Amir Maksyutov - Academia.edu (original) (raw)
Papers by Amir Maksyutov
Frontiers in Bioscience-Landmark
Background: A search for efficient graft rejection modulation techniques for the promotion of dur... more Background: A search for efficient graft rejection modulation techniques for the promotion of durable engraftment remains to be a matter of close study all over the world. Despite the variety of immunosuppressive drugs, the schemes currently used show a lack of selectivity and have a number of side effects. Here we investigated an approach for the induction of antigen-specific tolerance in a human "stimulator-responder" model in vitro, using dendritic cells (DCs) transfected with designed DNA constructs encoding the stimulator's major histocompatibility complex (MHC) epitopes. Methods: The object of the study is peripheral blood mononuclear cells (PBMCs) from 10 healthy donors. To induce antigen-specific tolerance, personalized DNA constructs were created for five responder-stimulator pairs, based on the sequences of donors' and recipients' MHCs. DNA sequencing was performed to select epitopes for incorporation into genetic constructs. A mixed lymphocyte culture assay was used (i) to assess the proliferative response in both directions for all possible stimulator-responder pairs (90 reactions) and (ii) to assess the tolerogenic properties of the generated transfected DCs (5 reactions). Results: A significant increase in the amounts of FoxP3 + CD4 + CD25 + cells and in IL-10 production was shown in culture of donor mononuclear cells after co-cultivation with the responder's dendritic cells transfected with donor-specific plasmids. The tolerogenic cultures generated using tolerogenic DCs transfected with MHC epitopes had a significantly greater ability to inhibit the proliferation of autologous MNCs in response to an allogeneic MHC stimulus. Conclusions: The produced DCs transfected with DNA constructs against HLA stimulating epitopes exhibited tolerogenic properties and may be used to develop antigen-specific tolerance. Thus, we proposed a perspective approach to the induction of antigen-specific tolerance, which should subsequently be studied for use in clinical practice.
Molecular Genetics Microbiology and Virology (Russian version), 2020
Федеральное бюджетное учреждение науки «Государственный научный центр вирусологии и биотехнологии... more Федеральное бюджетное учреждение науки «Государственный научный центр вирусологии и биотехнологии «Вектор» Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека, р.п. Кольцово, Новосибирская область, Россия; 2 Федеральное государственное бюджетное учреждение науки «Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наук», Новосибирск, Россия Резюме Введение. Меланома-злокачественная опухоль, образующаяся при перерождении пигментных клеток, продуцирующих меланины, является наиболее агрессивной разновидностью онкологических заболеваний кожи и характеризуется высокой резистентностью к химиотерапевтическим препаратам, что обусловливает необходимость поиска альтернативных методов терапии данного заболевания. В настоящее время активно развиваются новые направления в терапии рака, одним из которых является онколитическая иммунотерапия, заключающаяся в применении вирусов в качестве онкоселективных цитолитических агентов, способных стимулировать как опухоль-специфичный, так и неспецифичный иммунный ответ организма. Значительная доля современных научных работ посвящена повышению иммуностимулирующих свойств вирусов путем встройки в состав вирусных геномов генов, кодирующих белки-иммуномодуляторы или антигенные детерминанты, характерные для опухолевых клеток. Для меланомы идентифицировано наибольшее количество опухоль-ассоциированных антигенов (ОАА), на основе которых ведут разработки противоопухолевых ДНК-вакцин. Иммуногенность и эффективность таких препаратов остаются на низком уровне. Проблемой в использовании ДНК-вакцин для лечения рака может быть несовершенный дизайн полиэпитопной конструкции, а также неэффективная доставка терапевтических молекул непосредственно в целевые клетки. Частичным решением данной проблемы является использование в качестве способа доставки искусственных иммуногенов онколитических вирусов. Материал и методы. Рекомбинантный вирус осповакцины получен при помощи метода временной доминантной селекции. Цитолитическую эффективность полученного вируса оценивали в МТТ-тесте. Онколитическую эффективность полученного вируса оценивали с помощью мышиной модели ксенографтов с использованием злокачественных клеток SK-Mel-28. Результаты и обсуждение. В данной работе на основе вируса осповакцины штамма L-IVP был получен рекомбинантный вирус L-IVP_oncoM, представляющий собой онколитический вирус, обеспечивающий доставку противораковых терапевтических генов в клетки организма. С этой целью в геном вируса были встроены ген, кодирующий гранулоцит-макрофагальный колониестимулирующий фактор, и искусственный ген, кодирующий полиэпитопный иммуноген, состоящий из эпитопов антигенов, гиперэкспрессирующихся в клетках меланомы. Данные инсерции проведены в районе генов, кодирующих тимидинкиназу (J2R), и вирусный фактор роста (C11L) соответственно. Изучены свойства L-IVP_oncoM в экспериментах in vitro на культурах клеток различного генеза и in vivo в мышиной модели ксенографтов. Заключение. Проведены основные эксперименты по оценке биологических свойств полученного L-IVP_oncoM, которые являются необходимыми для характеризации онколитического вируса.
Molecular Genetics, Microbiology and Virology, 2020
Melanoma is a tumor that forms as a result of malignant transformation of melanin-producing pigme... more Melanoma is a tumor that forms as a result of malignant transformation of melanin-producing pigment cells (melanocytes). It is the most aggressive form of skin cancer and is characterized by high resistance to chemotherapeutic drugs, which results in a need to explore alternative methods for this disease therapy. Currently, new approaches to cancer treatment are being intensely developed, oncolytic immunotherapy being one of them. This approach consists in using viruses as targeted tumor-specific cytolytic agents capable of stimulating both tumor-specific and nonspecific immune responses. A considerable body of research is currently aimed on improving the immunostimulatory properties of viruses by inserting the genes encoding immunomodulatory proteins or tumor-specific antigenic determinants into viral genomes. For melanoma, the highest number of tumor-associated antigens (TAAs) has been identified, which serve as the basis for the development of anti-tumor DNA vaccines. The immunogenicity and efficacy of these drugs, however, remain low. The bottlenecks in using DNA vaccines to treat cancer are considered to be imperfect design of polyepitope constructs, as well as inefficient delivery of therapeutic molecules directly to the target cells. A partial solution to these problems may be represented by the use of oncolytic viruses as vectors for the delivery of artificial immunogens. The recombinant vaccinia virus was obtained by transient dominant selection. The cytolytic activity of the obtained virus was tested using the MTT assay. The oncolytic activity of the virus was assessed in the mouse xenograft model obtained using malignant SK-Mel-28 cells. This paper reports the production of a recombinant L-IVP_oncoM virus, the oncolytic virus for the delivery of anticancer therapeutic genes into the cells, on the basis of the vaccinia virus strain L-IVP. Toward this end, the gene encoding the Granulocyte-macrophage colony-stimulating factor (GM-CSF) and the artificial gene encoding a polyepitope immunogen containing the epitopes of the antigens over-expressed in melanoma cells were inserted into the virus genome. These insertions were located in the proximity of the genes encoding thymidine kinase (J2R) and viral growth factor (C11L), respectively. The properties of L-IVP_oncoM were studied in in vitro experiments using cell cultures of various origin and in the in vivo experiments using the mouse xenograft model. The basic experiments to assess the biological properties of the obtained L-IVP_oncoM, which are necessary to characterize the oncolytic virus, have been carried out.
Problems of Virology, 2020
Introduction. Currently, new directions in cancer therapy are actively developing, one of which i... more Introduction. Currently, new directions in cancer therapy are actively developing, one of which is oncolytic immunotherapy. This approach would be to use of viruses as cancer specific cytolytic agents capable of stimulating both the tumor-specific and non-specific immune response.The objective paper was obtain a recombinant vaccinia virus containing genes encoding immunostimulating molecules and study oncolytic and immunostimulating properties of recombinant virus.Material and methods. MTT test, ELISA, methods of transient dominant selection.Results. The recombinant vaccinia virus (L-IVP_oncoB) were obtained with deletion of the gene encoding thymidine kinase and had an integrated gene encoding GM-CSF. Also the virus have deletion of the gene encoding viral growth factor and integrated genes encoding synthetic tumor-specific polyepitopic immunogens. It was shown that the modifications made to the viral genome did not affect the growth characteristics of the virus when cultured on CV...
Typical scatter plots of the experimental sample showing the gating scheme. The experimental samp... more Typical scatter plots of the experimental sample showing the gating scheme. The experimental sample is the propidium iodide (PI)-labeled coculture of MCF-7 cells and HER2-specific cells. A – Scatter plot showing the distribution of all events corresponding to the emission parameters of the CFSE label in the FITC channel. Events corresponding CFSE-labeled cells are gated. B – Scatter plot showing the distribution of events from region of CFSE-labeled cells. Events corresponding to MCF-7 cells in terms of their phenotypic parameters (forward and side light scattering) are gated. C – Scatter plot showing the distribution of all events in the experimental sample with respect to parameters of forward and side light scattering. D – Scatter plot of the experimental sample (PI-labeled co-culture of MCF-7 cells and HER2-specific cells). E – Scatter plot of the control over spontaneous death of target cells (PI-labeled MCF-7 cells). (DOCX 160 kb)
A method for identification of fragments with high local similarity to human proteins within pote... more 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 involvement of such regions in immuno-pathogenesis 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 within HIV-1 proteins contain either T-cell (CD8+ CTL or CD4 + Th) or B-cell epitopes, or both of them simultaneously. The criteria for a design of safe polyepitopic antiviral vaccines that would allow us the...
L'invention concerne des constructions polyepitopiques immunogenes contenant des epitopes de ... more L'invention concerne des constructions polyepitopiques immunogenes contenant des epitopes de lymphocytes T cytotoxiques (CTL) et/ou de lymphocytes T auxiliaires (Th) et des sequences d'espacement optimisees qui permettent d'ameliorer le traitement et la presentation des epitopes, conduisant a l'induction d'un niveau eleve d'a la fois de reponses de lymphocytes T specifiques CD4+ et CD8+ et de types specifiques de cytokines, ainsi qu'un niveau eleve de protection et d'activite therapeutique.
Dendritic cells (DCs) loaded with tumor-associated antigens (TAA) are known to be the important a... more Dendritic cells (DCs) loaded with tumor-associated antigens (TAA) are known to be the important agents in antitumor response realization and still figure in lots of treatment schemes in cancer immunotherapy research. Here, we evaluated a cell-based protocol involving the use of original DNA constructs encoding the wide range of TAA epitopes expressed on different epithelial cancers. The constructs were transfected into ex-vivo-generated DCs of cancer patients (breast cancer, colorectal cancer, and non-small cell lung cancer). Direct cytotoxicity assay of effector cells, activated with the transfected DCs, showed a significant increase in the cytotoxicity against autologous tumor cells. The use of DNA-constructs encoding a large number of TAA’s for the in vitro DC loading to activate the T cell response could be a reliable and unified approach for immunotherapy and relapse prevention in patients with epithelial cancers.
The amino acid sequences of pMax DNA construct with N-terminal ubiquitin. N-terminal ubiquitin is... more The amino acid sequences of pMax DNA construct with N-terminal ubiquitin. N-terminal ubiquitin is underlined; C-terminal G replaced by V in ubiquitin for the protease cleavage site elimination. (DOCX 140 kb)
Cytokine, 2021
BACKGROUND B220+CD11c+plasmacytoid DCs(pDCs) are known to participate in the negative selection a... more BACKGROUND B220+CD11c+plasmacytoid DCs(pDCs) are known to participate in the negative selection and central tolerance induction by the capturing of self-antigens in peripheral tissues and further migration to the thymus using the CCL25-CCR9 chemotaxis axis. AIM Here we investigate the possibility of DCs migration stimulation to the thymus by the transfection with plasmid DNA-constructs encoding CCR9(pmaxCCR9) to develop a system for desired antigen delivery to the thymus for central tolerance induction. METHODS Dendritic cells(DCs) cultures were generated from UBC-GFP mice bone marrow cells expressing green fluorescent protein using the rmFlt3-L. DCs cultures were transfected with pmaxCCR9 by electroporation. The efficiency of electroporation was confirmed by RT-qPCR and flow cytometry. The migration of electroporated DCs was assessed in vitro and in vivo. RESULTS Dendritic cells(DCs) cultures obtained from UBC-GFP mice contained both B220+pDCs and SIRPa+cDC2. According to the RT-qP...
Russian journal of immunology: RJI: official journal of Russian Society of Immunology
A platform consisting of six oncolytic vaccinia viruses (VACV), expressing immunostimulatory and ... more A platform consisting of six oncolytic vaccinia viruses (VACV), expressing immunostimulatory and anti-cancer therapeutic molecules, was created. For address lysis of tumor cells the genes encoding thymidine kinase and viral growth factor in the VACV genome were inactivated. The disruption of these genes almost totally reduces the ability of the virus to replicate in normal cells. In place of these genes artificial genes were embedded that encode an immunostimulatory protein granulocyte-macrophage colony stimulating factor (GM-CSF) and the apoptosis-inducing protein TRAIL, with modified codon composition optimized for expression in mammalian cells, and a gene encoding green fluorescent protein for diagnostic use. Expression of GM-CSF will allow a better introduction of cancer antigens to the immune system of the organism, and the expression of the apoptosis-inducing protein will correct the mutations in the genome of tumor cells and activate apoptosis. Given the high tropism, recombi...
Vestnik Rossiĭskoĭ akademii meditsinskikh nauk / Rossiĭskaia akademiia meditsinskikh nauk, 1998
In the past 5 years, the investigators of the "VECTOR" SRB VB and the L.A. Tarasevich S... more In the past 5 years, the investigators of the "VECTOR" SRB VB and the L.A. Tarasevich State Institute of Standardization and Control of Medical Biological Preparations have jointly designed sera reference panels containing anti-HIV-1 IgG, anti-HCV IgG, and anti-HAV IgM which have been approved as national standard panels. The panels are intended for use in controlling the specificity and stability of the most widely used ELISA diagnostic kits and immunoblot test systems during production, control, and application stages. Some problems of development and production of these panels, including the representation of different sera in the panels and the selection of specific IgG concentrations in the different sera in the panel are described. The authors also attract attention to the stabilization of the specific characteristics of panel sera during storage and transportation.
Voprosy virusologii
Employment of radioimmunoassay led to the demonstration of serological crossing between tick-born... more Employment of radioimmunoassay led to the demonstration of serological crossing between tick-borne encephalitis (TBE) virus and Venezuelan equine encephalomyelitis (VEE) virus. Using hybridoma technology, three hybridomas were produced secreting monoclonal antibodies (MAb) cross-reacting with these two viruses. With MAb, the epitope of binding of these antibodies was shown to be located on protein E of TBE virus and protein E1 of VEE virus. Despite the low percentage (14%) of homology of amino acid sequences of these proteins, 12 areas with homology from 24% to 63% were demonstrated. Considering conservative replacements, homology of these areas was 53%-75%. The assumed existence of some of these areas in alpha-helical conformation may explain the observed immunological crossing.
Proc. 5th Int. Conf. on Bioinformatics of Genome Regulation and Structure (BGRS'2006), Novosibirsk, Russia, July 16–22, 2006, 2006
SUMMARY Motivation: Hemolysin II (HlyII) is one of several cytolytic proteins produced by Bacillu... more SUMMARY Motivation: Hemolysin II (HlyII) is one of several cytolytic proteins produced by Bacillus cereus, an opportunistic human pathogen. This toxin is secreted in a soluble form and cause its cytolytic effect by assembling into transmembrane pores. HlyII is proposed to be an important factor of B. cereus pathogenity and adaptivity. Results: We have built the homology model of the HlyII heptameric ionic channel. The model lacks the C-terminal domain of HlyII. Using de novo modeling, we have proposed a possible structure of the C- ...
Journal of Clinical Virology, 2004
Voprosy virusologii
Study of the prevalence of hepatitis C virus (HCV) markers in 153 patients of Municipal Infectiou... more Study of the prevalence of hepatitis C virus (HCV) markers in 153 patients of Municipal Infectious Diseases Clinical Hospital No. 1 in Novosibirsk revealed anti-HCV in 88.2% patients and viral RNA in 69.3% samples. For 93 Siberian HCV isolates 5'-UTR regions were amplified and sequenced. Comparison of these nucleotide sequences with databank showed that 63.4% HCV isolates belonged to subtype 1b, 7.5% to genotype 2, and 18.3% to genotype 3. In the rest HCV isolates the 5'-UTR sequences contained heretofore undescribed nucleotide substitutions, insertions, or deletions.
Typical scatter plots of gates used in DCs phenotyping analysis. DCs were analyzed by flow cytome... more Typical scatter plots of gates used in DCs phenotyping analysis. DCs were analyzed by flow cytometry in the region of large granular leukocytes. A – Events corresponding to large granular leukocytes in terms of their phenotypic parameters (forward and side light scattering) are gated. B – Events corresponding CD14-FITC-labeled cells are gated. C – Events corresponding CD83-APC-labeled cells are gated. D – Events corresponding CD86-PE-Cy7-labeled cells are gated. E – Events corresponding double-positive HLA-DR-PerCP-Cy5 and CD11c-PE-labeled cells are gated. (DOCX 785 kb)
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology, 2010
To prepare the monoclonal antibody (mAb) used for kits to detect the BNP32 antigen by means of do... more To prepare the monoclonal antibody (mAb) used for kits to detect the BNP32 antigen by means of double-antibody sandwich ELISA assay. Comparasion differences on detection BNP between double-antibody sandwich ELISA and Roche ECL. BALB/c mice were immunized with purified recombinant BNP32 protein and by routine hybridoma technique, Then comparasion differences on detection BNP between double-antibody sandwich ELISA and Roche ECL. 16 hybridoma cell lines secreting potent mAb against BNP32 were obtained. The subtype of these 16 mAb were found to be IgG1, IgG2a and IgM, then their cross-blocking properties were analyzed, when they reacted with the BNP32 protein in direct ELISA in order to offer the valuable data for selecting feasible pair of mAb in the detection of the BNP32 antigen. All the mAbs were used for the detection of BNP32 by double-antibody sandwich ELISA. In addition, the mAbs were purified and HRP-labelled in advance. Finally, we had successfully screened a pair of mAbs, whi...
Journal of Immunology Research
Background. Nonspecific immunosuppressive therapy for graft rejection and graft-versus-host disea... more Background. Nonspecific immunosuppressive therapy for graft rejection and graft-versus-host disease (GVHD) is often accompanied by severe side effects such as opportunistic infections and cancers. Several approaches have been developed to suppress transplantation reactions using tolerogenic cells, including induction of FoxP3+ Tregs with antigen-loaded dendritic cells (DCs) and induction of CD4+IL-10+ cells with interleukin IL-10-producing DCs. Here, we assessed the effectiveness of both approaches in the suppression of graft rejection and GVHD. Methods. IL-10-producing DCs were generated by the transfection of DCs with DNA constructs encoding mouse IL-10. Antigen-loaded DCs from C57BL/6 mice were generated by transfection with DNA constructs encoding antigenic determinants from the H2 locus of CBA mice which differ from the homologous antigenic determinants of C57BL/6 mice. Results. We found that both IL-10-producing DCs and antigen-loaded immature DCs could suppress graft rejectio...
Journal of Interferon & Cytokine Research
Frontiers in Bioscience-Landmark
Background: A search for efficient graft rejection modulation techniques for the promotion of dur... more Background: A search for efficient graft rejection modulation techniques for the promotion of durable engraftment remains to be a matter of close study all over the world. Despite the variety of immunosuppressive drugs, the schemes currently used show a lack of selectivity and have a number of side effects. Here we investigated an approach for the induction of antigen-specific tolerance in a human "stimulator-responder" model in vitro, using dendritic cells (DCs) transfected with designed DNA constructs encoding the stimulator's major histocompatibility complex (MHC) epitopes. Methods: The object of the study is peripheral blood mononuclear cells (PBMCs) from 10 healthy donors. To induce antigen-specific tolerance, personalized DNA constructs were created for five responder-stimulator pairs, based on the sequences of donors' and recipients' MHCs. DNA sequencing was performed to select epitopes for incorporation into genetic constructs. A mixed lymphocyte culture assay was used (i) to assess the proliferative response in both directions for all possible stimulator-responder pairs (90 reactions) and (ii) to assess the tolerogenic properties of the generated transfected DCs (5 reactions). Results: A significant increase in the amounts of FoxP3 + CD4 + CD25 + cells and in IL-10 production was shown in culture of donor mononuclear cells after co-cultivation with the responder's dendritic cells transfected with donor-specific plasmids. The tolerogenic cultures generated using tolerogenic DCs transfected with MHC epitopes had a significantly greater ability to inhibit the proliferation of autologous MNCs in response to an allogeneic MHC stimulus. Conclusions: The produced DCs transfected with DNA constructs against HLA stimulating epitopes exhibited tolerogenic properties and may be used to develop antigen-specific tolerance. Thus, we proposed a perspective approach to the induction of antigen-specific tolerance, which should subsequently be studied for use in clinical practice.
Molecular Genetics Microbiology and Virology (Russian version), 2020
Федеральное бюджетное учреждение науки «Государственный научный центр вирусологии и биотехнологии... more Федеральное бюджетное учреждение науки «Государственный научный центр вирусологии и биотехнологии «Вектор» Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека, р.п. Кольцово, Новосибирская область, Россия; 2 Федеральное государственное бюджетное учреждение науки «Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наук», Новосибирск, Россия Резюме Введение. Меланома-злокачественная опухоль, образующаяся при перерождении пигментных клеток, продуцирующих меланины, является наиболее агрессивной разновидностью онкологических заболеваний кожи и характеризуется высокой резистентностью к химиотерапевтическим препаратам, что обусловливает необходимость поиска альтернативных методов терапии данного заболевания. В настоящее время активно развиваются новые направления в терапии рака, одним из которых является онколитическая иммунотерапия, заключающаяся в применении вирусов в качестве онкоселективных цитолитических агентов, способных стимулировать как опухоль-специфичный, так и неспецифичный иммунный ответ организма. Значительная доля современных научных работ посвящена повышению иммуностимулирующих свойств вирусов путем встройки в состав вирусных геномов генов, кодирующих белки-иммуномодуляторы или антигенные детерминанты, характерные для опухолевых клеток. Для меланомы идентифицировано наибольшее количество опухоль-ассоциированных антигенов (ОАА), на основе которых ведут разработки противоопухолевых ДНК-вакцин. Иммуногенность и эффективность таких препаратов остаются на низком уровне. Проблемой в использовании ДНК-вакцин для лечения рака может быть несовершенный дизайн полиэпитопной конструкции, а также неэффективная доставка терапевтических молекул непосредственно в целевые клетки. Частичным решением данной проблемы является использование в качестве способа доставки искусственных иммуногенов онколитических вирусов. Материал и методы. Рекомбинантный вирус осповакцины получен при помощи метода временной доминантной селекции. Цитолитическую эффективность полученного вируса оценивали в МТТ-тесте. Онколитическую эффективность полученного вируса оценивали с помощью мышиной модели ксенографтов с использованием злокачественных клеток SK-Mel-28. Результаты и обсуждение. В данной работе на основе вируса осповакцины штамма L-IVP был получен рекомбинантный вирус L-IVP_oncoM, представляющий собой онколитический вирус, обеспечивающий доставку противораковых терапевтических генов в клетки организма. С этой целью в геном вируса были встроены ген, кодирующий гранулоцит-макрофагальный колониестимулирующий фактор, и искусственный ген, кодирующий полиэпитопный иммуноген, состоящий из эпитопов антигенов, гиперэкспрессирующихся в клетках меланомы. Данные инсерции проведены в районе генов, кодирующих тимидинкиназу (J2R), и вирусный фактор роста (C11L) соответственно. Изучены свойства L-IVP_oncoM в экспериментах in vitro на культурах клеток различного генеза и in vivo в мышиной модели ксенографтов. Заключение. Проведены основные эксперименты по оценке биологических свойств полученного L-IVP_oncoM, которые являются необходимыми для характеризации онколитического вируса.
Molecular Genetics, Microbiology and Virology, 2020
Melanoma is a tumor that forms as a result of malignant transformation of melanin-producing pigme... more Melanoma is a tumor that forms as a result of malignant transformation of melanin-producing pigment cells (melanocytes). It is the most aggressive form of skin cancer and is characterized by high resistance to chemotherapeutic drugs, which results in a need to explore alternative methods for this disease therapy. Currently, new approaches to cancer treatment are being intensely developed, oncolytic immunotherapy being one of them. This approach consists in using viruses as targeted tumor-specific cytolytic agents capable of stimulating both tumor-specific and nonspecific immune responses. A considerable body of research is currently aimed on improving the immunostimulatory properties of viruses by inserting the genes encoding immunomodulatory proteins or tumor-specific antigenic determinants into viral genomes. For melanoma, the highest number of tumor-associated antigens (TAAs) has been identified, which serve as the basis for the development of anti-tumor DNA vaccines. The immunogenicity and efficacy of these drugs, however, remain low. The bottlenecks in using DNA vaccines to treat cancer are considered to be imperfect design of polyepitope constructs, as well as inefficient delivery of therapeutic molecules directly to the target cells. A partial solution to these problems may be represented by the use of oncolytic viruses as vectors for the delivery of artificial immunogens. The recombinant vaccinia virus was obtained by transient dominant selection. The cytolytic activity of the obtained virus was tested using the MTT assay. The oncolytic activity of the virus was assessed in the mouse xenograft model obtained using malignant SK-Mel-28 cells. This paper reports the production of a recombinant L-IVP_oncoM virus, the oncolytic virus for the delivery of anticancer therapeutic genes into the cells, on the basis of the vaccinia virus strain L-IVP. Toward this end, the gene encoding the Granulocyte-macrophage colony-stimulating factor (GM-CSF) and the artificial gene encoding a polyepitope immunogen containing the epitopes of the antigens over-expressed in melanoma cells were inserted into the virus genome. These insertions were located in the proximity of the genes encoding thymidine kinase (J2R) and viral growth factor (C11L), respectively. The properties of L-IVP_oncoM were studied in in vitro experiments using cell cultures of various origin and in the in vivo experiments using the mouse xenograft model. The basic experiments to assess the biological properties of the obtained L-IVP_oncoM, which are necessary to characterize the oncolytic virus, have been carried out.
Problems of Virology, 2020
Introduction. Currently, new directions in cancer therapy are actively developing, one of which i... more Introduction. Currently, new directions in cancer therapy are actively developing, one of which is oncolytic immunotherapy. This approach would be to use of viruses as cancer specific cytolytic agents capable of stimulating both the tumor-specific and non-specific immune response.The objective paper was obtain a recombinant vaccinia virus containing genes encoding immunostimulating molecules and study oncolytic and immunostimulating properties of recombinant virus.Material and methods. MTT test, ELISA, methods of transient dominant selection.Results. The recombinant vaccinia virus (L-IVP_oncoB) were obtained with deletion of the gene encoding thymidine kinase and had an integrated gene encoding GM-CSF. Also the virus have deletion of the gene encoding viral growth factor and integrated genes encoding synthetic tumor-specific polyepitopic immunogens. It was shown that the modifications made to the viral genome did not affect the growth characteristics of the virus when cultured on CV...
Typical scatter plots of the experimental sample showing the gating scheme. The experimental samp... more Typical scatter plots of the experimental sample showing the gating scheme. The experimental sample is the propidium iodide (PI)-labeled coculture of MCF-7 cells and HER2-specific cells. A – Scatter plot showing the distribution of all events corresponding to the emission parameters of the CFSE label in the FITC channel. Events corresponding CFSE-labeled cells are gated. B – Scatter plot showing the distribution of events from region of CFSE-labeled cells. Events corresponding to MCF-7 cells in terms of their phenotypic parameters (forward and side light scattering) are gated. C – Scatter plot showing the distribution of all events in the experimental sample with respect to parameters of forward and side light scattering. D – Scatter plot of the experimental sample (PI-labeled co-culture of MCF-7 cells and HER2-specific cells). E – Scatter plot of the control over spontaneous death of target cells (PI-labeled MCF-7 cells). (DOCX 160 kb)
A method for identification of fragments with high local similarity to human proteins within pote... more 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 involvement of such regions in immuno-pathogenesis 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 within HIV-1 proteins contain either T-cell (CD8+ CTL or CD4 + Th) or B-cell epitopes, or both of them simultaneously. The criteria for a design of safe polyepitopic antiviral vaccines that would allow us the...
L'invention concerne des constructions polyepitopiques immunogenes contenant des epitopes de ... more L'invention concerne des constructions polyepitopiques immunogenes contenant des epitopes de lymphocytes T cytotoxiques (CTL) et/ou de lymphocytes T auxiliaires (Th) et des sequences d'espacement optimisees qui permettent d'ameliorer le traitement et la presentation des epitopes, conduisant a l'induction d'un niveau eleve d'a la fois de reponses de lymphocytes T specifiques CD4+ et CD8+ et de types specifiques de cytokines, ainsi qu'un niveau eleve de protection et d'activite therapeutique.
Dendritic cells (DCs) loaded with tumor-associated antigens (TAA) are known to be the important a... more Dendritic cells (DCs) loaded with tumor-associated antigens (TAA) are known to be the important agents in antitumor response realization and still figure in lots of treatment schemes in cancer immunotherapy research. Here, we evaluated a cell-based protocol involving the use of original DNA constructs encoding the wide range of TAA epitopes expressed on different epithelial cancers. The constructs were transfected into ex-vivo-generated DCs of cancer patients (breast cancer, colorectal cancer, and non-small cell lung cancer). Direct cytotoxicity assay of effector cells, activated with the transfected DCs, showed a significant increase in the cytotoxicity against autologous tumor cells. The use of DNA-constructs encoding a large number of TAA’s for the in vitro DC loading to activate the T cell response could be a reliable and unified approach for immunotherapy and relapse prevention in patients with epithelial cancers.
The amino acid sequences of pMax DNA construct with N-terminal ubiquitin. N-terminal ubiquitin is... more The amino acid sequences of pMax DNA construct with N-terminal ubiquitin. N-terminal ubiquitin is underlined; C-terminal G replaced by V in ubiquitin for the protease cleavage site elimination. (DOCX 140 kb)
Cytokine, 2021
BACKGROUND B220+CD11c+plasmacytoid DCs(pDCs) are known to participate in the negative selection a... more BACKGROUND B220+CD11c+plasmacytoid DCs(pDCs) are known to participate in the negative selection and central tolerance induction by the capturing of self-antigens in peripheral tissues and further migration to the thymus using the CCL25-CCR9 chemotaxis axis. AIM Here we investigate the possibility of DCs migration stimulation to the thymus by the transfection with plasmid DNA-constructs encoding CCR9(pmaxCCR9) to develop a system for desired antigen delivery to the thymus for central tolerance induction. METHODS Dendritic cells(DCs) cultures were generated from UBC-GFP mice bone marrow cells expressing green fluorescent protein using the rmFlt3-L. DCs cultures were transfected with pmaxCCR9 by electroporation. The efficiency of electroporation was confirmed by RT-qPCR and flow cytometry. The migration of electroporated DCs was assessed in vitro and in vivo. RESULTS Dendritic cells(DCs) cultures obtained from UBC-GFP mice contained both B220+pDCs and SIRPa+cDC2. According to the RT-qP...
Russian journal of immunology: RJI: official journal of Russian Society of Immunology
A platform consisting of six oncolytic vaccinia viruses (VACV), expressing immunostimulatory and ... more A platform consisting of six oncolytic vaccinia viruses (VACV), expressing immunostimulatory and anti-cancer therapeutic molecules, was created. For address lysis of tumor cells the genes encoding thymidine kinase and viral growth factor in the VACV genome were inactivated. The disruption of these genes almost totally reduces the ability of the virus to replicate in normal cells. In place of these genes artificial genes were embedded that encode an immunostimulatory protein granulocyte-macrophage colony stimulating factor (GM-CSF) and the apoptosis-inducing protein TRAIL, with modified codon composition optimized for expression in mammalian cells, and a gene encoding green fluorescent protein for diagnostic use. Expression of GM-CSF will allow a better introduction of cancer antigens to the immune system of the organism, and the expression of the apoptosis-inducing protein will correct the mutations in the genome of tumor cells and activate apoptosis. Given the high tropism, recombi...
Vestnik Rossiĭskoĭ akademii meditsinskikh nauk / Rossiĭskaia akademiia meditsinskikh nauk, 1998
In the past 5 years, the investigators of the "VECTOR" SRB VB and the L.A. Tarasevich S... more In the past 5 years, the investigators of the "VECTOR" SRB VB and the L.A. Tarasevich State Institute of Standardization and Control of Medical Biological Preparations have jointly designed sera reference panels containing anti-HIV-1 IgG, anti-HCV IgG, and anti-HAV IgM which have been approved as national standard panels. The panels are intended for use in controlling the specificity and stability of the most widely used ELISA diagnostic kits and immunoblot test systems during production, control, and application stages. Some problems of development and production of these panels, including the representation of different sera in the panels and the selection of specific IgG concentrations in the different sera in the panel are described. The authors also attract attention to the stabilization of the specific characteristics of panel sera during storage and transportation.
Voprosy virusologii
Employment of radioimmunoassay led to the demonstration of serological crossing between tick-born... more Employment of radioimmunoassay led to the demonstration of serological crossing between tick-borne encephalitis (TBE) virus and Venezuelan equine encephalomyelitis (VEE) virus. Using hybridoma technology, three hybridomas were produced secreting monoclonal antibodies (MAb) cross-reacting with these two viruses. With MAb, the epitope of binding of these antibodies was shown to be located on protein E of TBE virus and protein E1 of VEE virus. Despite the low percentage (14%) of homology of amino acid sequences of these proteins, 12 areas with homology from 24% to 63% were demonstrated. Considering conservative replacements, homology of these areas was 53%-75%. The assumed existence of some of these areas in alpha-helical conformation may explain the observed immunological crossing.
Proc. 5th Int. Conf. on Bioinformatics of Genome Regulation and Structure (BGRS'2006), Novosibirsk, Russia, July 16–22, 2006, 2006
SUMMARY Motivation: Hemolysin II (HlyII) is one of several cytolytic proteins produced by Bacillu... more SUMMARY Motivation: Hemolysin II (HlyII) is one of several cytolytic proteins produced by Bacillus cereus, an opportunistic human pathogen. This toxin is secreted in a soluble form and cause its cytolytic effect by assembling into transmembrane pores. HlyII is proposed to be an important factor of B. cereus pathogenity and adaptivity. Results: We have built the homology model of the HlyII heptameric ionic channel. The model lacks the C-terminal domain of HlyII. Using de novo modeling, we have proposed a possible structure of the C- ...
Journal of Clinical Virology, 2004
Voprosy virusologii
Study of the prevalence of hepatitis C virus (HCV) markers in 153 patients of Municipal Infectiou... more Study of the prevalence of hepatitis C virus (HCV) markers in 153 patients of Municipal Infectious Diseases Clinical Hospital No. 1 in Novosibirsk revealed anti-HCV in 88.2% patients and viral RNA in 69.3% samples. For 93 Siberian HCV isolates 5'-UTR regions were amplified and sequenced. Comparison of these nucleotide sequences with databank showed that 63.4% HCV isolates belonged to subtype 1b, 7.5% to genotype 2, and 18.3% to genotype 3. In the rest HCV isolates the 5'-UTR sequences contained heretofore undescribed nucleotide substitutions, insertions, or deletions.
Typical scatter plots of gates used in DCs phenotyping analysis. DCs were analyzed by flow cytome... more Typical scatter plots of gates used in DCs phenotyping analysis. DCs were analyzed by flow cytometry in the region of large granular leukocytes. A – Events corresponding to large granular leukocytes in terms of their phenotypic parameters (forward and side light scattering) are gated. B – Events corresponding CD14-FITC-labeled cells are gated. C – Events corresponding CD83-APC-labeled cells are gated. D – Events corresponding CD86-PE-Cy7-labeled cells are gated. E – Events corresponding double-positive HLA-DR-PerCP-Cy5 and CD11c-PE-labeled cells are gated. (DOCX 785 kb)
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology, 2010
To prepare the monoclonal antibody (mAb) used for kits to detect the BNP32 antigen by means of do... more To prepare the monoclonal antibody (mAb) used for kits to detect the BNP32 antigen by means of double-antibody sandwich ELISA assay. Comparasion differences on detection BNP between double-antibody sandwich ELISA and Roche ECL. BALB/c mice were immunized with purified recombinant BNP32 protein and by routine hybridoma technique, Then comparasion differences on detection BNP between double-antibody sandwich ELISA and Roche ECL. 16 hybridoma cell lines secreting potent mAb against BNP32 were obtained. The subtype of these 16 mAb were found to be IgG1, IgG2a and IgM, then their cross-blocking properties were analyzed, when they reacted with the BNP32 protein in direct ELISA in order to offer the valuable data for selecting feasible pair of mAb in the detection of the BNP32 antigen. All the mAbs were used for the detection of BNP32 by double-antibody sandwich ELISA. In addition, the mAbs were purified and HRP-labelled in advance. Finally, we had successfully screened a pair of mAbs, whi...
Journal of Immunology Research
Background. Nonspecific immunosuppressive therapy for graft rejection and graft-versus-host disea... more Background. Nonspecific immunosuppressive therapy for graft rejection and graft-versus-host disease (GVHD) is often accompanied by severe side effects such as opportunistic infections and cancers. Several approaches have been developed to suppress transplantation reactions using tolerogenic cells, including induction of FoxP3+ Tregs with antigen-loaded dendritic cells (DCs) and induction of CD4+IL-10+ cells with interleukin IL-10-producing DCs. Here, we assessed the effectiveness of both approaches in the suppression of graft rejection and GVHD. Methods. IL-10-producing DCs were generated by the transfection of DCs with DNA constructs encoding mouse IL-10. Antigen-loaded DCs from C57BL/6 mice were generated by transfection with DNA constructs encoding antigenic determinants from the H2 locus of CBA mice which differ from the homologous antigenic determinants of C57BL/6 mice. Results. We found that both IL-10-producing DCs and antigen-loaded immature DCs could suppress graft rejectio...
Journal of Interferon & Cytokine Research