Résumé: In this chapter, we show an example of how an epitope database can be integrated to other database resources using the Distributed Annotation System (DAS) (Dowell
et al. 2001). For that we describe the TEPIDAS server, a DAS Annotation Server of
HLA I-restricted CD8 T-cell epitopes specific of human pathogenic organisms.
Mots-clés: Cumulative phenotypic frequency, Distributed Annotation System (DAS), Human
leukocyte antigen class I, Position-specific scoring matrix Immunoinformatics, Bioinformatics
Résumé: ADP-ribosyltransferases (ADPRTs) form an interesting class of enzymes with well-established roles as potent bacterial toxins and metabolic regulators. ADPRTs catalyze the transfer of the ADP-ribose moiety from NAD(+) onto specific substrates including proteins. ADP-ribosylation usually inactivates the function of the target. ADPRTs have become adapted to
function in extra- and intracellular settings. Regulation of ADPRT activity can be mediated by ligand binding to associated regulatory domains, proteolytic cleavage, disulphide bond reduction, and association with other proteins. Crystallisation has revealed a conserved core set of elements that define an unusual minimal scaffold of the catalytic domain with remarkably plastic sequence requirements--only a single glutamic acid residue critical to catalytic activity is invariant. These inherent properties of ADPRTs suggest that the ADPRT catalytic fold is an attractive, malleable subject for protein design.
Résumé: The EGF-like domain of smallpox growth factor (SPGF) targets human ErbB-1, inducing tyrosine phosphorylation of certain host cellular substrates via activation of the receptor's kinase domain and thereby facilitating viral replication. Given these findings, low molecular weight organic
inhibitors of ErbB-1 kinases might function as antiviral agents against smallpox. Here we show that CI-1033 and related 4-anilinoquinazolines inhibit SPGF-induced human cellular DNA synthesis, protein tyrosine kinase activation, and c-Cbl association with ErbB-1 and resultant internalization. Infection of monkey kidney BSC-40 and VERO-E6 cells in vitro by variola strain Solaimen is blocked by CI-1033, primarily at the level of secondary viral spreading. In an in vivo lethal vaccinia virus pneumonia model, CI-1033 alone promotes survival of animals, augments systemic T cell immunity and, in conjunction with a single dose of anti-L1R intracellular mature virus particle-specific mAb, fosters virtually complete viral clearance of the lungs of infected mice by the eighth day after infection. Collectively, these findings show that chemical inhibitors of host-signaling pathways exploited by viral pathogens may represent potent antiviral therapies.
Résumé: Variola, the causative agent of smallpox, is a highly infectious double-stranded DNA virus of the orthopox genus that replicates within the cytoplasm of infected cells. For unknown reasons prominent skin
manifestations, including "pox," mark the course of this systemic human disease. Here we characterized smallpox growth factor (SPGF), a protein containing an epidermal growth factor (EGF)-like domain that is conserved among orthopox viral genomes, and investigated its possible mechanistic link. We show that after recombinant expression, refolding, and purification, the EGF domain of SPGF binds exclusively to the broadly expressed cellular receptor, erb-B1 (EGF receptor), with subnanomolar affinity, stimulating the growth of primary human keratinocytes and fibroblasts. High affinity monoclonal antibodies specific for SPGF reveal in vivo immunoprotection in a murine vaccinia pneumonia model by a mechanism distinct from viral neutralization. These findings suggest that blockade of pathogenic factor actions, in general, may be advantageous to the infected host.
Résumé: We have cloned, sequenced and expressed the Trypanosoma cruzi gene encoding the bifunctional protein dihydrofolate reductase-thymidylate synthase (DHFR-TS). The strategy followed for the isolation of positive clones from a genomic library was based on the construction of a probe by the amplification of highly conserved
sequences of the TS domain by the polymerase chain reaction. Translation of the open reading frame of 1563 bp yields a polypeptide of 521 amino acids with a molecular mass of 58829 Da. For heterologous expression of T. cruzi DHFR-TS in Escherichia coli, the entire coding sequence was amplified by polymerase chain reaction and cloned into the plasmid vector pKK223.3. The presence of catalytically active DHFR-TS was demonstrated by complementation of the Thy- E. coli strain chi 2913 and the DHFR- Thy- E. coli strain PA414. The gene is expressed as an active protein which constitutes approximately 2% of the total cell soluble protein. Recombinant bifunctional enzyme and the DHFR domain have been purified by methotrexate-Sepharose chromatography to yield 1-2 mg of active DHFR-TS per litre of culture. Southern and electrophoretic analyses using the coding sequence as probe indicated that the T. cruzi enzyme is encoded by a single copy gene which maps to two bands of approximately 990 kb and 1047 kb. It appears that T. cruzi is diploid for the DHFR-TS gene which is located on two different-sized homologous chromosomes.
Résumé: Identification of peptides that can bind to major histocompatibility complex (MHC) molecules is important for anticipation of T-cell epitopes and for the design of epitope-based vaccines. Population coverage of epitope vaccines is, however, compromised by the extreme polymorphism of MHC molecules, which is in fact the basis for their differential peptide binding. Therefore,
grouping of MHC molecules into supertypes according to peptide-binding specificity is relevant for optimizing the composition of epitope-based vaccines. Despite the fact that the peptide-binding specificity of MHC molecules is linked to their specific amino acid sequences, it is unclear how amino sequence differences correlate with peptide-binding specificities. In this chapter, we detail a method for defining MHC supertypes based on the analysis and subsequent clustering of their peptide-binding repertoires.
Résumé: The functional consequences of glycan structural changes associated with cellular differentiation are ill defined. Herein, we investigate the role of glycan adducts to the O-glycosylated polypeptide stalk tethering the CD8alphabeta coreceptor to the thymocyte surface. We show that immature CD4(+)CD8(+) double-positive thymocytes bind MHCI tetramers more avidly than
mature CD8 single-positive thymocytes, and that this differential binding is governed by developmentally programmed O-glycan modification controlled by the ST3Gal-I sialyltransferase. ST3Gal-I induction and attendant core 1 sialic acid addition to CD8beta on mature thymocytes decreases CD8alphabeta-MHCI avidity by altering CD8alphabeta domain-domain association and/or orientation. Hence, glycans on the CD8beta stalk appear to modulate the ability of the distal binding surface of the dimeric CD8 globular head domains to clamp MHCI.
