target population. This article reviews state-of-the-art algorithms and computational tools to guide EV design through all the stages of the process: epitope prediction, epitope selection and vaccine assembly, while optimizing vaccine immunogenicity and coping with genetic variation in humans and pathogens. PMID:26211755

  13. HLA-DQ molecules as affinity matrix for identification of gluten T cell epitopes.

    PubMed

    Dørum, Siri; Bodd, Michael; Fallang, Lars-Egil; Bergseng, Elin; Christophersen, Asbjørn; Johannesen, Marie K; Qiao, Shuo-Wang; Stamnaes, Jorunn; de Souza, Gustavo A; Sollid, Ludvig M

    2014-11-01

    Even though MHC class II is a dominant susceptibility factor for many diseases, culprit T cell epitopes presented by disease-associated MHC molecules remain largely elusive. T cells of celiac disease lesions recognize cereal gluten epitopes presented by the disease-associated HLA molecules DQ2.5, DQ2.2, or DQ8. Employing celiac disease and complex gluten Ag digests as a model, we tested the feasibility of using DQ2.5 and DQ2.2 as an affinity matrix for identification of disease-relevant T cell epitopes. Known gluten T cell epitope peptides were enriched by DQ2.5, whereas a different set of peptides was enriched by DQ2.2. Of 86 DQ2.2-enriched peptides, four core sequences dominated. One of these core sequences is a previously known epitope and two others are novel epitopes. The study provides insight into the selection of gluten epitopes by DQ2.2. Furthermore, the approach presented is relevant for epitope identification in other MHC class II-associated disorders. PMID:25261484

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

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

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

  15. Introduced Amino Terminal Epitopes Can Reduce Surface Expression of Neuronal Nicotinic Receptors

    PubMed Central

    Bracamontes, John R.; Akk, Gustav; Steinbach, Joe Henry

    2016-01-01

    Epitopes accessible on the surface of intact cells are extremely valuable in studies of membrane proteins, allowing quantification and determination of the distribution of proteins as well as identification of cells expressing large numbers of proteins. However for many membrane proteins there are no suitable antibodies to native sequences, due to lack of availability, low affinity or lack of specificity. In these cases the use of an introduced epitope at specific sites in the protein of interest can often provide a suitable tool for studies. However, the introduction of the epitope sequence has the potential to affect protein expression, the assembly of multisubunit proteins or transport to the surface membrane. We find that surface expression of heteromeric neuronal nicotinic receptors containing the α4 and β4 subunits can be affected by introduced epitopes when inserted near the amino terminus of a subunit. The FLAG epitope greatly reduces surface expression when introduced into either α4 or β4 subunits, the V5 epitope has little effect when placed in either, while the Myc epitope reduces expression more when inserted into β4 than α4. These results indicate that the extreme amino terminal region is important for assembly of these receptors, and demonstrate that some widely used introduced epitopes may severely reduce surface expression. PMID:26963253

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

    PubMed Central

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

    2015-01-01

    Development of a prophylactic vaccine against hepatitis C virus (HCV) has been hampered by the extraordinary viral diversity and the poor host immune response. Scaffolding, by grafting an epitope onto a heterologous protein scaffold, offers a possible solution to epitope vaccine design. In this study, we designed and characterized epitope vaccine antigens for the antigenic sites of HCV envelope glycoproteins E1 (residues 314–324) and E2 (residues 412–423), for which neutralizing antibody-bound structures are available. We first combined six structural alignment algorithms in a “scaffolding meta-server” to search for diverse scaffolds that can structurally accommodate the HCV epitopes. For each antigenic site, ten scaffolds were selected for computational design, and the resulting epitope scaffolds were analyzed using structure-scoring functions and molecular dynamics simulation. We experimentally confirmed that three E1 and five E2 epitope scaffolds bound to their respective neutralizing antibodies, but with different kinetics. We then investigated a “multivalent scaffolding” approach by displaying 24 copies of an epitope scaffold on a self-assembling nanoparticle, which markedly increased the avidity of antibody binding. Our study thus demonstrates the utility of a multi-scale scaffolding strategy in epitope vaccine design and provides promising HCV immunogens for further assessment in vivo. PMID:26238798

  17. Insert engineering and solubility screening improves recovery of virus-like particle subunits displaying hydrophobic epitopes.

    PubMed

    Abidin, R S; Lua, L H L; Middelberg, A P J; Sainsbury, F

    2015-11-01

    The Polyomavirus coat protein, VP1 has been developed as an epitope presentation system able to provoke humoral immunity against a variety of pathogens, such as Influenza and Group A Streptococcus. The ability of the system to carry cytotoxic T cell epitopes on a surface-exposed loop and the impact on protein solubility has not been examined. Four variations of three selected epitopes were cloned into surface-exposed loops of VP1, and expressed in Escherichia coli. VP1 pentamers, also known as capsomeres, were purified via a glutathione-S-transferase tag. Size exclusion chromatography indicated severe aggregation of the recombinant VP1 during enzymatic tag removal resulting from the introduction the hydrophobic epitopes. Inserts were modified to possess double aspartic acid residues at each end of the hydrophobic epitopes and a high-throughput buffer condition screen was implemented with protein aggregation monitored during tag removal by spectrophotometry and dynamic light scattering. These analyses showed that the insertion of charged residues at the extremities of epitopes could improve solubility of capsomeres and revealed multiple windows of opportunity for further condition optimization. A combination of epitope design, pH optimization, and the additive l-arginine permitted the recovery of soluble VP1 pentamers presenting hydrophobic epitopes and their subsequent assembly into virus-like particles. PMID:26401641

  18. Complete epitopes for vaccine design derived from a crystal structure of the broadly neutralizing antibodies PGT128 and 8ANC195 in complex with an HIV-1 Env trimer.

    PubMed

    Kong, Leopold; Torrents de la Peña, Alba; Deller, Marc C; Garces, Fernando; Sliepen, Kwinten; Hua, Yuanzi; Stanfield, Robyn L; Sanders, Rogier W; Wilson, Ian A

    2015-10-01

    The HIV-1 envelope gp160 glycoprotein (Env) is a trimer of gp120 and gp41 heterodimers that mediates cell entry and is the primary target of the humoral immune response. Broadly neutralizing antibodies (bNAbs) to HIV-1 have revealed multiple epitopes or sites of vulnerability, but mapping of most of these sites is incomplete owing to a paucity of structural information on the full epitope in the context of the Env trimer. Here, a crystal structure of the soluble BG505 SOSIP gp140 trimer at 4.6 Å resolution with the bNAbs 8ANC195 and PGT128 reveals additional interactions in comparison to previous antibody-gp120 structures. For 8ANC195, in addition to previously documented interactions with gp120, a substantial interface with gp41 is now elucidated that includes extensive interactions with the N637 glycan. Surprisingly, removal of the N637 glycan did not impact 8ANC195 affinity, suggesting that the antibody has evolved to accommodate this glycan without loss of binding energy. PGT128 indirectly affects the N262 glycan by a domino effect, in which PGT128 binds to the N301 glycan, which in turn interacts with and repositions the N262 glycan, thereby illustrating the important role of neighboring glycans on epitope conformation and stability. Comparisons with other Env trimer and gp120 structures support an induced conformation for glycan N262, suggesting that the glycan shield is allosterically modified upon PGT128 binding. These complete epitopes of two broadly neutralizing antibodies on the Env trimer can now be exploited for HIV-1 vaccine design. PMID:26457433

  19. PrP Conformational Transitions Alter Species Preference of a PrP-specific Antibody*

    PubMed Central

    Zou, Wen-Quan; Langeveld, Jan; Xiao, Xiangzhu; Chen, Shugui; McGeer, Patrick L.; Yuan, Jue; Payne, Michael C.; Kang, Hae-Eun; McGeehan, John; Sy, Man-Sun; Greenspan, Neil S.; Kaplan, David; Wang, Gong-Xian; Parchi, Piero; Hoover, Edward; Kneale, Geoff; Telling, Glenn; Surewicz, Witold K.; Kong, Qingzhong; Guo, Jian-Ping

    2010-01-01

    The epitope of the 3F4 antibody most commonly used in human prion disease diagnosis is believed to consist of residues Met-Lys-His-Met (MKHM) corresponding to human PrP-(109–112). This assumption is based mainly on the observation that 3F4 reacts with human and hamster PrP but not with PrP from mouse, sheep, and cervids, in which Met at residue 112 is replaced by Val. Here we report that, by brain histoblotting, 3F4 did not react with PrP of uninfected transgenic mice expressing elk PrP; however, it did show distinct immunoreactivity in transgenic mice infected with chronic wasting disease. Compared with human PrP, the 3F4 reactivity with the recombinant elk PrP was 2 orders of magnitude weaker, as indicated by both Western blotting and surface plasmon resonance. To investigate the molecular basis of these species- and conformer-dependent preferences of 3F4, the epitope was probed by peptide membrane array and antigen competition experiments. Remarkably, the 3F4 antibody did not react with MKHM but reacted strongly with KTNMK (corresponding to human PrP-(106–110)), a sequence that is also present in cervids, sheep, and cattle. 3F4 also reacted with elk PrP peptides containing KTNMKHV. We concluded that the minimal sequence for the 3F4 epitope consists of residues KTNMK, and the species- and conformer-dependent preferences of 3F4 arise largely from the interactions between Met112 (human PrP) or Val115 (cervid PrP) and adjacent residues. PMID:20194495

  20. HIV Control Is Mediated in Part by CD8+ T-Cell Targeting of Specific Epitopes

    PubMed Central

    Pereyra, Florencia; Heckerman, David; Carlson, Jonathan M.; Kadie, Carl; Soghoian, Damien Z.; Karel, Daniel; Goldenthal, Ariel; Davis, Oliver B.; DeZiel, Charles E.; Lin, Tienho; Peng, Jian; Piechocka, Alicja; Carrington, Mary

    2014-01-01

    ABSTRACT We investigated the hypothesis that the correlation between the class I HLA types of an individual and whether that individual spontaneously controls HIV-1 is mediated by the targeting of specific epitopes by CD8+ T cells. By measuring gamma interferon enzyme-linked immunosorbent spot (ELISPOT) assay responses to a panel of 257 optimally defined epitopes in 341 untreated HIV-infected persons, including persons who spontaneously control viremia, we found that the correlation between HLA types and control is mediated by the targeting of specific epitopes. Moreover, we performed a graphical model-based analysis that suggested that the targeting of specific epitopes is a cause of such control—that is, some epitopes are protective rather than merely associated with control—and identified eight epitopes that are significantly protective. In addition, we use an in silico analysis to identify protein regions where mutations are likely to affect the stability of a protein, and we found that the protective epitopes identified by the ELISPOT analysis correspond almost perfectly to such regions. This in silico analysis thus suggests a possible mechanism for control and could be used to identify protective epitopes that are not often targeted in natural infection but that may be potentially useful in a vaccine. Our analyses thus argue for the inclusion (and exclusion) of specific epitopes in an HIV vaccine. IMPORTANCE Some individuals naturally control HIV replication in the absence of antiretroviral therapy, and this ability to control is strongly correlated with the HLA class I alleles that they express. Here, in a large-scale experimental study, we provide evidence that this correlation is mediated largely by the targeting of specific CD8+ T-cell epitopes, and we identify eight epitopes that are likely to cause control. In addition, we provide an in silico analysis indicating that control occurs because mutations within these epitopes change the stability of the

  1. Towards conformal loop quantum gravity

    NASA Astrophysics Data System (ADS)

    H-T Wang, Charles

    2006-03-01

    A discussion is given of recent developments in canonical gravity that assimilates the conformal analysis of gravitational degrees of freedom. The work is motivated by the problem of time in quantum gravity and is carried out at the metric and the triad levels. At the metric level, it is shown that by extending the Arnowitt-Deser-Misner (ADM) phase space of general relativity (GR), a conformal form of geometrodynamics can be constructed. In addition to the Hamiltonian and Diffeomorphism constraints, an extra first class constraint is introduced to generate conformal transformations. This phase space consists of York's mean extrinsic curvature time, conformal three-metric and their momenta. At the triad level, the phase space of GR is further enlarged by incorporating spin-gauge as well as conformal symmetries. This leads to a canonical formulation of GR using a new set of real spin connection variables. The resulting gravitational constraints are first class, consisting of the Hamiltonian constraint and the canonical generators for spin-gauge and conformorphism transformations. The formulation has a remarkable feature of being parameter-free. Indeed, it is shown that a conformal parameter of the Barbero-Immirzi type can be absorbed by the conformal symmetry of the extended phase space. This gives rise to an alternative approach to loop quantum gravity that addresses both the conceptual problem of time and the technical problem of functional calculus in quantum gravity.

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

    PubMed Central

    Lu, Yudong; Li, Zhong; Teng, Huan; Xu, Hongke; Qi, Songnan; He, Jian’an; Gu, Dayong; Chen, Qijun; Ma, Hongwei

    2015-01-01

    Linear B-cell epitopes are ideal biomarkers for the serodiagnosis of infectious diseases. However, the long-predicted diagnostic value of epitopes has not been realized. Here, we demonstrated a method, diagnostic epitopes in four steps (DEIFS), that delivers a combination of epitopes for the serodiagnosis of infectious diseases with a high success rate. Using DEIFS for malaria, we identified 6 epitopes from 8 peptides and combined them into 3 chimeric peptide constructs. Along with 4 other peptides, we developed a rapid diagnostic test (RDT), which is able to differentiate Plasmodium falciparum (P. falciparum) from Plasmodium vivax (P. vivax) infections with 95.6% overall sensitivity and 99.1% overall specificity. In addition to applications in diagnosis, DEIFS could also be used in the diagnosis of virus and bacterium infections, discovery of vaccine candidates, evaluation of vaccine potency, and study of disease progression. PMID:26293607

  3. High-throughput epitope binning of therapeutic monoclonal antibodies: why you need to bin the fridge.

    PubMed

    Brooks, Benjamin D; Miles, Adam R; Abdiche, Yasmina N

    2014-08-01

    Analytical tools are evolving to meet the need for the higher-throughput characterization of therapeutic monoclonal antibodies. An antibody's epitope is arguably its most important property because it underpins its functional activity but, because epitope selection is innate, it remains an empirical process. Here, we focus on the emergence of label-free biosensors with throughput capabilities orders of magnitude higher than the previous state-of-the-art, which can facilitate large assays such as epitope binning so that they can be incorporated alongside functional activity screens, enabling the rapid identification of leads that exhibit unique and functional epitopes. In addition to streamlining the drug development process by saving time and cost, the information from epitope binning assays could provide the basis for intellectual property protection. PMID:24880105

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

    PubMed

    Lu, Yudong; Li, Zhong; Teng, Huan; Xu, Hongke; Qi, Songnan; He, Jian'an; Gu, Dayong; Chen, Qijun; Ma, Hongwei

    2015-01-01

    Linear B-cell epitopes are ideal biomarkers for the serodiagnosis of infectious diseases. However, the long-predicted diagnostic value of epitopes has not been realized. Here, we demonstrated a method, diagnostic epitopes in four steps (DEIFS), that delivers a combination of epitopes for the serodiagnosis of infectious diseases with a high success rate. Using DEIFS for malaria, we identified 6 epitopes from 8 peptides and combined them into 3 chimeric peptide constructs. Along with 4 other peptides, we developed a rapid diagnostic test (RDT), which is able to differentiate Plasmodium falciparum (P. falciparum) from Plasmodium vivax (P. vivax) infections with 95.6% overall sensitivity and 99.1% overall specificity. In addition to applications in diagnosis, DEIFS could also be used in the diagnosis of virus and bacterium infections, discovery of vaccine candidates, evaluation of vaccine potency, and study of disease progression. PMID:26293607

  5. Destructive processing by asparagine endopeptidase limits presentation of a dominant T cell epitope in MBP.

    PubMed

    Manoury, Bénédicte; Mazzeo, Daniela; Fugger, Lars; Viner, Nick; Ponsford, Mary; Streeter, Heather; Mazza, Graziella; Wraith, David C; Watts, Colin

    2002-02-01

    Little is known about the processing of putative human autoantigens and why tolerance is established to some T cell epitopes but not others. Here we show that a principal human HLA-DR2-restricted epitope--amino acids 85-99 of myelin basic protein, MBP(85-99)--contains a processing site for the cysteine protease asparagine endopeptidase (AEP). Presentation of this epitope by human antigen-presenting cells is inversely proportional to the amount of cellular AEP activity: inhibition of AEP in living cells greatly enhances presentation of the MBP(85-99) epitope, whereas overexpression of AEP diminishes presentation. These results indicate that central tolerance to this encephalitogenic MBP epitope may not be established because destructive processing limits its display in the thymus. Consistent with this hypothesis, AEP is expressed abundantly in thymic antigen-presenting cells. PMID:11812994

  6. Diosgenone: a second P21 polymorph

    PubMed Central

    Hernández Linares, María-Guadalupe; Guerrero-Luna, Gabriel; Bernès, Sylvain; Flores-Alamo, Marcos; Fernández-Herrera, María A.

    2012-01-01

    Diosgenone [(20S,22R,25R)-spirost-4-en-3-one, C27H40O3] has been proposed as a new therapeutic alternative for the treatment of malaria. The first X-ray structure report for diosgenone was by Piro et al. [(2002). Z. Naturforsch. Teil C, 57, 947–950] in the space group P21 (Z′ = 2). We now report a new polymorph in the same space group, with two mol­ecules in the asymmetric unit. Both mol­ecules have similar conformations, characterized by a skewed envelope A ring, which contains the C=C bond conjugated with the ketone functionality at C3. The dimorphism results from a modification of the relative orientation of the mol­ecules in the asymmetric unit: two independent mol­ecules were arranged anti­parallel in the Piro report, while they are parallel in the present determination. PMID:22904824

  7. The immune epitope database (IEDB) 3.0.

    PubMed

    Vita, Randi; Overton, James A; Greenbaum, Jason A; Ponomarenko, Julia; Clark, Jason D; Cantrell, Jason R; Wheeler, Daniel K; Gabbard, Joseph L; Hix, Deborah; Sette, Alessandro; Peters, Bjoern

    2015-01-01

    The IEDB, www.iedb.org, contains information on immune epitopes--the molecular targets of adaptive immune responses--curated from the published literature and submitted by National Institutes of Health funded epitope discovery efforts. From 2004 to 2012 the IEDB curation of journal articles published since 1960 has caught up to the present day, with >95% of relevant published literature manually curated amounting to more than 15,000 journal articles and more than 704,000 experiments to date. The revised curation target since 2012 has been to make recent research findings quickly available in the IEDB and thereby ensure that it continues to be an up-to-date resource. Having gathered a comprehensive dataset in the IEDB, a complete redesign of the query and reporting interface has been performed in the IEDB 3.0 release to improve how end users can access this information in an intuitive and biologically accurate manner. We here present this most recent release of the IEDB and describe the user testing procedures as well as the use of external ontologies that have enabled it. PMID:25300482

  8. T Cell Epitope Peptide Therapy for Allergic Diseases.

    PubMed

    O'Hehir, Robyn E; Prickett, Sara R; Rolland, Jennifer M

    2016-02-01

    Careful selection of dominant T cell epitope peptides of major allergens that display degeneracy for binding to a wide array of MHC class II molecules allows induction of clinical and immunological tolerance to allergen in a refined treatment strategy. From the original concept of peptide-induced T cell anergy arising from in vitro studies, proof-of-concept murine models and flourishing human trials followed. Current randomized, double-blind, placebo-controlled clinical trials of mixtures of T cell-reactive short allergen peptides or long contiguous overlapping peptides are encouraging with intradermal administration into non-inflamed skin a preferred delivery. Definitive immunological mechanisms are yet to be resolved but specific anergy, Th2 cell deletion, immune deviation, and Treg induction seem implicated. Significant efficacy, particularly with short treatment courses, in a range of aeroallergen therapies (cat, house dust mite, grass pollen) with inconsequential non-systemic adverse events likely heralds a new class of therapeutic for allergy, Synthetic Peptide Immuno-Regulatory Epitopes (SPIRE). PMID:26768622

  9. Improving wheat to remove coeliac epitopes but retain functionality

    PubMed Central

    Shewry, Peter R.; Tatham, Arthur S.

    2016-01-01

    Coeliac disease is an intolerance triggered by the ingestion of wheat gluten proteins. It is of increasing concern to consumers and health professionals as its incidence appears to be increasing. The amino acid sequences in gluten proteins that are responsible for triggering responses in sensitive individuals have been identified showing that they vary in distribution among and between different groups of gluten proteins. Conventional breeding may therefore be used to select for gluten protein fractions with lower contents of coeliac epitopes. Molecular breeding approaches can also be used to specifically down-regulate coeliac-toxic proteins or mutate coeliac epitopes within individual proteins. A combination of these approaches may therefore be used to develop a “coeliac-safe” wheat. However, this remains a formidable challenge due to the complex multigenic control of gluten protein composition. Furthermore, any modified wheats must retain acceptable properties for making bread and other processed foods. Not surprisingly, such coeliac-safe wheats have not yet been developed despite over a decade of research. PMID:26937068

  10. Logarithmic conformal field theory

    NASA Astrophysics Data System (ADS)

    Gainutdinov, Azat; Ridout, David; Runkel, Ingo

    2013-12-01

    Conformal field theory (CFT) has proven to be one of the richest and deepest subjects of modern theoretical and mathematical physics research, especially as regards statistical mechanics and string theory. It has also stimulated an enormous amount of activity in mathematics, shaping and building bridges between seemingly disparate fields through the study of vertex operator algebras, a (partial) axiomatisation of a chiral CFT. One can add to this that the successes of CFT, particularly when applied to statistical lattice models, have also served as an inspiration for mathematicians to develop entirely new fields: the Schramm-Loewner evolution and Smirnov's discrete complex analysis being notable examples. When the energy operator fails to be diagonalisable on the quantum state space, the CFT is said to be logarithmic. Consequently, a logarithmic CFT is one whose quantum space of states is constructed from a collection of representations which includes reducible but indecomposable ones. This qualifier arises because of the consequence that certain correlation functions will possess logarithmic singularities, something that contrasts with the familiar case of power law singularities. While such logarithmic singularities and reducible representations were noted by Rozansky and Saleur in their study of the U (1|1) Wess-Zumino-Witten model in 1992, the link between the non-diagonalisability of the energy operator and logarithmic singularities in correlators is usually ascribed to Gurarie's 1993 article (his paper also contains the first usage of the term 'logarithmic conformal field theory'). The class of CFTs that were under control at this time was quite small. In particular, an enormous amount of work from the statistical mechanics and string theory communities had produced a fairly detailed understanding of the (so-called) rational CFTs. However, physicists from both camps were well aware that applications from many diverse fields required significantly more

  11. Conservation of HIV-1 T cell epitopes across time and clades: validation of immunogenic HLA-A2 epitopes selected for the GAIA HIV vaccine.

    PubMed

    Levitz, Lauren; Koita, Ousmane A; Sangare, Kotou; Ardito, Matthew T; Boyle, Christine M; Rozehnal, John; Tounkara, Karamoko; Dao, Sounkalo M; Koné, Youssouf; Koty, Zoumana; Buus, Soren; Moise, Leonard; Martin, William D; De Groot, Anne S

    2012-12-14

    HIV genomic sequence variability has complicated efforts to generate an effective globally relevant vaccine. Regions of the viral genome conserved in sequence and across time may represent the "Achilles' heel" of HIV. In this study, highly conserved T-cell epitopes were selected using immunoinformatics tools combining HLA-A2 supertype binding predictions with relative global conservation. Analysis performed in 2002 on 10,803 HIV-1 sequences, and again in 2009, on 43,822 sequences, yielded 38 HLA-A2 epitopes. These epitopes were experimentally validated for HLA binding and immunogenicity with PBMCs from HIV-infected patients in Providence, Rhode Island, and/or Bamako, Mali. Thirty-five (92%) stimulated an IFNγ response in PBMCs from at least one subject. Eleven of fourteen peptides (79%) were confirmed as HLA-A2 epitopes in both locations. Validation of these HLA-A2 epitopes conserved across time, clades, and geography supports the hypothesis that such epitopes could provide effective coverage of virus diversity and would be appropriate for inclusion in a globally relevant HIV vaccine. PMID:23102976

  12. 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

    SciTech Connect

    Yu Hua; Jiang Lifang . E-mail: jianglf909@yahoo.com.cn; Fang Danyun; Yan Huijun; Zhou Jingjiao; Zhou Junmei; Liang Yu; Gao Yang; Zhao, Wei; Long Beiguo

    2007-03-15

    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.

  13. Conservation of HIV-1 T cell epitopes across time and clades: Validation of immunogenic HLA-A2 epitopes selected for the GAIA HIV vaccine

    PubMed Central

    Levitz, Lauren; Koita, Ousmane A.; Sangare, Kotou; Ardito, Matthew T.; Boyle, Christine M.; Rozehnal, John; Tounkara, Karamoko; Dao, Sounkalo M.; Koné, Youssouf; Koty, Zoumana; Buus, Soren; Moise, Leonard; Martin, William D.; De Groot, Anne S.

    2012-01-01

    HIV genomic sequence variability has complicated efforts to generate an effective globally relevant vaccine. Regions of the viral genome conserved in sequence and across time may represent the “Achilles’ heel” of HIV. In this study, highly conserved T-cell epitopes were selected using immunoinformatics tools combining HLA-A2 supertype binding predictions with relative global conservation. Analysis performed in 2002 on 10,803 HIV-1 sequences, and again in 2009, on 43,822 sequences, yielded 38 HLA-A2 epitopes. These epitopes were experimentally validated for HLA binding and immunogenicity with PBMCs from HIV-infected patients in Providence, Rhode Island, and/or Bamako, Mali. Thirty-five (92%) stimulated an IFNγ response in PBMCs from at least one subject. Eleven of fourteen peptides (79%) were confirmed as HLA-A2 epitopes in both locations. Validation of these HLA-A2 epitopes conserved across time, clades, and geography supports the hypothesis that such epitopes could provide effective coverage of virus diversity and would be appropriate for inclusion in a globally relevant HIV vaccine. PMID:23102976

  14. The presence of prolines in the flanking region of an immunodominant HIV‐2 gag epitope influences the quality and quantity of the epitope generated

    PubMed Central

    Leligdowicz, Aleksandra; Kramer, Holger B.; Onyango, Clayton; Cotten, Matthew; Wright, Cynthia; Whittle, Hilton C.; McMichael, Andrew; Dong, Tao; Kessler, Benedikt M.; Rowland‐Jones, Sarah L.

    2015-01-01

    Both the recognition of HIV‐infected cells and the immunogenicity of candidate CTL vaccines depend on the presentation of a peptide epitope at the cell surface, which in turn depends on intracellular antigen processing. Differential antigen processing maybe responsible for the differences in both the quality and the quantity of epitopes produced, influencing the immunodominance hierarchy of viral epitopes. Previously, we showed that the magnitude of the HIV‐2 gag‐specific T‐cell response is inversely correlated with plasma viral load, particularly when responses are directed against an epitope, 165DRFYKSLRA173, within the highly conserved Major Homology Region of gag‐p26. We also showed that the presence of three proline residues, at positions 119, 159 and 178 of gag‐p26, was significantly correlated with low viral load. Since this proline motif was also associated with stronger gag‐specific CTL responses, we investigated the impact of these prolines on proteasomal processing of the protective 165DRFYKSLRA173 epitope. Our data demonstrate that the 165DRFYKSLRA173 epitope is most efficiently processed from precursors that contain two flanking proline residues, found naturally in low viral‐load patients. Superior antigen processing and enhanced presentation may account for the link between infection with HIV‐2 encoding the “PPP‐gag” sequence and both strong gag‐specific CTL responses as well as lower viral load. PMID:26018465

  15. CONFORMANCE IMPROVEMENT USING GELS

    SciTech Connect

    Randall S. Seright

    2002-02-28

    This technical progress report describes work performed from June 20 through December 19, 2001, for the project, ''Conformance Improvement Using Gels''. Interest has increased in some new polymeric products that purport to substantially reduce permeability to water while causing minimum permeability reduction to oil. In view of this interest, we are currently studying BJ's Aqua Con. Results from six corefloods revealed that the Aqua Con gelant consistently reduced permeability to water more than that to oil. However, the magnitude of the disproportionate permeability reduction varied significantly for the various experiments. Thus, as with most materials tested to date, the issue of reproducibility and control of the disproportionate permeability remains to be resolved. Concern exists about the ability of gels to resist washout after placement in fractures. We examined whether a width constriction in the middle of a fracture would cause different gel washout behavior upstream versus downstream of the constriction. Tests were performed using a formed Cr(III)-acetate-HPAM gel in a 48-in.-long fracture with three sections of equal length, but with widths of 0.08-, 0.02-, and 0.08-in., respectively. The pressure gradients during gel extrusion (i.e., placement) were similar in the two 0.08-in.-wide fracture sections, even though they were separated by a 0.02-in.-wide fracture section. The constriction associated with the middle fracture section may have inhibited gel washout during the first pulse of brine injection after gel placement. However, during subsequent phases of brine injection, the constriction did not inhibit washout in the upstream fracture section any more than in the downstream section.

  16. Detection of newly antibody-defined epitopes on HLA class I alleles reacting with antibodies induced during pregnancy.

    PubMed

    Duquesnoy, R J; Hönger, G; Hösli, I; Marrari, M; Schaub, S

    2016-08-01

    The determination of HLA mismatch acceptability at the epitope level can be best performed with epitopes that have been verified experimentally with informative antibodies. The website-based International Registry of HLA Epitopes (http://www.epregistry.com.br) has a list of 81 antibody-verified HLA-ABC epitopes but more epitopes need to be added. Pregnancy offers an attractive model to study antibody responses to mismatched HLA epitopes which can be readily determined from the HLA types of child and mother. This report describes a HLAMatchmaker-based analysis of 16 postpregnancy sera tested in single HLA-ABC allele binding assays. Most sera reacted with alleles carrying epitopes that have been antibody-verified, and this study focused on the reactivity of additional alleles that share other epitopes corresponding to eplets and other amino acid residue configurations. This analysis led in the identification of 16 newly antibody-defined epitopes, seven are equivalent to eplets and nine correspond to combinations of eplets in combination with other nearby residue configurations. These epitopes will be added to the repertoire of antibody-verified epitopes in the HLA Epitope Registry. PMID:27312793

  17. Conformational changes of adsorbed proteins

    NASA Astrophysics Data System (ADS)

    Allen, Scott

    2005-03-01

    The adsorption of bovine serum albumin (BSA) and pepsin to gold surfaces has been studied using surface plasmon resonance (SPR). Proteins are adsorbed from solution onto a gold surface and changes in the conformation of the adsorbed proteins are induced by changing the buffer solution. We selected pH and ionic strength values for the buffer solutions that are known from our circular dichroism measurements to cause conformational changes of the proteins in bulk solution. We find that for both BSA and pepsin the changes in conformation are impeded by the interaction of the protein with the gold surface.

  18. Immunologically relevant strain polymorphism in the Amastigote Surface Protein 2 of Trypanosoma cruzi.

    PubMed

    Claser, Carla; Espíndola, Noeli Maria; Sasso, Gisela; Vaz, Adelaide José; Boscardin, Silvia B; Rodrigues, Mauricio M

    2007-07-01

    Several evidences suggest that the Amastigote Surface Protein-2 (ASP-2) of Trypanosoma cruzi is an important target for immunity during infection. Based on this, we considered it important to evaluate its strain polymorphism. Initially, we observed the presence of conserved cross-reactive epitopes in amastigotes of all parasite strains tested. In addition, the predicted amino acid sequences of the genes isolated from the cDNA of amastigotes of CL-Brener, Tulahuen, Colombian and G strains displayed a high degree of identity (>80%) to the previously described genes of ASP-2. Unexpectedly, Sylvio X10/4 and G strains expressed a new isoform of ASP-2 with limited identity to the previously described genes, but with a high degree of identity when compared to each other. Immunological studies confirmed the presence of cross-reactive epitopes between recombinant proteins representing the different isoforms of ASP-2. However, the genetic vaccination of mice with the new isoform of asp-2 gene expressed by the G strain failed to provide the same degree of protective immunity to a challenge by parasites of the Y strain as did asp-2 genes of Y or CL-Brener strains. In summary, we found that few strains can express different isoforms of ASP-2 which may not share cross-protective epitopes. PMID:17548222

  19. Identification and validation of T-cell epitopes in outer membrane protein (OMP) of Salmonella typhi.

    PubMed

    Tanu, Arifur Rahman; Ashraf, Mohammad Arif; Hossain, Md Faruk; Ismail, Md; Shekhar, Hossain Uddin

    2014-01-01

    This study aims to design epitope-based peptides for the utility of vaccine development by targeting outer membrane protein F (Omp F), because two available licensed vaccines, live oral Ty21a and injectable polysaccharide, are 50% to 80% protective with a higher rate of side effects. Conventional vaccines take longer time for development and have less differentiation power between vaccinated and infected cells. On the other hand, Peptide-based vaccines present few advantages over other vaccines, such as stability of peptide, ease to manufacture, better storage, avoidance of infectious agents during manufacture, and different molecules can be linked with peptides to enhance their immunogenicity. Omp F is highly conserved and facilitates attachment and fusion of Salmonella typhi with host cells. Using various databases and tools, immune parameters of conserved sequences from Omp F of different isolates of Salmonella typhi were tested to predict probable epitopes. Binding analysis of the peptides with MHC molecules, epitopes conservancy, population coverage, and linear B cell epitope prediction were analyzed. Among all those predicted peptides, ESYTDMAPY epitope interacted with six MHC alleles and it shows highest amount of interaction compared to others. The cumulative population coverage for these epitopes as vaccine candidates was approximately 70%. Structural analysis suggested that epitope ESYTDMAPY fitted well into the epitope-binding groove of HLA-C*12:03, as this HLA molecule was common which interact with each and every predicted epitopes. So, this potential epitope may be linked with other molecules to enhance its immunogenicity and used for vaccine development. PMID:25258481

  20. Identification and validation of T-cell epitopes in outer membrane protein (OMP) of Salmonella typhi

    PubMed Central

    Tanu, Arifur Rahman; Ashraf, Mohammad Arif; Hossain, Md Faruk; Ismail, Md; Shekhar, Hossain Uddin

    2014-01-01

    This study aims to design epitope-based peptides for the utility of vaccine development by targeting outer membrane protein F (Omp F), because two available licensed vaccines, live oral Ty21a and injectable polysaccharide, are 50% to 80% protective with a higher rate of side effects. Conventional vaccines take longer time for development and have less differentiation power between vaccinated and infected cells. On the other hand, Peptide-based vaccines present few advantages over other vaccines, such as stability of peptide, ease to manufacture, better storage, avoidance of infectious agents during manufacture, and different molecules can be linked with peptides to enhance their immunogenicity. Omp F is highly conserved and facilitates attachment and fusion of Salmonella typhi with host cells. Using various databases and tools, immune parameters of conserved sequences from Omp F of different isolates of Salmonella typhi were tested to predict probable epitopes. Binding analysis of the peptides with MHC molecules, epitopes conservancy, population coverage, and linear B cell epitope prediction were analyzed. Among all those predicted peptides, ESYTDMAPY epitope interacted with six MHC alleles and it shows highest amount of interaction compared to others. The cumulative population coverage for these epitopes as vaccine candidates was approximately 70%. Structural analysis suggested that epitope ESYTDMAPY fitted well into the epitope-binding groove of HLA-C*12:03, as this HLA molecule was common which interact with each and every predicted epitopes. So, this potential epitope may be linked with other molecules to enhance its immunogenicity and used for vaccine development. PMID:25258481

  1. Bioinformatic prediction of epitopes in the Emy162 antigen of Echinococcus multilocularis

    PubMed Central

    LI, YANHUA; LIU, XIANFEI; ZHU, YUEJIE; ZHOU, XIAOTAO; CAO, CHUNBAO; HU, XIAOAN; MA, HAIMEI; WEN, HAO; MA, XIUMIN; DING, JIAN-BING

    2013-01-01

    The aim of the present study was to predict the secondary structure and the T- and B-cell epitopes of the Echinococcus multilocularis Emy162 antigen, in order to reveal the dominant epitopes of the antigen. The secondary structure of the protein was analyzed using the Gamier-Robson method, and the improved self-optimized prediction method (SOPMA) server. The T- and B-cell epitopes of Emy162 were predicted using Immune Epitope Database (IEDB), Syfpeithi, Bcepred and ABCpred online software. The characteristics of hydrophilicity, flexibility, antigenic propensity and exposed surface area were predicted. The tertiary structure of the Emy162 protein was predicted by the 3DLigandSite server. The results demonstrated that random coils and β sheets accounted for 34.64 and 21.57% of the secondary structure of the Emy162 protein, respectively. This was indicative of the presence of potential dominant antigenic epitopes in Emy162. Following bioinformatic analysis, numerous distinct antigenic epitopes of Emy162 were identified. The high-scoring T-cell epitopes were located at positions 16–29, 36–39, 97–103, 119–125 and 128–135, whilst the likely B-cell epitopes were located at positions 8–10, 19–25, 44–50, 74–81, 87–93, 104–109 and 128–136. In conclusion, five T-cell and seven B-cell dominant epitopes of the Emy162 antigen were revealed by the bioinformatic methods, which may be of use in the development of a dominant epitope vaccine. PMID:24137185

  2. Recognition of Zinc Transporter 8 and MAP3865c Homologous Epitopes by Hashimoto's Thyroiditis Subjects from Sardinia: A Common Target with Type 1 Diabetes?

    PubMed Central

    Masala, Speranza; Cossu, Davide; Palermo, Mario; Sechi, Leonardo Antonio

    2014-01-01

    Mycobacterium avium subspecies paratuberculosis (MAP) asymptomatic infection has been previously linked to Type 1 diabetes (T1D) and Multiple Sclerosis. An association between MAP infection and Hashimoto's thyroiditis (HT) was also proposed only in a case report. This study aimed to investigate the robustness of the latter association, testing a large cohort of HT and healthy control (HCs) subjects, all from Sardinia. Prevalence of anti-MAP3865c Abs was assessed by indirect enzyme-linked immunosorbent assay (ELISA). Moreover, given that human ZnT8 is specifically expressed in the pancreatic β-cells, in the follicle epithelial cells and in the parafollicular cells of the thyroid gland, we also tested ZnT8 epitopes homologues to the MAP3865c immunodominant peptides previously identified. Indeed, Abs targeting MAP3865c and ZnT8 homologous regions display similar frequencies in patients and controls, thus suggesting that Abs recognizing these epitopes could be cross-reactive. A statistically significant difference was found between HT patients and HCs when analyzing the humoral response mounted against MAP3865c/ZnT8 homologues epitopes. To our knowledge, this is the first report, which provides statistically significant evidence sustaining the existence of an association between MAP sero-reactivity and HT. Further studies are required to investigate the relevance of MAP to HT, aimed at deciphering if this pathogen can be at play in triggering this autoimmune disease. Likewise, genetic polymorphism of the host, and other environmental factors need to be investigated. PMID:24830306

  3. Conformational Landscape of Nicotinoids: Solving the "conformational - Rity" of Anabasine

    NASA Astrophysics Data System (ADS)

    Lesarri, Alberto; Cocinero, Emilio J.; Evangelisti, Luca; Suenram, Richard D.; Caminati, Walther; Grabow, Jens-Uwe

    2010-06-01

    The conformational landscape of the alkaloid anabasine (neonicotine) has been investigated using rotational spectroscopy and ab initio calculations. The results allow a detailed comparison of the structural properties of the prototype piperidinic and pyrrolidinic nicotinoids (anabasine vs. nicotine). Anabasine adopts two most stable conformations in isolation conditions, for which we determined accurate rotational and nuclear quadrupole coupling parameters. The preferred conformations are characterized by an equatorial pyridine moiety and additional N-H equatorial stereochemistry at the piperidine ring (Eq-Eq). The two rings of anabasine are close to a bisecting arrangement, with the observed conformations differing in a ca. 180° rotation of the pyridine subunit, denoted either Syn or Anti. The preference of anabasine for the Eq-Eq-Syn conformation has been established by relative intensity measurements (Syn/Anti˜5(2)). The conformational preferences of free anabasine are directed by a N\\cdot\\cdot\\cdotH-C weak hydrogen bond interaction between the nitrogen lone pair at piperidine and the closest hydrogen bond in pyridine, with N\\cdot\\cdot\\cdotN distances ranging from 4.750 Å (Syn) to 4.233 Å (Anti). R. J. Lavrich, R. D. Suenram, D. F. Plusquellic and S. Davis, 58^th OSU Int. Symp. on Mol. Spectrosc., Columbus, OH, 2003, Comm. RH13.

  4. Inhibitory and blocking monoclonal antibody epitopes on merozoite surface protein 1 of the malaria parasite Plasmodium falciparum.

    PubMed

    Uthaipibull, C; Aufiero, B; Syed, S E; Hansen, B; Guevara Patiño, J A; Angov, E; Ling, I T; Fegeding, K; Morgan, W D; Ockenhouse, C; Birdsall, B; Feeney, J; Lyon, J A; Holder, A A

    2001-04-13

    Merozoite surface protein 1 (MSP-1) is a precursor to major antigens on the surface of Plasmodium spp. merozoites, which are involved in erythrocyte binding and invasion. MSP-1 is initially processed into smaller fragments; and at the time of erythrocyte invasion one of these of 42 kDa (MSP-1(42)) is subjected to a second processing, producing 33 kDa and 19 kDa fragments (MSP-1(33) and MSP-1(19)). Certain MSP-1-specific monoclonal antibodies (mAbs) react with conformational epitopes contained within the two epidermal growth factor domains that comprise MSP-1(19), and are classified as either inhibitory (inhibit processing of MSP-1(42) and erythrocyte invasion), blocking (block the binding and function of the inhibitory mAb), or neutral (neither inhibitory nor blocking). We have mapped the epitopes for inhibitory mAbs 12.8 and 12.10, and blocking mAbs such as 1E1 and 7.5 by using site-directed mutagenesis to change specific amino acid residues in MSP-1(19) and abolish antibody binding, and by using PEPSCAN to measure the reaction of the antibodies with every octapeptide within MSP-1(42). Twenty-six individual amino acid residue changes were made and the effect of each on the binding of mAbs was assessed by Western blotting and BIAcore analysis. Individual changes had either no effect, or reduced, or completely abolished the binding of individual mAbs. No two antibodies had an identical pattern of reactivity with the modified proteins. Using PEPSCAN each mAb reacted with a number of octapeptides, most of which were derived from within the first epidermal growth factor domain, although 1E1 also reacted with peptides spanning the processing site. When the single amino acid changes and the reactive peptides were mapped onto the three-dimensional structure of MSP-1(19), it was apparent that the epitopes for the mAbs could be defined more fully by using a combination of both mutagenesis and PEPSCAN than by either method alone, and differences in the fine specificity of

  5. Conformational nanobodies reveal tethered epidermal growth factor receptor involved in EGFR/ErbB2 predimers.

    PubMed

    Nevoltris, Damien; Lombard, Benjamin; Dupuis, Elodie; Mathis, Gérard; Chames, Patrick; Baty, Daniel

    2015-02-24

    The epidermal growth factor receptor (EGFR) is a cell-surface receptor with a single transmembrane domain and tyrosine kinase activity carried by the intracellular domain. This receptor is one of the four members of the ErbB family including ErbB2, ErbB3, and ErbB4. Ligand binding, like EGF binding, induces a conformational rearrangement of the receptor and induces a homo/hetero dimerization essentially with ErbB family receptors that leads to the phosphorylation of the kinase domain, triggering a signaling cascade. EGFR can also form inactive dimers in a ligand-independent way through interactions between cytoplasmic domains. To date, the conformation of EGFR extracellular domain engaged in these inactive dimers remains unclear. In this study, we describe the successful selection and characterization of llama anti-EGFR nanobodies and their use as innovative conformational sensors. We isolated three different specific anti-EGFR clones binding to three distinct epitopes. Interestingly, the binding of all three nanobodies was found highly sensitive to ligand stimulation. Two nanobodies, D10 and E10, can only bind the ligand-free EGFR conformation characterized by an intramolecular tether between domains II and IV, whereas nanobody G10 binds both ligand-free and ligand activated EGFR, with an 8-fold higher affinity for the extended conformation in the presence of ligand. Here we took advantage of these conformational probes to reveal the existence of tethered EGFR in EGFR/ErbB2 predimers. These biosensors represent important tools allowing the determination of EGFR conformations and should help the design of relevant inhibitors. PMID:25603171

  6. Identification of the Rheumatoid Arthritis Shared Epitope Binding Site on Calreticulin

    PubMed Central

    Ling, Song; Cheng, Andrew; Pumpens, Paul; Michalak, Marek; Holoshitz, Joseph

    2010-01-01

    Background The rheumatoid arthritis (RA) shared epitope (SE), a major risk factor for severe disease, is a five amino acid motif in the third allelic hypervariable region of the HLA-DRβ chain. The molecular mechanisms by which the SE affects susceptibility to – and severity of - RA are unknown. We have recently demonstrated that the SE acts as a ligand that interacts with cell surface calreticulin (CRT) and activates innate immune signaling. In order to better understand the molecular basis of SE-RA association, here we have undertaken to map the SE binding site on CRT. Principal Findings Surface plasmon resonance (SPR) experiments with domain deletion mutants suggested that the SE binding site is located in the P-domain of CRT. The role of this domain as a SE-binding region was further confirmed by a sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azido-benzamido) hexanoamido] ethyl-1,3-dithiopropionate (sulfo-SBED) photoactive cross-linking method. In silico analysis of docking interactions between a conformationally intact SE ligand and the CRT P-domain predicted the region within amino acid residues 217–224 as a potential SE binding site. Site-directed mutagenesis demonstrated involvement of residues Glu217 and Glu223 - and to a lesser extent residue Asp220 - in cell-free SPR-based binding and signal transduction assays. Significance We have characterized here the molecular basis of a novel ligand-receptor interaction between the SE and CRT. The interaction represents a structurally and functionally well-defined example of cross talk between the adaptive and innate immune systems that could advance our understanding of the pathogenesis of autoimmunity. PMID:20661469

  7. Logarithmic conformal field theory

    NASA Astrophysics Data System (ADS)

    Gainutdinov, Azat; Ridout, David; Runkel, Ingo

    2013-12-01

    Conformal field theory (CFT) has proven to be one of the richest and deepest subjects of modern theoretical and mathematical physics research, especially as regards statistical mechanics and string theory. It has also stimulated an enormous amount of activity in mathematics, shaping and building bridges between seemingly disparate fields through the study of vertex operator algebras, a (partial) axiomatisation of a chiral CFT. One can add to this that the successes of CFT, particularly when applied to statistical lattice models, have also served as an inspiration for mathematicians to develop entirely new fields: the Schramm-Loewner evolution and Smirnov's discrete complex analysis being notable examples. When the energy operator fails to be diagonalisable on the quantum state space, the CFT is said to be logarithmic. Consequently, a logarithmic CFT is one whose quantum space of states is constructed from a collection of representations which includes reducible but indecomposable ones. This qualifier arises because of the consequence that certain correlation functions will possess logarithmic singularities, something that contrasts with the familiar case of power law singularities. While such logarithmic singularities and reducible representations were noted by Rozansky and Saleur in their study of the U (1|1) Wess-Zumino-Witten model in 1992, the link between the non-diagonalisability of the energy operator and logarithmic singularities in correlators is usually ascribed to Gurarie's 1993 article (his