PVS: a web server for protein sequence variability analysis tuned to facilitate conserved epitope discovery

We have developed PVS (Protein Variability Server), a web-based tool that uses several variability metrics to compute the absolute site variability in multiple protein-sequence alignments (MSAs). The variability is then assigned to a user-selected reference sequence consisting of either the first sequence in the alignment or a consensus sequence. Subsequently, PVS performs tasks that are relevant for structure-function studies, such as plotting and visualizing the variability in a relevant 3D-structure. Neatly, PVS also implements some other tasks that are thought to facilitate the design of epitope discovery-driven vaccines against pathogens where sequence variability largely contributes to immune evasion. Thus, PVS can return the conserved fragments in the MSA—as defined by a user-provided variability threshold—and locate them in a relevant 3D-structure. Furthermore, PVS can return a variability-masked sequence, which can be directly submitted to the RANKPEP server for the prediction of conserved T-cell epitopes. PVS is freely available at: http://imed.med.ucm.es/PVS/

Using simple artificial intelligence methods for predicting amyloidogenesis in antibodies

<p>Abstract</p> <p>Background</p> <p>All polypeptide backbones have the potential to form amyloid fibrils, which are associated with a number of degenerative disorders. However, the likelihood that amyloidosis would actually occur under physiological conditions depends largely on the amino acid composition of a protein. We explore using a naive Bayesian classifier and a weighted decision tree for predicting the amyloidogenicity of immunoglobulin sequences.</p> <p>Results</p> <p>The average accuracy based on leave-one-out (LOO) cross validation of a Bayesian classifier generated from 143 amyloidogenic sequences is 60.84%. This is consistent with the average accuracy of 61.15% for a holdout test set comprised of 103 AM and 28 non-amyloidogenic sequences. The LOO cross validation accuracy increases to 81.08% when the training set is augmented by the holdout test set. In comparison, the average classification accuracy for the holdout test set obtained using a decision tree is 78.64%. Non-amyloidogenic sequences are predicted with average LOO cross validation accuracies between 74.05% and 77.24% using the Bayesian classifier, depending on the training set size. The accuracy for the holdout test set was 89%. For the decision tree, the non-amyloidogenic prediction accuracy is 75.00%.</p> <p>Conclusions</p> <p>This exploratory study indicates that both classification methods may be promising in providing straightforward predictions on the amyloidogenicity of a sequence. Nevertheless, the number of available sequences that satisfy the premises of this study are limited, and are consequently smaller than the ideal training set size. Increasing the size of the training set clearly increases the accuracy, and the expansion of the training set to include not only more derivatives, but more alignments, would make the method more sound. The accuracy of the classifiers may also be improved when additional factors, such as structural and physico-chemical data, are considered. The development of this type of classifier has significant applications in evaluating engineered antibodies, and may be adapted for evaluating engineered proteins in general.</p

Specific Binding of the Pathogenic Prion Isoform: Development and Characterization of a Humanized Single-Chain Variable Antibody Fragment

Murine monoclonal antibody V5B2 which specifically recognizes the pathogenic form of the prion protein represents a potentially valuable tool in diagnostics or therapy of prion diseases. As murine antibodies elicit immune response in human, only modified forms can be used for therapeutic applications. We humanized a single-chain V5B2 antibody using variable domain resurfacing approach guided by computer modelling. Design based on sequence alignments and computer modelling resulted in a humanized version bearing 13 mutations compared to initial murine scFv. The humanized scFv was expressed in a dedicated bacterial system and purified by metal-affinity chromatography. Unaltered binding affinity to the original antigen was demonstrated by ELISA and maintained binding specificity was proved by Western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, humanized scFv specific for the pathogenic form of the prion protein might become a potential therapeutic reagent

Antibody Complementarity-Determining Regions (CDRs) Can Display Differential Antimicrobial, Antiviral and Antitumor Activities

Background: Complementarity-determining regions (CDRs) are immunoglobulin (Ig) hypervariable domains that determine specific antibody (Ab) binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. the possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here.Methodology/Principal Findings: CDR-based synthetic peptides of murine and human monoclonal Abs directed to: a) a protein epitope of Candida albicans cell wall stress mannoprotein; b) a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c) a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. the inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains.Conclusions/Significance: the high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. the easy production and low cost of small sized synthetic peptides representing Ig CDRs and the possibility of peptide engineering and chemical optimization associated to new delivery mechanisms are expected to give rise to a new generation of therapeutic agents.Department of Education, Universities and Research, Basque GovermentFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Istituto Superiore di Sanita, National Research Project on A.I.D.S.Cariparma Banking FoundationBrazilian National Research CouncilUniv Parma, Sez Microbiol, Dipartimento Patol, I-43100 Parma, ItalyUniv Basque Country, Fac Med Odontol, Dept Inmunol, Microbiol Parasitol, Bilbao, SpainUniv Basque Country, Dept Enfermeria I, Bilbao, SpainUniv Milan, Dipartimento Sci Cliniche L Sacco, Sez Malattie Infettive Immunopatol, Milan, ItalyUniv Studi Parma, Dipartimento Clin Med, Nefrol Sci Prev, Parma, ItalyUniversidade Federal de São Paulo, Departamento Microbiol, Imunol Parasitol, Unidade Oncol Expt, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilUniversidade Federal de São Paulo, Departamento Microbiol, Imunol Parasitol, Unidade Oncol Expt, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, São Paulo, BrazilDepartment of Education, Universities and Research, Basque Goverment: IT-264-07FAPESP: 06/50634-2Istituto Superiore di Sanita, National Research Project on A.I.D.S.: 50G.30Istituto Superiore di Sanita, National Research Project on A.I.D.S.: 40D.14Cariparma Banking Foundation: 2004.0190Brazilian National Research Council: research fellowshipWeb of Scienc

Antibody Vh Repertoire Differences between Resolving and Chronically Evolving Hepatitis C Virus Infections

Despite the production of neutralizing antibodies to hepatitis C virus (HCV), many patients fail to clear the virus and instead develop chronic infection and long-term complications. To understand how HCV infection perturbs the antibody repertoire and to identify molecular features of antibody genes associated with either viral clearance or chronic infection, we sequenced the V(D)J region of naïve and memory B cells of 6 persons who spontaneously resolved an HCV infection (SR), 9 patients with a newly diagnosed chronically evolving infection (CE), and 7 healthy donors. In both naïve and memory B cells, the frequency of use of particular antibody gene subfamilies and segments varied among the three clinical groups, especially between SR and CE. Compared to CE, SR antibody genes used fewer VH, D and JH gene segments in naïve B cells and fewer VH segments in memory B cells. SR and CE groups significantly differed in the frequency of use of 7 gene segments in naïve B cell clones and 3 gene segments in memory clones. The nucleotide mutation rates were similar among groups, but the pattern of replacement and silent mutations in memory B cell clones indicated greater antigen selection in SR than CE. Greater clonal evolution of SR than CE memory B cells was revealed by analysis of phylogenetic trees and CDR3 lengths. Pauciclonality of the peripheral memory B cell population is a distinguishing feature of persons who spontaneously resolved an HCV infection. This finding, previously considered characteristic only of patients with HCV-associated lymphoproliferative disorders, suggests that the B cell clones potentially involved in clearance of the virus may also be those susceptible to abnormal expansion

Antibody Repertoires in Humanized NOD-scid-IL2Rγnull Mice and Human B Cells Reveals Human-Like Diversification and Tolerance Checkpoints in the Mouse

Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγnull engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγnull mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential DH-JH pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors intrinsic to human B cells and are principally unaltered by differences between mouse and human stromal microenvironments