141,028 research outputs found
Immobilization by surface conjugation of cyclic peptides for effective mimicry of the HCV-envelope E2 protein as a strategy toward synthetic vaccines
Mimicry of the binding interface of antibody-antigen interactions using peptide-based modulators (i.e. epitope mimics) has promising applications for vaccine design. These epitope mimics can be synthesized in a streamlined and straightforward fashion, thereby allowing for high-throughput analysis. The design of epitope mimics is highly influenced by their spatial configuration and structural conformation. It is widely assumed that for proper mimicry sufficient conformational constraints have to be implemented. This paper describes the synthesis of bromide derivatives functional-ized with a flexible TEG linker equipped with a thiol-moiety that could be used to support cyclic or linear peptides. The cyclic and linear epitope mimics were covalently conjugated via the free thiol-moiety on maleimide-activated plate sur-faces. The resulting covalent, uniform, and oriented coated surface of cyclic or linear epitope mimics were subjected to an ELISA to investigate the effect of peptide cyclization with respect to mimicry of an antigen-antibody interaction of the HCV E2 glycoprotein. To our knowledge, the benefit of cyclized peptides over linear peptides has been clearly demon-strated here for the first time. Cyclic epitope mimics, and not the linear epitope mimics, demonstrated specificity towards their monoclonal antibodies HC84.1 and V3.2, respectively. The described strategy for the construction of epitope mimics shows potential for high-throughput screening of key-binding residues by simply changing the amino-acid sequences within synthetic peptides. In this way, leucine-438 has been identified as a key-binding residue for binding monoclonal antibody V3.2
CD4+ T-cell responses to Epstein-Barr virus (EBV) latent-cycle antigens and the recognition of EBV-transformed lymphoblastoid cell lines
There is considerable interest in the potential of Epstein-Barr virus (EBV) latent antigen-specific CD4 T cells to act as direct effectors controlling EBV-induced B lymphoproliferations. Such activity would require direct CD4 T-cell recognition of latently infected cells through epitopes derived from endogenously expressed viral proteins and presented on the target cell surface in association with HLA class II molecules. It is therefore important to know how often these conditions are met. Here we provide CD4 epitope maps for four EBV nuclear antigens, EBNA1, -2, -3A, and -3C, and establish CD4 T-cell clones against 12 representative epitopes. For each epitope we identify the relevant HLA class II restricting allele and determine the efficiency with which epitope-specific effectors recognize the autologous EBV-transformed B-lymphoblastoid cell line (LCL). The level of recognition measured by gamma interferon release was consistent among clones to the same epitope but varied between epitopes, with values ranging from 0 to 35% of the maximum seen against the epitope peptide-loaded LCL. These epitope-specific differences, also apparent in short-term cytotoxicity and longer-term outgrowth assays on LCL targets, did not relate to the identity of the source antigen and could not be explained by the different functional avidities of the CD4 clones; rather, they appeared to reflect different levels of epitope display at the LCL surface. Thus, while CD4 T-cell responses are detectable against many epitopes in EBV latent proteins, only a minority of these responses are likely to have therapeutic potential as effectors directly recognizing latently infected target cells
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Determinant of HIV-1 mutational escape from cytotoxic T lymphocytes.
CD8+ class I-restricted cytotoxic T lymphocytes (CTLs) usually incompletely suppress HIV-1 in vivo, and while analogous partial suppression induces antiretroviral drug-resistance mutations, epitope escape mutations are inconsistently observed. However, escape mutation depends on the net balance of selective pressure and mutational fitness costs, which are poorly understood and difficult to study in vivo. Here we used a controlled in vitro system to evaluate the ability of HIV-1 to escape from CTL clones, finding that virus replicating under selective pressure rapidly can develop phenotypic resistance associated with genotypic changes. Escape varied between clones recognizing the same Gag epitope or different Gag and RT epitopes, indicating the influence of the T cell receptor on pressure and fitness costs. Gag and RT escape mutations were monoclonal intra-epitope substitutions, indicating limitation by fitness constraints in structural proteins. In contrast, escape from Nef-specific CTL was more rapid and consistent, marked by a polyclonal mixture of epitope point mutations and upstream frameshifts. We conclude that incomplete viral suppression by CTL can result in rapid emergence of immune escape, but the likelihood is strongly determined by factors influencing the fitness costs of the particular epitope targeted and the ability of responding CTL to recognize specific epitope variants
Review of Immunoinformatic approaches to in-silico B-cell epitope prediction
In this paper, the current state of in-silico, B-cell epitope prediction is discussed. Recommendations for improving some of the approaches encountered are outlined, along with the presentation of an entirely novel technique, which uses molecular mechanics for epitope classification, evaluation and prediction
A biophysical model of cell adhesion mediated by immunoadhesin drugs and antibodies
A promising direction in drug development is to exploit the ability of
natural killer cells to kill antibody-labeled target cells. Monoclonal
antibodies and drugs designed to elicit this effect typically bind cell-surface
epitopes that are overexpressed on target cells but also present on other
cells. Thus it is important to understand adhesion of cells by antibodies and
similar molecules. We present an equilibrium model of such adhesion,
incorporating heterogeneity in target cell epitope density and epitope
immobility. We compare with experiments on the adhesion of Jurkat T cells to
bilayers containing the relevant natural killer cell receptor, with adhesion
mediated by the drug alefacept. We show that a model in which all target cell
epitopes are mobile and available is inconsistent with the data, suggesting
that more complex mechanisms are at work. We hypothesize that the immobile
epitope fraction may change with cell adhesion, and we find that such a model
is more consistent with the data. We also quantitatively describe the parameter
space in which binding occurs. Our results point toward mechanisms relating
epitope immobility to cell adhesion and offer insight into the activity of an
important class of drugs.Comment: 13 pages, 5 figure
Selection of neutralizing antibody escape mutants with type A influenza virus HA-specific polyclonal antisera: possible significance for antigenic drift
Ten antisera were produced in rabbits by two or three intravenous injections of inactivated whole influenza type A virions. All contained haemagglutination-inhibition (HI) antibody directed predominantly to an epitope in antigenic site B and, in addition, various amounts of antibodies to an epitope in site A and in site D. The ability of untreated antisera to select neutralization escape mutants was investigated by incubating virus possessing the homologous haemagglutinin with antiserum adjusted to contain anti-B epitope HI titres of 100, 1000 and 10000 HIU/ml. Virus-antiserum mixtures were inoculated into embryonated hen's eggs, and progeny virus examined without further selection. Forty percent of the antisera at a titre of 1000 HIU/ml selected neutralizing antibody escape mutants as defined by their lack of reactivity to Mab HC10 (site B), and unchanged reactivity to other Mabs to site A and site D epitopes. All escape mutant-selecting antisera had a ratio of anti-site B (HC10)-epitope antibody[ratio]other antibodies of [gt-or-equal, slanted]2·0[ratio]1. The antiserum with the highest ratio (7·4[ratio]1) selected escape mutants in all eggs tested in four different experiments. No antiserum used at a titre of 10000 HIU/ml allowed multiplication of any virus. All antisera used at a titre of 100 HIU/ml permitted virus growth, but this was wild-type (wt) virus. We conclude that a predominant epitope-specific antibody response, a titre of [gt-or-equal, slanted]1000 HIU/ml, and a low absolute titre of other antibodies ([less-than-or-eq, slant]500 HIU/ml) are three requirements for the selection of escape mutants. None of the antisera in this study could have selected escape mutants without an appropriate dilution factor, so the occurrence of an escape mutant-selecting antiserum in nature is likely to be a rare event
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