Artificial COVID-19 T-Cell Immunogen.

An artificial T-cell immunogen consisting of conserved fragments of different proteins of the SARS-CoV-2 virus and its immunogenic properties were studied in BALB/c mice. To create a T-cell immunogen, we used an approach based on the design of artificial antigens that combine many epitopes from the main proteins of the SARS-CoV-2 virus in the one molecule. The gene of the engineered immunogen protein was cloned as part of the pVAX1 plasmid in two versions: with an N-terminal ubiquitin and without it. The obtained plasmids were analyzed for their ability to provide the synthesis of the immunogen protein in vitro and in vivo. It has been shown that protein product of the created artificial genes is actively processed in HEK293T cells and induces cellular immunity in mice.

Immunogenicity of the DNA/Protein Combined Vaccine against COVID-19.

During the COVID-19 pandemic, the development of prophylactic vaccines, including those based on new platforms, became highly relevant. One such platform is the creation of vaccines combining DNA and protein components in one construct. For the creation of DNA vaccine, we chose the full-length spike protein (S) of the SARS-CoV-2 virus and used the recombinant receptor-binding domain (RBD) of the S protein produced in CHO-K1 cells as a protein component. The immunogenicity of the developed combined vaccine and its individual components was compared and the contribution of each component to the induction of the immune response was analyzed. The combined DNA/protein vaccine possesses the advantages of both underlying approaches and is capable of inducing both humoral (similar to subunit vaccines) and cellular (similar to DNA vaccines) immunity.

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.

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.

[A mathematical model of the intracellular reproduction of the influenza virus].

A mathematical model of the intracellular reproduction of the influenza virus has been constructed, which describes the regulation of transcription, translation, replication, and assembly of the virus in a sensitive noninterferonogenic cell. A structural and parametric verification of the model has been performed, which involves an analysis of alternative mechanisms of regulation of the key events of the reproduction of the influenza virus at the stages of the primary and secondary transcription. It was shown that the dynamics of the concentrations of the system components is in qualitative agreement with the experimental data. An analysis of the model parameters showed a high sensitivity of the model to changes in the parameters that control the efficiency of the binding of the viral polymerase to virus-specific RNAs at the stages of initiation and elongation of transcription and replication of the viral genome. The results obtained suggest that the stages of the virus reproduction sensitive to changes in the parameters are potential targets for drugs. Using the model, a minimal multiplicity of infection at which the formation of viral particles is observed was predicted.

[Different systems of delivery of HIV-1 DNA vaccine encoding the multiepitope CTL-immunogene].

We used, within the case study, virus-like particles (VLP) and attenuated strains of salmonella for the delivery of HIV-1 DNA vaccine encoding the multiepitope CTL-immunogene. The immunogenicity of the thus obtained vaccine constructions was comparatively analyzed. All constructions were shown to be able of inducing, in immunized animals, both the specific T-cell responses and the synthesis of virus-specific antibodies. The lowest level of immune response was registered in animals immunized by "naked" plasmid DNA. The delivery by plasmid DNA involving VLP or the attenuated strain of salmonella enhances the efficiency of the DNA-vaccine presentation to the immune system.

[Artificial anti-HIV immunogens and methods of their delivery]. / Iskusstvennye anti-VICH-immonogeny i sposoby ikh dostavki.

Elaboration of an anti-HIV vaccine is a highly important task because there is a need to arrest or at least to slow-down the rapid spread of AIDS throughout the world. Regrettably, no attempts to create an effective vaccine resulted in success. Nonetheless, the available data contribute to building up the confidence in that the set purpose can be achieved provided extra resources are found for working out a potential anti-HIV vaccine. The paper contains some results of research conducted by the "Vector" Research Center for Virology and Biotechnology in the field of artificial polyepitope immunogens, which could be potential anti-HIV vaccines, and in the field of creating various system for their delivery. The immunogenic properties of the thus obtained vaccine structures were tested on mice BALB/c. The delivery systems were experimentally demonstrated to ensure the induction of specific antibodies against HIV-1, with such anti-bodies having a virus-neutralizing activity; the above systems also induce the cellular immunity.

[Searching for local similarities between HIV-1 and human proteins. Application to vaccines]. / Poisk v belkakh VICh-1 raionov, lokal'no skhodnykh s belkami cheloveka. Primenenie k vaktsinam.

A method for identification of fragments with a high local similarity to human proteins within potentially immunopathogenic regions of HIV-1 proteins was developed. The method is based on the use of an original matrix of antigenic similarity of amino acids. The regions, whose fragments are frequent in human proteins, and regions, exhibiting a high similarity to the proteins responsible for important physiological functions, were identified in HIV-1 proteins. A possibility of participation of such regions in immunopathogenesis of HIV-infection due either to induction of cross-reacting effectors of immune system or through molecular mimicry of physiologically important human proteins, leading to an alteration of homeostasis of the organism, is discussed. Most of regions, identified in HIV-1 proteins, contain either T-cell (CD8+ CTL or CD4+ Th) or B-cell epitopes, or both of them simultaneously. The criteria for the design of safe polyepitopic antiviral vaccines which enable the exclusion of epitopes, having the (immuno)pathogenic potential, are discussed. According to these criteria, polyepitopic immunogens should be free of the virus protein regions, whose fragments are frequent in human proteins, as well as of regions exhibiting a pronounced local similarity to proteins that provide for important physiological functions.

Interferon-induced antiviral resistance. A mathematical model of regulation of Mx1 protein induction and action.

Influenza A virus and various single-stranded RNA viruses have been reported to be blocked by IFN-stimulated Mx protein. Here we present a mathematical model of regulation of mouse Mx1 protein induction and action under influenza infection. Parameter estimates are derived from published experimental data. Numerical solutions of the model equations completely correspond to experimental data. The model is used to analyse the role of virus- and interferon-mediated expression of Mx1 in maintenance of antiviral state. The study suggests that virus- and IFN-induced Mx1 proteins act on different stages of intracellular ontogenesis of influenza virus and these actions result in different efficacy of cell protection. The model demonstrates that the synergistic action of inteferon and virus in regulation of Mx1 gene expression is the important factor of antiviral resistance. The results of simulation permit to assume that the active form of Mx1 protein is trimer.

[Molecular genetic aspects of interferon induction and antiviral action]. / Molekuliarno-geneticheskie aspekty induktsii i protivovirusnogo deistviia interferona.

Interferons (IFN) are cytokines with a wide range of biological activities. The most important function of IFN is associated with the induction of host resistance to viral infections. This paper discusses the well-known experimental data and the results of the authors' theoretical studies of the regularities of type 1 interferon induction and its antiviral action. The mechanisms both of interferon gene expression and transcription regulation of IFN-stimulated genes by IFNs and viruses are analyzed. The molecular mechanisms of IFN-induced resistance and the well-known strategies acquired by viruses to overcome IFN action are discussed. The authors' analyze possible regulation by IFN of its own expression and the role of IFN- and virus-induced expression of genes of antiviral response in the development of resistance to infection.

Theoretical analysis of the regulation of interferon expression during priming and blocking.

Interferon superinduction, in the case of cell pretreatment with low doses of interferon (priming), may be explained by activation of 2',5'-oligoadenylate synthetase and endonuclease L, since the latter, as expected, leads to a more rapid amplification of the standard scheme of interferon induction based on the antirepression mechanism. In the given case, endonuclease L will further increase the degradation rates for messages, which encode repressor proteins controlling interferon gene expression. Under ordinary induction, these messages are destroyed only by short-lived nuclease activated by double-stranded RNA. Cell pretreatment with high doses of interferon (blocking) considerably increases the concentrations of protein kinase and 2',5'-oligoadenylate synthetase in the cell. However, it seems that during blocking protein kinase plays the main role in inhibition of interferon synthesis, and this leads to almost complete depression of translation in the cell. When protein kinase is not sufficiently activated, blocking does not occur since treatment of cells with high concentrations of interferon does not hinder priming induced by 2',5'-oligoadenylate synthetase and endonuclease L. The proposed model is consistent with the findings that both interferon-treated primed and blocked cells are able to produce interferon more rapidly than normal cells. The analysis, based on a computer simulation model, suggests that priming and blocking of interferon may be based on processes controlling its induction and antiviral activity.

A computer system for analysis and integrated description of regulation of the molecular-genetic system of interferon induction and action.

A new theoretical approach to elaboration of an information-analytical integrated knowledge base containing data on regulation and function of biological systems is presented. The knowledge base incorporates: (i) a reference database containing experimental data on the structural-functional organization of a biological system; (ii) a dynamic mathematical model for analysis of the evolution of the system over time; and (iii) an interpretation module of simulation results. Application of this approach to theoretical investigation of the interferon system in the case of viral infection is discussed. The approach is specific in that it uses mathematical modeling technology, which allows one to generate mathematical models of different degrees of complexity in the analysis of the diverse aspects of biological system behavior. This approach allows one not only to store and to treat available experimental data, but also to acquire new knowledge about the behavior of a biological system. The proposed approach is implemented as a computer system for the IBM PC and compatibles.

Mathematical model of antiviral immune response regulation. II. Mathematical formalization of the modelled processes. Imitation of acute course of hepatitis B.

The mathematical formalization of the conceptual model for antiviral immune response regulation described in the preceding report was carried out. The mathematical model is presented as a system of 30 ordinary nonlinear differential equations with delays. The algorithm for numerical integration of the mathematical model is based on Gear's methods of variable step and variable order. Initial conditions and parameters, as well as intervals of plausible values for them, were chosen for adaptation of the model for description of acute hepatitis B.