Large-scale computational identification of HIV T-cell epitopes

Bioinformatics-driven T-cell epitope-identification methods can enhance vaccine target selection significantly. We evaluated three unrelated computational methods to screen Pol, Gag and Env sequences extracted from the Los Alamos HIV database for HLA-A*0201 and HLA-B*3501 T-cell epitope candidates. The hidden Markov model predicted 389 HLA-B*3501-restricted candidates from 374 HIV-1 and 97 HIV-2 sequences. The artificial neural network (ANN) model, and Bioinformatics and Molecular Analysis Section (BIMAS) quantitative matrix predictions for A*0201 yielded 1122 HIV-1 and 548 HIV-2 candidates. The overall sequence coverage of the predicted A*0201 T-cell epitopes was 2.7% (HIV-1)and 3.0% (HIV-2). HLA-B*3501-predicted epitopes covered 0.9% (HIV-1) and 1.4% (HIV-2) of the total sequence. Comparison of 890 ANN- and 397 BIMAS-derived HIV-1 A*0201- restricted epitope candidates showed that only 13-19% of the predicted and 26% of the experimentally confirmed T-cell epitopes were captured by both methods. Extrapolating these results, we estimated that at least 247 predicted HIV-1 epitopes are yet to be discovered as active A*0201-restricted T-cell epitopes. Adequate comparison and combined usage of various predictive bioinformatics methods, rather than uncritical use of any single prediction method, will enable cost-effective and efficient T-cell epitope screening.     

Rapid determination of antigenic epitopes in human NGAL using NMR

The recent remarkable rise in biomedical applications of antibodies and their recombinant constructs has shifted the interest in determination of antigenic epitopes in target proteins from the areas of protein science and molecular immunology to the vast fields of modern biotechnology. In this article, we demonstrated that measuring binding induced changes in two‐dimensional NMR spectra enables rapid determination of antibody binding footprints on target protein antigens. Such epitopes recognized by six high‐affinity monoclonal murine antibodies (mAbs) against human neutrophil gelatinase‐associated lipocalin (NGAL) were determined by measuring chemical shifts or broadening of peaks in ¹H‐¹⁵N‐TROSY HSQC and ¹H‐¹³C HSQC spectra of isotope‐labeled NGAL occurring upon its binding to the antibodies. Locations of the epitopes defined by the NMR studies are in good agreement with the results of antibody binding pairing observed by dual‐color fluorescence cross‐correlation spectroscopy. In all six cases, the antibodies recognize conformational epitopes in regions of relatively rigid structure on the protein. None of the antibodies interact with the more flexible funnel‐like opening of the NGAL calyx. All determined epitope areas in NGAL reflect the dimensions of respective antibody binding surface (paratopes) and contain amino acid residues that provide strong interactions. This NMR‐based approach offers comprehensive information on antigenic epitopes and can be applied to numerous protein targets of diagnostic or therapeutic interest. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 657–667, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Coupling of inflammatory cytokine signaling pathways probed by measurements of extracellular acidification rate

There is a growing interest in the mechanisms of how cells integrate the multitude of signals that emanate during inflammatory stimuli, such as the hepatic acute phase response to burn or trauma. We have used measurements of extracellular acidification rate (ECAR) of HepG2 cells cultured on microporous membranes to probe the coupling between signaling pathways for gp130 family cytokines (interleukin-6, oncostatin M) and IL-1, each of which is considered to play a significant role in the hepatic acute phase response. We found that brief (30 min or less) exposure to any of these cytokines desensitized the HepG2 cells to subsequent exposure with the same cytokine. Furthermore, we found that this property serves as a probe of the coupling of signaling pathways: exposure to IL-1 did not desensitize the cells to exposure to OSM and vice versa. However, cells exposed to IL-6 with soluble gp80, which together share with OSM the use of gp130 as a signal transducing receptor, were subsequently unable to respond to OSM, and vice versa. Simultaneous exposure of cells to moderate concentrations (near their respective EC50 values) of both IL-1 and OSM resulted in synergistic effects on the ECAR, but simultaneous exposure to saturating concentrations of IL-1 and OSM resulted in a response that tracked that of OSM alone. These results suggest that the signaling pathways of IL-1 and OSM may be simultaneously activated in HepG2 cells under moderate inflammatory cytokine challenge but that the cells must prioritize their response under extreme cytokine challenges.     

Rapid extracellular acidification induced by glucose metabolism in non-proliferating cells of Serratia marcescens.

The addition of glucose or other sugars to resting cells of Serratia maurcescens induced rapid acidification of the extracellular medium. This acidification was due to the catabolism of sugars. The rate of acidification depended on the carbon source and its concentration. HPLC analysis of the supernatants demonstrated that the progressive fall in pH resulted from the rapid production of lactic, acetic, pyruvic and citric acids. Other microorganisms were tested for their ability to produce this rapid acidification of the medium. This study may provide a rapid and simple method for metabolism studies.     

Distinct epitopes recognized by I-Ad-restricted T-cell clones within antigenic site E on influenza virus hemagglutinin.

A total of 14 I-Ad-restricted helper T-cell clones specific for the hemagglutinin (HA) molecule of influenza virus were isolated from spleens of BALB/c or (BALB/c X C57BL/10)F1 mice immunized with the H3 subtype influenza virus A/Memphis/71 (Mem 71) and from lymph nodes of BALB/c mice primed with purified HA. The specificity of these T-cell clones was assessed in proliferation assays by reactivity with naturally occurring strains of viruses that arose by antigenic drift and contain known amino acid sequence changes in HA and with a panel of monoclonal antibody (MAb)-selected mutants of Mem 71 with single amino acid substitutions in HA. The HA genes of those mutant viruses that failed to stimulate one or more of the T-cell clones were sequenced. The clones could be allocated to at least four groups, each group having a distinct pattern of reactivity with the panel of natural field strains. The epitopes recognized by the four groups of clones were found, by reactivity with MAb-selected mutants, to be in very close proximity to one another and probably overlapping. All of the distinct epitopes recognized by the T-cell clones were adversely affected by a single amino acid substitution, either at residue 60 or at residue 63 in the HA1 polypeptide chain, within the region known from antibody-binding studies as site E. Some, but not all, of the epitopes may be influenced by the addition of a carbohydrate side chain to the HA of a particular MAb-selected mutant and certain field strains containing an Asp----Asn substitution at residue 63. Site E is therefore a major site of H-2d helper T-cell recognition on the H3 HA.     

Expression of extracellular matrix genes by cultured human cells: localization of messenger RNAs and antigenic epitopes

Simplified and expedient methodologies for examination of cellular gene expression at the mRNA and protein levels, utilizing in situ hybridization and peroxidase-anti-peroxidase immunodetection, were developed. These techniques were first optimized for the detection of extracellular matrix genes expressed by cultured human skin fibroblasts and keratinocytes, the two principal cell types of human skin. In situ hybridizations and Northern transfer analyses with human-sequence-specific cDNAs encoding collagenous and noncollagenous protein sequences demonstrated selective expression of different matrix genes by these two cell types, indicating different biosynthetic capacities of these cells and attesting to the specificity of the hybridizations. The utility of in situ hybridization was also demonstrated in mixed primary cell cultures established from cutaneous neurofibromas consisting of Schwann cells, perineurial cells, and fibroblasts. The methodologies developed here were further utilized for simultaneous detection of fibronectin mRNA and immunoreactive protein in fibroblast cultures. This procedure allowed detection of grains representative of radioactively labeled cDNA-mRNA hybrids and protein epitopes, as visualized by peroxidase-anti-peroxidase immunodetection on the same cells. This methodology, with appropriate modifications, may be applicable to other cell types as well as tissue specimens.     

Analysis of the human T-cell response to picornaviruses: identification of T-cell epitopes close to B-cell epitopes in poliovirus.

Little is known about the nature and specificity of T-cell-mediated responses to picornaviruses in humans. In this study, the nature of the T-cell response to seven picornaviruses, including polioviruses, coxsackieviruses B3 and B4, human rhinovirus 14, and encephalomyocarditis virus, was determined. Twenty-nine individuals responded to poliovirus type 3, coxsackievirus B3, and encephalomyocarditis virus by proliferation of T cells, and from such cultures, 130 virus-specific T-cell lines were established. T-cell lines generated in response to encephalomyocarditis virus were exclusively strain specific. However, the majority of T-cell lines established in response to viruses, other than encephalomyocarditis virus, were cross-reactive to each other. Their cross-reactivity was confirmed in 2 of the 30 picornavirus-specific clonally derived T-cell lines from two subjects, but the majority of these lines were serotype specific. T-cell epitopes adjacent to each of the B-cell antigenic sites in VP1 of poliovirus type 3 were identified. The response to the region adjacent to B-cell antigenic site 1 (residues 97 to 114) was dominant between individuals. The localization of this major CD4 T-cell epitope may permit the construction of chimeric viruses utilizing the natural picornavirus T-cell response to augment production of antibody specific for inserted sequences.     

HCV抗原表位预测

应用网络生物信息资源查找丙型肝炎病毒基因组全序列,用软件Lasergene中的EditSeq将来自中国河北株mRNA序列翻译为氨基酸序列,尔后用程序Protean进行氨基酸序列分析,对HCV各区段的B细胞抗原指数进行预测。同时又在两个网站对中国汉族人中频率较高的HLA基因型进行CD8和CD4T细胞表位预测。B细胞和T细胞抗原表位预测结果对于HCV诊断试剂和疫苗研制有重要的指导意义。     

On-line identification of P-glycoprotein substrates by monitoring of extracellular acidification and respiration rates in living cells

The influence of P-glycoprotein (ABCB1) in drug resistance as well as drug absorption and disposition is an important factor to be considered during the development of new drugs. Thus, the early identification and exclusion of compounds showing a high affinity towards P-glycoprotein can help to select drug candidates. The aim of our study was to implement a label-free assay for the identification of P-glycoprotein substrates in living cells. For this approach, a multiparametric, chip-based sensor system was used to determine extracellular acidification, cell respiration and adhesion upon stimulation with P-glycoprotein substrates. Using L-MDR1 cells, a human P-glycoprotein overexpressing cell line, the influence of P-glycoprotein activity was determined for seven different compounds, demonstrating the applicability of the system for P-glycoprotein substrate identification. Effects were concentration dependent, as shown for the P-glycoprotein substrate verapamil, and were associated with cellular acidification and respiration. P-glycoprotein ATPase activation by verapamil could be described by a Michaelis-Menten type kinetic profile showing saturation at high substrate concentrations. The Michaelis-Menten constants K(M) were determined to be 0.92μM (calculated based on extracellular acidification) and 4.9μM (calculated based on cellular respiration). Control experiments using 100nM of the P-glycoprotein inhibitor elacridar indicated that the observed effects were related to P-glycoprotein ATPase activity. In contrast, wild-type LLC-PK1 cells not expressing P-glycoprotein were not responsive towards stimulation with different P-glycoprotein substrates. Summarizing these findings, the used microsensor system is a generic system suitable for the identification of P-glycoprotein substrates. In contrast to biochemical P-glycoprotein assays, activation of the drug efflux pump can be monitored on-line in living cells to identify P-glycoprotein substrates and to study the molecular mechanisms of adenosintriphosphate-dependent active transport.     

A strategy for the identification of T-cell epitopes on Leishmania cysteine proteinases

In this study computational analysis was used to compile sequence alignments, construct a dendrogram and calculate physical data in order to predict potential T-cell epitopes of the Leishmania cysteine proteinase. Using multiple alignment of human and Leishmania proteinase sequences deposited on data bank sequences, it was possible to predict that the extreme C-terminus of cysteine proteinase (Cyspep, 355-444) contained three peptides (pI 361-370, pII 415-422 and pIII 431-444) with charge score, hydrophobicity and isoelectric points compatible for human leucocyte-associated antigen (HLA) class II binding. The prediction was confirmed in vitro through the ability of synthetic peptides corresponding to the predicted regions to stimulate peripheral blood mononuclear cells of patients with leishmaniasis.     

Patterns of predicted T-cell epitopes associated with antigenic drift in influenza H3N2 hemagglutinin

Antigenic drift allowing escape from neutralizing antibodies is an important feature of transmission and survival of influenza viruses in host populations. Antigenic drift has been studied in particular detail for influenza A H3N2 and well defined antigenic clusters of this virus documented. We examine how host immunogenetics contributes to determination of the antibody spectrum, and hence the immune pressure bringing about antigenic drift. Using uTOPE™ bioinformatics analysis of predicted MHC binding, based on amino acid physical property principal components, we examined the binding affinity of all 9-mer and 15-mer peptides within the hemagglutinin 1 (HA1) of 447 H3N2 virus isolates to 35 MHC-I and 14 MHC-II alleles. We provide a comprehensive map of predicted MHC-I and MHC-II binding affinity for a broad array of HLA alleles for the H3N2 influenza HA1 protein. Each HLA allele exhibited a characteristic predicted binding pattern. Cluster analysis for each HLA allele shows that patterns based on predicted MHC binding mirror those described based on antibody binding. A single amino acid mutation or position displacement can result in a marked difference in MHC binding and hence potential T-helper function. We assessed the impact of individual amino acid changes in HA1 sequences between 10 virus isolates from 1968–2002, representative of antigenic clusters, to understand the changes in MHC binding over time. Gain and loss of predicted high affinity MHC-II binding sites with cluster transitions were documented. Predicted high affinity MHC-II binding sites were adjacent to antibody binding sites. We conclude that host MHC diversity may have a major determinant role in the antigenic drift of influenza A H3N2.     

Regulation of inflammation by extracellular acidification and proton-sensing GPCRs

Under ischemic and inflammatory circumstances, such as allergic airway asthma, rheumatoid arthritis, atherosclerosis, and tumors, extracellular acidification occurs due to the stimulation of anaerobic glycolysis. An acidic microenvironment has been shown to modulate pro-inflammatory or anti-inflammatory responses, including cyclooxygenase-2 (COX-2) expression, prostaglandin synthesis, and cytokine expression, in a variety of cell types, and thereby to exacerbate or ameliorate inflammation. However, molecular mechanisms underlying extracellular acidic pH-induced actions have not been fully understood. Recent studies have shown that ovarian cancer G protein-coupled receptor 1 (OGR1)-family G protein-coupled receptors (GPCRs) can sense extracellular pH or protons, which in turn stimulates intracellular signaling pathways and subsequent diverse cellular responses. In the present review, I discuss extracellular acidic pH-induced inflammatory responses and related responses in inflammatory cells, such as macrophages and neutrophils, and non-inflammatory cells, such as smooth muscle cells and endothelial cells, focusing especially on proton-sensing GPCRs.     

Prediction of antigenic epitopes on protein surfaces by consensus scoring

Background  

Prediction of antigenic epitopes on protein surfaces is important for vaccine design. Most existing epitope prediction methods focus on protein sequences to predict continuous epitopes linear in sequence. Only a few structure-based epitope prediction algorithms are available and they have not yet shown satisfying performance.     

Cell orientation induced by extracellular signals

Cells like fibroblasts and osteoblasts are oriented by different extracellular guiding signals like an electric field, a bent surface, and a periodically stretched surface. An automatic controller is responsible for the cell alignment. The controller contains both a deterministic and a stochastic signal. The following machine properties were determined: (1) The angle dependence of the cellular signal transformer is cos 2(psi 0 - psi). (2) The set point of the automatic controller is psi 0 = +/- 90 degrees. The cells like to orient their long axis perpendicular to the direction of the applied guiding signal. (3) The signal transformer measures the extracellular signal in a quadratic fashion. The cells cannot register the sign of the guiding field. (4) The stochastic signal in the automatic controller can be quantified by a characteristic time (approximately 130 min for fibroblasts). (5) The extracellular signal is registered in cell-made standards (ratio of the deterministic and stochastic signal equals one): 0.3 +/- 0.05 V/mm for human fibroblasts (electric field) and 85 +/- 3 microns for human fibroblasts and osteoblasts (cyclindrically bent surface). (6) The lag-time in the signal transduction system of fibroblasts is approximately 4 min.     

Regulation of Par6 by extracellular signals

The critical regulator of polarity, Par6, is a key member of a multi-component polarity complex that controls a variety of cellular processes such as asymmetric cell division, establishment of epithelial apico-basal polarity, and polarized cell migration. Recently, we have come to understand how regulation of the Par6 interactome by extracellular cues such as integrin and transforming growth factor beta signalling regulates cell motility and tight junction dissolution. These studies have begun to elucidate how signalling to the polarity complex might regulate pathological processes such as tumour cell invasion and metastasis.     

Location of antigenic epitopes on antibody molecules

Using X-ray crystallographic co-ordinates of immunoglobulins, surface regions accessible to a large spherical probe, comparable in size to an antibody domain, were computed. Locations of these exposed regions were compared with those of experimentally determined antigenic sites, i.e. idiotypic, allotypic and isotypic serological markers. In all cases, an excellent agreement was found. The most prominent computed epitopes correspond to convex parts of antibody surface made by reverse turn segments of the polypeptide chain. The computed epitopes occur in homologous positions in all the immunoglobulin domains, and most of the beta-sheet surfaces on the domains are poorly antigenic. The CH2 domain (Fc fragment) has many more antigenic sites than the Fab fragments (antigen-binding fragments). Variable domain epitopes (idiotypes) involve both hypervariable and framework residues, and only about 25% of the hypervariable residues are strongly antigenic. The results indicate that, in a vertebrate body, each antibody molecule may be recognized, and its concentration regulated, by at least 40 complementary anti-immunoglobulin antibodies; therefore, a possibility of an "immune network" with much higher connectivity than is generally assumed should be seriously contemplated.     

Inhibition of superoxide anion production by extracellular acidification in neutrophils

Extracellular acidification inhibited formyl-Met-Leu-Phe- or C5a-induced superoxide anion (O₂⁻) production in differentiated HL-60 neutrophil-like cells and human neutrophils. A cAMP-increasing agonist, prostaglandin E₁, also inhibited the formyl peptide-induced O₂⁻ production. The inhibitory action on the O₂⁻ production by extracellular acidic pH was associated with cAMP accumulation and partly attenuated by H89, a protein kinase A inhibitor. A significant amount of mRNAs for T-cell death-associated gene 8 (TDAG8) and other proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1)-family receptors is expressed in these cells. These results suggest that cAMP/protein kinase A, possibly through proton-sensing G-protein-coupled receptors, may be involved in extracellular acidic pH-induced inhibition of O₂⁻ production.     

Regulation of the epithelial Na(+) channel by extracellular acidification

The effect of extracellular acidification wastested on the native epithelial Na⁺ channel (ENaC) in A6epithelia and on the cloned ENaC expressed in Xenopusoocytes. Channel activity was determined utilizing blocker-inducedfluctuation analysis in A6 epithelia and dual electrode voltage clampin oocytes. In A6 cells, a decrease of extracellular pH(pH_o) from 7.4 to 6.4 caused a slow stimulation of theamiloride-sensitive short-circuit current (I_Na)by 68.4 ± 11% (n = 9) at 60 min. This increaseof I_Na was attributed to an increase of openchannel and total channel (N_T) densities. Similar changes were observed with pH_o 5.4. The effects ofpH_o were blocked by buffering intracellularCa²⁺ with 5 µM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Inoocytes, pH_o 6.4 elicited a small transient increase of theslope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min)followed by a decrease to 83.7 ± 11.7% of control at 60 min (n = 6). Thus small decreases of pH_ostimulate the native ENaC by increasing N_T butdo not appreciably affect ENaC expressed in Xenopus oocytes.These effects are distinct from those observed with decreasingintracellular pH with permeant buffers that are known to inhibit ENaC.

     

Poliovirus-specific major histocompatibility complex class I-restricted cytolytic T-cell epitopes in mice localize to neutralizing antigenic regions.

A major histocompatibility complex (MHC) class I-restricted cytotoxic T-lymphocyte (CTL) response is induced in BALB/c mice upon immunization with poliovirus serotype 1 (Mahoney strain). A similar class I-restricted response is also induced upon immunization with purified VP1 capsid proteins. Thus, poliovirus-specific MHC class I CTL responses can be induced independently of viral infection in murine hosts. In experiments using recombinant vaccinia virus vectors expressing different segments of the poliovirus capsid proteins and synthetic peptides, two regions of the VP1 capsid protein appear to contain epitopes recognized by this bulk CTL population. These epitope regions contain a Kd-restricted peptide-binding motif. Interestingly, each of these CTL epitopes is located near previously defined neutralizing antigenic sites.