Denis Antonets

Genome-wide analysis of A-to-I RNA editing by single-molecule sequencing in Drosophila

The accurate and thorough genome-wide detection of adenosine-to-inosine editing, a biologically indispensable process, has proven challenging. Here, we present a discovery pipeline in adult Drosophila, with 3,581 high-confidence editing sites identified with an estimated accuracy of 87%. The target genes and specific sites highlight global biological properties and functions of RNA editing, including hitherto-unknown editing in well-characterized classes of noncoding RNAs and 645 sites that cause amino acid substitutions, usually at conserved positions. The spectrum of functions that these gene targets encompass suggests that editing participates in a diverse set of cellular processes. Editing sites in Drosophila exhibit sequence-motif preferences and tend to be concentrated within a small subset of total RNAs. Finally, editing regulates expression levels of target mRNAs and strongly correlates with alternative splicing.

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family

BackgroundVariola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein.FindingsDe novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins.ConclusionsOur results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

Rational design based synthetic polyepitope DNA vaccine for eliciting HIV-specific CD8+ T cell responses

Advances in defining HIV-1 CD8+ T cell epitopes and understanding endogenous MHC class I antigen processing enable the rational design of polyepitope vaccines for eliciting broadly targeted CD8+ T cell responses to HIV-1. Here we describe the construction and comparison of experimental DNA vaccines consisting of ten selected HLA-A2 epitopes from the major HIV-1 antigens Env, Gag, Pol, Nef, and Vpr. The immunogenicity of designed gene constructs was assessed after double DNA prime, single vaccinia virus boost immunization of HLA-A2 transgenic mice. We compared a number of parameters including different strategies for fusing ubiquitin to the polyepitope and including spacer sequences between epitopes to optimize proteasome liberation and TAP transport. It was demonstrated that the vaccine construct that induced in vitro the largest number of [peptide-MHC class I] complexes was also the most immunogenic in the animal experiments. This most immunogenic vaccine construct contained the N-terminal ubiquitin for targeting the polyepitope to the proteasome and included both proteasome liberation and TAP transport optimized spacer sequences that flanked the epitopes within the polyepitope construct. The immunogenicity of determinants was strictly related to their affinities for HLA-A2. Our finding supports the concept of rational vaccine design based on detailed knowledge of antigen processing.

Novel approaches in polyepitope T-cell vaccine development against HIV-1

RV144 clinical trial was modestly effective in preventing HIV infection. New alternative approaches are needed to design improved HIV-1 vaccines and their delivery strategies. One of these approaches is construction of synthetic polyepitope HIV-1 immunogen using protective T- and B-cell epitopes that can induce broadly neutralizing antibodies and responses of cytotoxic (CD8+ CTL) and helpers (CD4+ Th) T-lymphocytes. This approach seems to be promising for designing of new generation of vaccines against HIV-1, enables in theory to cope with HIV-1 antigenic variability, focuses immune responses on protective determinants and enables to exclude from the vaccine compound that can induce autoantibodies or antibodies enhancing HIV-1 infectivity. Herein, the authors will focus on construction and rational design of polyepitope T-cell HIV-1 immunogens and their delivery, including: advantages and disadvantages of existing T-cell epitope prediction methods; features of organization of polyepitope immunogens, which can generate high-level CD8+ and CD4+ T-lymphocyte responses; the strategies to optimize efficient processing, presentation and immunogenicity of polyepitope constructs; original software to design polyepitope immunogens; and delivery vectors as well as mucosal strategies of vaccination. This new knowledge may bring us a one step closer to developing an effective T-cell vaccine against HIV-1, other chronic viral infections and cancer.

Functional annotation of the vlinc class of non-coding RNAs using systems biology approach

Functionality of the non-coding transcripts encoded by the human genome is the coveted goal of the modern genomics research. While commonly relied on the classical methods of forward genetics, integration of different genomics datasets in a global Systems Biology fashion presents a more productive avenue of achieving this very complex aim. Here we report application of a Systems Biology-based approach to dissect functionality of a newly identified vast class of very long intergenic non-coding (vlinc) RNAs. Using highly quantitative FANTOM5 CAGE dataset, we show that these RNAs could be grouped into 1542 novel human genes based on analysis of insulators that we show here indeed function as genomic barrier elements. We show that vlincRNAs genes likely function in cis to activate nearby genes. This effect while most pronounced in closely spaced vlincRNA–gene pairs can be detected over relatively large genomic distances. Furthermore, we identified 101 vlincRNA genes likely involved in early embryogenesis based on patterns of their expression and regulation. We also found another 109 such genes potentially involved in cellular functions also happening at early stages of development such as proliferation, migration and apoptosis. Overall, we show that Systems Biology-based methods have great promise for functional annotation of non-coding RNAs.

PolyCTLDesigner: a computational tool for constructing polyepitope T-cell antigens

BackgroundConstruction of artificial polyepitope antigens is one of the most promising strategies for developing more efficient and safer vaccines evoking T-cell immune responses. Epitope rearrangements and utilization of certain spacer sequences have been proven to greatly influence the immunogenicity of polyepitope constructs. However, despite numerous efforts towards constructing and evaluating artificial polyepitope immunogens as well as despite numerous computational methods elaborated to date for predicting T-cell epitopes, peptides binding to TAP and for antigen processing prediction, only a few computational tools were currently developed for rational design of polyepitope antigens.FindingsHere we present a PolyCTLDesigner program that is intended for constructing polyepitope immunogens. Given a set of either known or predicted T-cell epitopes the program selects N-terminal flanking sequences for each epitope to optimize its binding to TAP (if necessary) and joins resulting oligopeptides into a polyepitope in a way providing efficient liberation of potential epitopes by proteasomal and/or immunoproteasomal processing. And it also tries to minimize the number of non-target junctional epitopes resulting from artificial juxtaposition of target epitopes within the polyepitope. For constructing polyepitopes, PolyCTLDesigner utilizes known amino acid patterns of TAP-binding and proteasomal/immunoproteasomal cleavage specificity together with genetic algorithm and graph theory approaches. The program was implemented using Python programming language and it can be used either interactively or through scripting, which allows users familiar with Python to create custom pipelines.ConclusionsThe developed software realizes a rational approach to designing poly-CTL-epitope antigens and can be used to develop new candidate polyepitope vaccines. The current version of PolyCTLDesigner is integrated with our TEpredict program for predicting T-cell epitopes, and thus it can be used not only for constructing the polyepitope antigens based on preselected sets of T-cell epitopes, but also for predicting cytotoxic and helper T-cell epitopes within selected protein antigens. PolyCTLDesigner is freely available from the project’s web site: http://tepredict.sourceforge.net/PolyCTLDesigner.html.

TEpredict: Software for T-Cell epitope prediction

The program TEpredict was developed for T-cell epitope prediction. The used models for T-cell epitope prediction were constructed by the partial least squares regression method using the data extracted from the IEDB (Immune Epitope Database), the most complete resource of experimental peptide-MHC binding data. TEpredict is also able to predict proteasomal processing of protein antigens and the ability of produced oligopeptides to bind to the transporters associated with antigen processing, to discard the peptides sharing local similarity with human proteins from the set of predicted epitopes, and to estimate the expected population coverage by the selected peptides using the data on HLA allele genotypic frequencies. The main part of the constructed models demonstrated a high prediction sensitivity (0.50–0.80) in combination with a high specificity (0.75–0.99). Comparative testing demonstrated that TEpredict was competitive with or even superior to the programs ProPred1, SVRMHC, SVMHC, and SYFPEITHI. Thus, TEpredict can become an efficient tool for developing polyepitope vaccines, including the vaccines against various human pathogens, such as HIV and the influenza virus. TEpredict and the source code are available at http://tepredict.source- forge.net.

Design and Evaluation of Optimized Artificial HIV-1 Poly-T Cell-Epitope Immunogens

A successful HIV vaccine in addition to induction of antibody responses should elicit effective T cell responses. Here we described possible strategies for rational design of T-cell vaccine capable to induce high levels of both CD4+ and CD8+ T- cell responses. We developed artificial HIV-1 polyepitope T-cell immunogens based on the conserved natural CD8+ and CD4+ T cell epitopes from different HIV-1 strains and restricted by the most frequent major human leukocyte antigen (HLA) alleles. Designed immunogens contain optimized core polyepitope sequence and additional “signal” sequences which increase epitope processing and presentation to CD8+ and CD4+ T-lymphocytes: N-terminal ubiquitin, N-terminal signal peptide and C-terminal tyrosine motif of LAMP-1 protein. As a result we engineered three T cell immunogens – TCI-N, TCI-N2, and TCI-N3, with different combinations of signal sequences. All designed immunogens were able to elicit HIV-specific CD4+ and CD8+ T cell responses following immunization. Attachment of either ubiquitin or ER-signal/LAMP-1 sequences increased both CD4+ and CD8+ mediated HIV-specific T cell responses in comparison with polyepitope immunogen without any additional signal sequences. Moreover, TCI-N3 polyepitope immunogen with ubiquitin generated highest magnitude of HIV-specific CD4+ and CD8+ T cell responses in our study. Obtained data suggests that attachment of signal sequences targeting polyepitope immunogens to either MHC class I or MHC class II presentation pathways may improve immunogenicity of T-cell vaccines. These results support the strategy of the rational T cell immunogen design and contribute to the development of effective HIV-1 vaccine.

3D structure modeling of complexes formed by CrmB TNF-binding proteins of Variola and cowpox viruses with murine and human TNFs

Orthopoxviral genomes bear genes for a series of homologous secreted proteins binding tumor necrosis factor (TNF). Orthopoxvirus species have different sets of these proteins. Variola virus has only one protein of this series, CrmB. Although CrmB protein sequences are similar to each other, their physicochemical and biological properties show certain species-specific features. We constructed 3D models of complexes formed by TNF-binding domains of variola and cowpox viruses with murine and human TNFs. We also constructed corresponding models with a mutant human TNF. In this mutant TNF, the arginine residue at position 31 involved in receptor binding was replaced by glutamine, characteristic of murine TNF. Analysis of the models showed that the least stable complex should be that formed by cowpox virus CrmB with human TNF, and the Arg31/Gln substitution should significantly stabilize the interaction between cowpox CrmB and mutant human TNF. Experimental comparison of the abilities of recombinant variola and cowpox CrmB proteins to inhibit the cytotoxic action of TNFs confirmed the predictions.

Prediction of antigenically active regions in the OmpF-like porin of Yersinia pseudotuberculosis.

124 Nonspecific porins forming channels for diffusion of low-molecular-weight compounds are the main proteins of the outer membrane of gram-negative bacteria. Porins exist in the outer bacterial membrane in the form of oligomers (most frequently trimers) [1]. Bacterial porins are the products of conserved chromosomal genes and, as a result, have a high extent of homology and similar spatial organization [2].