DNA Vaccine Encoding the Artificial T-Cell Polyepitope Immunogen of Tick-Borne Encephalitis Virus.

A promising approach to the development of new means for preventing infection caused by tick-borne encephalitis virus can be DNA vaccines encoding polyepitope T-cell immunogens. A DNA vaccine pVAX-AG4-ub encoding an artificial polyepitope immunogen that includes cytotoxic and T-helper epitopes from the NS1, NS3, NS5, and E proteins of the tick-borne encephalitis virus has been obtained. The developed construct ensured the synthesis of the corresponding mRNAs in transfected eukaryotic cells. Immunization of mice with pVAX-AG4-ub induced the formation of a virus-specific T-cell response providing 50% protection from lethal infection with the virus.

Design of Artificial Immunogens Containing Melanoma-associated T-cell Epitopes.

OBJECTIVE: Immunotherapy based on induction of T-cell responses is a promising approach to cancer treatment. The study aims to design artificial epitope-based immunogens, DNA vaccine candidates against melanoma and evaluate their ability to stimulate tumor cytotoxicity of ex vivo generated T-cells. METHODS: The original computational methods were used for predicting T-cell epitopes and designing polyepitope melanoma antigens. Artificial genes encoding the target antigens were cloned into DNA vaccine plasmid vector. Target gene expression was confirmed both at transcriptional and translational level in HEK-293T cells transfected with DNA-vaccine constructs. Dendritic cells were generated from adherent peripheral blood mononuclear cells of HLA-A*02:01+ donors. Cytotoxic activity of effector lymphocytes stimulated in co-culture with autologous antigen-presenting dendritic cells towards melanoma Mel Is cells was assessed with lactate dehydrogenase release assay. The proportion of granzyme B producing CD8+ T-cells was estimated using intracellular cytokine staining and flow cytometry. RESULTS: Two DNA vaccine constructions were created - pMEL-TCI and pMEL-A0201 - encoding polypeptides containing T-cell epitopes of six immunodominant melanoma antigens (NY-ESO-1, MART1, MAGE-A1, MAGE-A11, MAGE-A3, and MAGE-C1). Dendritic cells transfected with DNA vaccine constructs were found to stimulate both tumor cytotoxicity mediated by autologous lymphocytes and granzyme B production by CD8+ T-cells, and pMEL-A0201 was found to be the most efficient. CONCLUSION: The described approach may become a common platform for designing immunotherapeutic vaccines against oncological diseases.

Design of Polyepitope DNA Vaccine against Breast Carcinoma Cells and Analysis of Its Expression in Dendritic Cells.

Polyepitope DNA vaccine inducing T-cell-mediated immune response against cancer-specific antigens is a promising tool for selective elimination of tumor cells. Breast cancer-specific polyepitope DNA vaccine was designed using TEpredict and PolyCTLDesigner software on the basis of immunogenic peptides of HER2 and Mammaglobin-1 (Mam) tumor antigens. LPS-free preparations of plasmid DNA encoding polyepitope T-cell antigen and full-length copies of HER2 and Mam antigens were obtained. TaqMan-PCR systems for evaluation of the expression of immunogens in cells were created. The protocol of vaccine DNA delivery into dendritic cells was optimized. Expression of the target immunogens in dendritic cells derived from human peripheral blood mononuclear fraction after transfection with plasmid DNA preparations is demonstrated.

[Gene therapy of arthritis].

Gene therapy can offer a new approach to arthritis treatment which acts at an inflammation site. Numerous studies show high efficacy of gene therapy in different models of arthritis in humans. Even a single injection of a recombinant vector results in a stable prolonged expression of a therapeutic gene and a longterm therapeutic effect. In contrast to biologic therapy involving numerous systemic injections of recombinant anti-inflammatory proteins, gene therapy does not produce systemic side effects. Vectors based on retroviruses, adenoviruses, adeno-associated viruses, and recombinant plasmids could provide delivery of target genes. Of significant importance is the development of noninvasive methods of gene therapy: intranasal and peroral. The current state of research in arthritis gene therapy is discussed in this review.

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.

[TEpredict: software for T-cell epitope prediction].

A program named TEpredict was developed for T-cell epitope prediction. Original models for T-cell epitope prediction were constructed by means of Partial Least Squares regression method on the basis of data, extracted from the IEDB (Immune Epitope Database)--the most complete resource of experimental peptide-MHC binding data known to date. TEpredict is also able to predict proteasomal processing of protein antigens, and the ability of produced oligopeptides to bind to TAP (Tansporters Associated with Processing). TEpredict could exclude peptides, shearing local similarity with human proteins, from the set of predicted T-cell epitopes. It is also able to estimate expected population coverage by selected peptides, using known HLA allele genotypic frequencies data. The majority of produced models demonstrated high sensitivity of predictions (0.50-0.80) concurrent with high specificity (0.75-0.99). TEpredict was shown to be highly competitive or even superior in comparison with such programs as ProPred1, SVRMHC, SVMHC and SYFPEITHI. TEpredict demonstrated high quality of predictions and we expect that it could become a useful tool in the development ofpolyepitope vaccines against dangerous human pathogens, including HIV, influenza etc. The program and its source code could be freely downloaded from the project web-site: http://tepredict.sourceforge.net.

[Modeling spatial structures of variola and cowpox virus TNF-binding CrmB proteins bound to murine or human TNF].

Orthopoxviruses bear in their genomes several genes coding for homologous secreted proteins able to bind tumor necrosis factor. Different species of the genus possess different sets of these tumor necrosis factor-binding proteins. Viriola virus encodes the only one of them named CrmB. Despite sharing high sequence identity, CrmB proteins belonging to distinct orthopoxviral species were shown to significantly differ by their physico-chemical and biological properties. We modeled spatial structures of tumor necrosis factor receptor domains of variola and cowpox virus CrmB proteins bound to either murine, or human or mutated human tumor necrosis factor. In the sequence of last the arginine residue at position 31 is substituted with glutamine that is characteristic for murine tumor necrosis factor. Theoretical analysis of modeled ligand-receptor complexes revealed that the least stable should be the complex of cowpox virus CrmB with human tumor necrosis factor, and that arginine to glutamine substitution at position 31 should significantly stabilize binding of corresponding human tumor necrosis factor mutant to cowpox virus CrmB. Experimental evaluation of recombinant variola and cowpox virus CrmB efficiencies in inhibiting cytotoxic effect of all these tumor necrosis factors have approved our predictions.