Papers by Sergei Shchelkunov
Infekciâ i Immunitet, Mar 24, 2021
автор для переписки Сергеев А. А., к.б.н., ведущий научный сотрудник Кабанов А. С., к.б.н., старш... more автор для переписки Сергеев А. А., к.б.н., ведущий научный сотрудник Кабанов А. С., к.б.н., старший научный сотрудник Якубицкий С. Н., младший научный сотрудник Бауэр Т. В., младший научный сотрудник Пьянков С. А., ведущий научный сотрудник Название статьи: ПАТОГЕННОСТЬ И ИММУНОГЕННОСТЬ ВАРИАНТОВ ВИРУСА ОСПОВАКЦИНЫ ПРИ РАЗНЫХ СПОСОБАХ ИХ ВВЕДЕНИЯ МЫШАМ 14 страниц, 4 рисунка.
Аннотация. Фактор некроза опухолей (TNF)-один из основных цитокинов, медиаторов иммунной системы,... more Аннотация. Фактор некроза опухолей (TNF)-один из основных цитокинов, медиаторов иммунной системы, обеспечивающих защиту организма человека от вирусных инфекций. В процессе эволюции антропогенный вирус натуральной оспы (Variola virus, VARV) освоил эффективные механизмы преодоления иммунологических барьеров человека, кодируя в своем геноме белки, способные взаимодействовать с рецепторами цитокинов организма-хозяина, блокируя таким образом их активность. В частности, ген G2R этого вируса кодирует белок CrmB, который эффективно связывает TNF человека и мышей. При этом данный белок является двухдоменным и наряду с TNF-связывающим N-концевым доменом содержит С-концевой хемокин-связывающий домен. При использовании методологии молекулярного клонирования нами ранее получен рекомбинантный бакуловирус, продуцирующий в клетках насекомых рекомбинантный белок CrmB вируса натуральной оспы (VARV-CrmB), и показан его TNF-нейтрализующий потенциал в различных тестах in vitro и in vivo. С целью снижения иммуногенности этого вирусного белка при его многократном введении для терапии хронических воспалительных заболеваний получена рекомбинантная плазмида, направляющая в клетках Escherichia coli биосинтез укороченного однодоменного TNF-связывающего (TNF-BD) белка VARV. Методом металл-хелатной аффинной хроматографии из клеток выделен рекомбинантный белок TNF-BD. Терапевтический потенциал белка TNF-BD изучен в экспериментальной модели септического шока, индуцированного в организме мышей введением бактериального липополисахарида (ЛПС). После индукции септического шока животным вводили разные дозы рекомбинантного белка TNF-BD и регистрировали их гибель в течение 7 сут. Все мыши, не получавшие препарат белка TNF-BD после инъекции ЛПС, погибли через 3 сут, а в группах животных, которым вводили TNF-BD, в зависимости от дозы этого белка выжили 30, 40 или 60 % мышей. Результаты исследования демонстрируют наличие специфической фармакологической активности у рекомбинантного белка TNF-BD, синтезированного в бактериальных клетках, в мышиной модели ЛПС-индуцированного септического шока. Ключевые слова: белок VARV-CrmB; вирус натуральной оспы; ЛПС; септический шок; TNF-связывающий домен.
Acta naturae, Jan 20, 2023
International Journal of Molecular Sciences
The protein encoded by the vaccinia virus D4R gene has base excision repair uracil–DNA N-glycosyl... more The protein encoded by the vaccinia virus D4R gene has base excision repair uracil–DNA N-glycosylase (vvUNG) activity and also acts as a processivity factor in the viral replication complex. The use of a protein unlike PolN/PCNA sliding clamps is a unique feature of orthopoxviral replication, providing an attractive target for drug design. However, the intrinsic processivity of vvUNG has never been estimated, leaving open the question whether it is sufficient to impart processivity to the viral polymerase. Here, we use the correlated cleavage assay to characterize the translocation of vvUNG along DNA between two uracil residues. The salt dependence of the correlated cleavage, together with the similar affinity of vvUNG for damaged and undamaged DNA, support the one-dimensional diffusion mechanism of lesion search. Unlike short gaps, covalent adducts partly block vvUNG translocation. Kinetic experiments show that once a lesion is found it is excised with a probability ~0.76. Varying ...
Vavilov Journal of Genetics and Breeding
The monkeypox epidemic, which became unusually widespread among humans in 2022, has brought aware... more The monkeypox epidemic, which became unusually widespread among humans in 2022, has brought awareness about the necessity of smallpox vaccination of patients in the risk groups. The modern smallpox vaccine variants are introduced either intramuscularly or by skin scarification. Intramuscular vaccination cannot elicit an active immune response, since tissues at the vaccination site are immunologically poor. Skin has evolved into an immunologically important organ in mammals; therefore, intradermal delivery of a vaccine can ensure reliable protective immunity. Historically, vaccine inoculation into scarified skin (the s.s. route) was the first immunization method. However, it does not allow accurate vaccine dosing, and high-dose vaccines need to be used to successfully complete this procedure. Intradermal (i.d.) vaccine injection, especially low-dose one, can be an alternative to the s.s. route. This study aimed to compare the s.s. and i.d. smallpox immunization routes in a mouse mode...
Russian Journal of Infection and Immunity, 2021
Smallpox eradication and lack of adequate animal model for smallpox infection underlies a necessi... more Smallpox eradication and lack of adequate animal model for smallpox infection underlies a necessity to assess immunogenic and protective properties of genetic engineering-created live attenuated smallpox vaccines in several animal models of orthopoxviral infections. Here we compared immunogenic and protective properties of the recombinant vaccinia virus (VACV) LIVP-GFP intradermally (i.d.) inoculated to mice, guinea pigs and rabbits. LIVP-GFP immunization in all animal species was applied at dose of 2 × 104 or 2 × 106 PFU. Control animals were injected with saline. Blood sampling was performed on day 28 after virus LIVP-GFP or saline inoculation. Blood samples were taken intravitally from the retro-orbital venous sinus in mice, heart in guinea pigs or marginal ear vein in rabbits. Serum samples were isolated by precipitating blood cells via centrifugation. The serum anti-VACV IgG titers were determined by ELISA. On day 30 post-immunization animals were intranasally challenged with l...
Viruses
Considering that vaccination against smallpox with live vaccinia virus led to serious adverse eff... more Considering that vaccination against smallpox with live vaccinia virus led to serious adverse effects in some cases, the WHO, after declaration of the global eradication of smallpox in 1980, strongly recommended to discontinue the vaccination in all countries. This led to the loss of immunity against not only smallpox but also other zoonotic orthopoxvirus infections in humans over the past years. An increasing number of human infections with zoonotic orthopoxviruses and, first of all, monkeypox, force us to reconsider a possible re-emergence of smallpox or a similar disease as a result of natural evolution of these viruses. The review contains a brief analysis of the results of studies on genomic organization and evolution of human pathogenic orthopoxviruses, development of modern methods for diagnosis, vaccination, and chemotherapy of smallpox, monkeypox, and other zoonotic human orthopoxvirus infections.
Russian Journal of Genetics: Applied Research, 2012
Fundamentals of the development of transplastome plants and structural features of genetic con st... more Fundamentals of the development of transplastome plants and structural features of genetic con structs for incorporation into chloroplast genomes are reviewed. Major achievements in the transformation and expression of alien genes in chloroplasts are considered. The potential of this method for significant increase in the production of alien proteins in transplastome plants is substantiated.
Doklady Biochemistry and Biophysics, 2015
Viruses
The conventional live smallpox vaccine based on the vaccinia virus (VACV) cannot be widely used t... more The conventional live smallpox vaccine based on the vaccinia virus (VACV) cannot be widely used today because it is highly reactogenic. Therefore, there is a demand for designing VACV variants possessing enhanced immunogenicity, making it possible to reduce the vaccine dose and, therefore, significantly eliminate the pathogenic effect of the VACV on the body. In this study, we analyzed the development of the humoral and T cell-mediated immune responses elicited by immunizing mice with low-dose VACV variants carrying the mutant A34R gene (which increases production of extracellular virions) or the deleted A35R gene (whose protein product inhibits antigen presentation by the major histocompatibility complex class II). The VACV LIVP strain, which is used as a smallpox vaccine in Russia, and its recombinant variants LIVP-A34R*, LIVP-dA35R, and LIVP-A34R*-dA35R, were compared upon intradermal immunization of BALB/c mice at a dose of 104 pfu/animal. The strongest T cell-mediated immunity ...
Vavilov Journal of Genetics and Breeding
Due to cessation of mass smallpox vaccination in 1980, the collective immunity of humans against ... more Due to cessation of mass smallpox vaccination in 1980, the collective immunity of humans against orthopoxvirus infections has virtually been lost. Therefore, the risk of spreading zoonotic human orthopoxvirus infections caused by monkeypox and cowpox viruses has increased in the world. First-generation smallpox vaccines based on Vaccinia virus (VAC) are reactogenic and therefore not suitable for mass vaccination under current conditions. This necessitates the development of modern safe live vaccines based on VAC using genetic engineering. We created the VACΔ6 strain by transient dominant selection. In the VACΔ6 genome, five virulence genes were intentionally deleted, and one gene was inactivated by inserting a synthetic DNA fragment. The virus was passaged 71 times in CV-1 cells to obtain the VACΔ6 strain from the VAC LIVP clonal variant. Such a long passage history might have led to additional off-target mutations in VACΔ6 compared to the original LIVP variant. To prevent this, we ...
Russian Journal of Genetics, 2016
Gene therapy can offer a new approach to arthritis treatment which acts at an inflammation site. ... more 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.
Doklady Akademii nauk / [Rossiĭskaia akademii nauk], 1996
Vavilov Journal of Genetics and Breeding, 2021
Vaccination is the most simple and reliable approach of protection to virus infections. The most ... more Vaccination is the most simple and reliable approach of protection to virus infections. The most effective agents are live vaccines, usually low-virulence organisms for humans and closely related to pathogenic viruses or attenuated as a result of mutations/deletions in the genome of pathogenic virus. Smallpox vaccination with live vaccinia virus (VACV) closely related to smallpox virus played a key role in the success of the global smallpox eradication program carried out under the World Health Organization auspices. As a result of the WHO decision as of 1980 to stop smallpox vaccination, humankind has lost immunity not only to smallpox, but also to other zoonotic, orthopoxviruscaused human infections. This new situation allows orthopoxviruses to circulate in the human population and, as a consequence, to alter several established concepts of the ecology and range of sensitive hosts for various orthopoxvirus species. Classic VACV-based live vaccine for vaccination against orthopoxvi...
Medical Immunology (Russia), 2022
At the present time, vast majority of human population lacks immunity against orthopoxvirus infec... more At the present time, vast majority of human population lacks immunity against orthopoxvirus infections caused by variola (smallpox), monkeypox, cowpox, or buffalopox viruses. More and more mass outbreaks of orthopoxvirus infections are yearly registered among humans on different continents. To prevent transition of these outbreaks to widespread epidemics, we should develop appropriate immunoprophylaxis strategies. Currently, massive usage of the classic live vaccine based on vaccinia virus is not acceptable, due to its high reactogenicity. Therefore, it is necessary to develop the variants of vaccinia virus with reduced virulence and increased immunogenicity/protectivity. The aim of this work was to study protective effects against a lethal orthopoxvirus infection occuring after low-dose immunization of mice with vaccinia virus variants, i.e., carrying mutant A34R gene causing increased production of extracellular virions, or a A35R gene deletion encoding protein product inhibiting ...
Vavilovskij Žurnal Genetiki i Selekcii, 2015
The study was performed with complete nucleotide sequences of the mitochondrial DNA of Wistar rat... more The study was performed with complete nucleotide sequences of the mitochondrial DNA of Wistar rats and prematurely aging OXYS rats, sensitive to the cataractogenic effect of galactose, kept at the Gene pool of laboratory animals shared access center, Institute of Cytology and Genetics (ICG) of the Siberian Branch of the Russian Academy of Sciences. The nucleotide sequences were submitted to GenBank. It was shown that two additional unique nucleotide substitutions between rats OXYS (ICG) and Wistar (ICG) were absent from other known nucleotide sequences of rat mitochondrial DNA of rats and that they did not cause amino acid substitutions, so that all phenotypic differences between the lines were determined only by differences in their nuclear DNA. The comparative analysis showed that Wistar (ICG) rats had a significant number of nucleotide substitutions compared to rat strains derived from an outbred Wistar colony. This observation contradicts the conjectured relationship of Wistar (...
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WHO wishes to express its appreciation to all those who have contributed to this document, as fol... more WHO wishes to express its appreciation to all those who have contributed to this document, as follows:
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Papers by Sergei Shchelkunov