Amir Maksyutov

Searching for Local Similarities between HIV-1 and Human Proteins. Application to Vaccines

A method for identification of fragments with 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 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 either through induction of cross-reacting effectors of the immune system or through molecular mimicry of physiologically important human proteins, leading to alteration of homeostasis of the organism, is discussed. Most of the 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 that would allow exclusion of epitopes with (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 pronounced local similarity to proteins that provide for important physiological functions.

Induction of an antitumor response using dendritic cells transfected with DNA constructs encoding the HLA-A*02:01-restricted epitopes of tumor-associated antigens in culture of mononuclear cells of breast cancer patients.

Advances in oncoimmunology related to the definition of the basic mechanisms of the formation of antitumor immune response, as well as the opening of tumor-associated antigens recognized by immune cells, allowed to start developing ways to influence the effector cells of the immune system to generate effective antitumor cytotoxic response. We investigated the possibility to stimulate an antitumor response in a culture of mononuclear cells of breast cancer patients by dendritic cells transfected with HLA-A*02:01-restricted DNA constructs. We isolated dendritic cells from peripheral blood monocytes and delivered our constructs to these cells by magnetic transfection. Additionally, a series of experiments with loading of dendritic cells with autologous tumor cell lysate antigens was conducted. We have shown that dendritic cells transfected with the HLA-A*02:01-restricted DNA constructs are effective in inducing an antitumor response in a culture of mononuclear cells of breast cancer patients. Dendritic cells transfected with DNA constructor dendritic cells loaded with lysate antigens revealed a comparable stimulated cytotoxic response of mononuclear cells to these two ways of antigen delivery. We conclude that using DNA constructs in conjunction with patient stratification by HLA type allows the application of transfected DCs as an effective method to stimulate antitumor immunity in vitro.

Genotyping of hepatitis B and C virus Russian isolates for reference serum panel construction.

Approximately 2% and 5% of the world human population is estimated to be infected with HCV and HBV, respectively. Reference panels of HCV and HBV serum samples with defined genotypes and serotypes is necessary for monitoring of the specificity and sensitivity of diagnostic test kits. The aim of this study was to determine genotypes/serotypes of HBV and HCV circulating in Russia in order to construct a panel of reference sera containing these HCV genotypes and HBV serotypes. A total of 343 HBsAg‐positive and 207 anti‐HCV positive serum samples were collected from patients with HBV and HCV infection from different cities between years 2002 and 2010 in St. Petersburg, Krasnodar, Nizhny Novgorod, Novosibirsk, Barnaul, Gorno‐Altaisk, and Khabarovsk. HBV DNA was found in 76.4% of HBsAg positive samples by PCR for the S gene and HCV RNA was found in 71.5, 70.0, and 64.7% of anti‐HCV positive samples in the 5′UTR, Core, and NS5B regions, respectively. The prevalence and proportion of HBV genotype/serotype associations were as follows: A/adw2, 2.1%; D/ayw2, 54.0%; D/ayw3, 43.1%; D/adw2, 0.7%. A new combination of genotype D and adw2 serotype was discovered. The distribution of HCV genotypes was the following: 43.6%, b; 3.8%, 2a; and 52.6%, 3a. Russian National reference panels of HBV and HCV lyophilized sera were developed to monitor specificity and sensitivity of approved kits and for the certification of newly developed assays. J. Med. Virol. 87:1192–1198, 2015.

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].

Generation of populations of antigen-specific cytotoxic T cells using DCs transfected with DNA construct encoding HER2/neu tumor antigen epitopes

BackgroundRecent fundamental and clinical studies have confirmed the effectiveness of utilizing the potential of the immune system to remove tumor cells disseminated in a patient’s body. Cytotoxic T lymphocytes (CTLs) are considered the main effectors in cell-mediated antitumor immunity. Approaches based on antigen presentation to CTLs by dendritic cells (DCs) are currently being intensively studied, because DCs are more efficient in tumor antigen presentation to T cells through their initiation of strong specific antitumor immune responses than other types of antigen-presenting cells. Today, it has become possible to isolate CTLs specific for certain antigenic determinants from heterogeneous populations of mononuclear cells. This enables direct and specific cell-mediated immune responses against cells carrying certain antigens. The aim of the present study was to develop an optimized protocol for generating CTL populations specific for epitopes of tumor-associated antigen HER2/neu, and to assess their cytotoxic effects against the HER2/neu-expressing MCF-7 tumor cell line.MethodsThe developed protocol included sequential stages of obtaining mature DCs from PBMCs from HLA A*02-positive healthy donors, magnet-assisted transfection of mature DCs with the pMax plasmid encoding immunogenic peptides HER2 p369–377 (E75 peptide) and HER2 p689–697 (E88 peptide), coculture of antigen-activated DCs with autologous lymphocytes, magnetic-activated sorting of CTLs specific to HER2 epitopes, and stimulation of isolated CTLs with cytokines (IL-2, IL-7, and IL-15).ResultsThe resulting CTL populations were characterized by high contents of CD8+ cells (71.5% in cultures of E88-specific T cells and 90.2% in cultures of E75-specific T cells) and displayed strong cytotoxic effects against the MCF-7 cell line (percentages of damaged tumor cells in samples under investigation were 60.2 and 65.7% for E88- and E75-specific T cells, respectively; level of spontaneous death of target cells was 17.9%).ConclusionsThe developed protocol improves the efficiency of obtaining HER2/neu-specific CTLs and can be further used to obtain cell-based vaccines for eradicating targeted tumor cells to prevent tumor recurrence after the major tumor burden has been eliminated and preventing metastasis in patients with HER2-overexpressing tumors.

Introduction of foreign peptides in surface loops of alkaline phosphatase

The introduction of foreign peptides into alkaline phosphatase that have a minimal influence on the enzymatic activity of a protein is described in the work. Calculated based on the methods of molecular dynamics the longest surface loops of alkaline phosphatase, which are not adjacent to both active center of a dimeric enzyme and contact surface between its monomers, were used as sites of the introduction of foreign peptides. The length of loops was estimated by several methods. Two loops were used to genetically engineer the introduction of peptides. Experiments demonstrated that the introduction of several peptides after the Ala218 residue of alkaline phosphatase insignificantly influences on the enzyme activity. According to experimental data, the selection of the loop for introducing the foreign peptide can be determined using the methods of molecular dynamics based on calculating the mobility of dihedral angles. In order to create macromolecules with new features by introducing foreign polypeptides in enzymatically active proteins, it is possible in practice to use an estimation of the loop length by means of the methods of molecular dynamics.

P17-02. PolyCTLDesigner: the software for constructing highly efficient polyepitope immunogens. Application to HIV-1

Background Design of the artificial polyepitope immunogens capable of eliciting high levels of the CD8+ CTL responses to the most of contained epitopes is a promising approach in creation of an efficient vaccine against HIV-1. When designing such immunogens, it is necessary to provide their processing to liberate the epitopes and present them to the immune system. We have demonstrated that DNA vaccine construct encoding poly-CTL-epitope immunogen which contains N-terminal ubiquitin and residues ensuring the proteasome processing of polyepitope construct and transport liberated determinants through the endoplasmic reticulum where they bind to MHC class I molecules is most optimal for stimulating the maximal CD8+ CTL responses. These results inspired us to create PolyCTLDesigner software, intended for designing optimal polyepitope antigens.