Browsing by Author "Starodubova, Elizaveta S."

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    The Immunogenicity in Mice of HCV Core Delivered as DNA Is Modulated by Its Capacity to Induce Oxidative Stress and Oxidative Stress Response
    (2019-02-28) Jansons, Juris; Sominskaya, Irina; Petrakova, Natalia; Starodubova, Elizaveta S.; Smirnova, Olga A.; Alekseeva, Ekaterina; Bruvere, Ruta; Eliseeva, Olesja; Skrastina, Dace; Kashuba, Elena; Mihailova, Marija; Kochetkov, Sergey N.; Ivanov, Alexander V.; Isaguliants, Maria G.; Department of Pathology
    HCV core is an attractive HCV vaccine target, however, clinical or preclinical trials of core-based vaccines showed little success. We aimed to delineate what restricts its immunogenicity and improve immunogenic performance in mice. We designed plasmids encoding full-length HCV 1b core and its variants truncated after amino acids (aa) 60, 98, 152, 173, or up to aa 36 using virus-derived or synthetic polynucleotides (core191/60/98/152/173/36_191v or core152s DNA, respectively). We assessed their level of expression, route of degradation, ability to trigger the production of reactive oxygen species/ROS, and to activate the components of the Nrf2/ARE antioxidant defense pathway heme oxygenase 1/HO-1 and NAD(P)H: quinone oxidoreductase/Nqo-1. All core variants with the intact N-terminus induced production of ROS, and up-regulated expression of HO-1 and Nqo-1. The capacity of core variants to induce ROS and up-regulate HO-1 and Nqo-1 expression predetermined their immunogenicity in DNA-immunized BALB/c and C57BL/6 mice. The most immunogenic was core 152s, expressed at a modest level and inducing moderate oxidative stress and oxidative stress response. Thus, immunogenicity of HCV core is shaped by its ability to induce ROS and oxidative stress response. These considerations are important in understanding the mechanisms of viral suppression of cellular immune response and in HCV vaccine design.
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    Oxidative Stress during HIV Infection : Mechanisms and Consequences
    (2016) Ivanov, Alexander V.; Valuev-Elliston, Vladimir T.; Ivanova, Olga N.; Kochetkov, Sergey N.; Starodubova, Elizaveta S.; Bartosch, Birke; Isaguliants, Maria G.; Rīga Stradiņš University
    It is generally acknowledged that reactive oxygen species (ROS) play crucial roles in a variety of natural processes in cells. If increased to levels which cannot be neutralized by the defense mechanisms, they damage biological molecules, alter their functions, and also act as signaling molecules thus generating a spectrum of pathologies. In this review, we summarize current data on oxidative stress markers associated with human immunodeficiency virus type-1 (HIV-1) infection, analyze mechanisms by which this virus triggers massive ROS production, and describe the status of various defense mechanisms of the infected host cell. In addition, we have scrutinized scarce data on the effect of ROS on HIV-1 replication. Finally, we present current state of knowledge on the redox alterations as crucial factors of HIV-1 pathogenicity, such as neurotoxicity and dementia, exhaustion of CD4+/CD8+ T-cells, predisposition to lung infections, and certain side effects of the antiretroviral therapy, and compare them to the pathologies associated with the nitrosative stress.
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    Prokaryotic expression, purification and immunogenicity in rabbits of the small antigen of hepatitis delta virus
    (2016-10-01) Tunitskaya, Vera L.; Eliseeva, Olesja V.; Valuev-Elliston, Vladimir T.; Tyurina, Daria A.; Zakirova, Natalia F.; Khomich, Olga A.; Kalis, Martins; Latyshev, Oleg E.; Starodubova, Elizaveta S.; Ivanova, Olga N.; Kochetkov, Sergey N.; Isaguliants, Maria G.; Ivanov, Alexander V.; Institute of Microbiology and Virology
    Hepatitis delta virus (HDV) is a viroid-like blood-borne human pathogen that accompanies hepatitis B virus infection in 5% patients. HDV has been studied for four decades; however, the knowledge on its life-cycle and pathogenesis is still sparse. The studies are hampered by the absence of the commercially-available HDV-specific antibodies. Here, we describe a set of reproducible methods for the expression in E. coli of His-tagged small antigen of HDV (S-HDAg), its purification, and production of polyclonal anti-S-HDAg antibodies in rabbits. S-HDAg was cloned into a commercial vector guiding expression of the recombinant proteins with the C-terminal His-tag. We optimized S-HDAg protein purification procedure circumventing a low affinity of the His-tagged S-HDAg to the Ni-nitrilotriacetyl agarose (Ni-NTA-agarose) resin. Optimization allowed us to obtain S-HDAg with >90% purity. S-HDAg was used to immunize Shinchilla grey rabbits which received 80 µg of S-HDAg in two subcutaneous primes in the complete, followed by four 40 µg boosts in incomplete Freunds adjuvant. Rabbits were bled two weeks post each boost. Antibody titers determined by indirect ELISA exceeded 107. Anti-S-HDAg antibodies detected the antigen on Western blots in the amounts of up-to 100 pg. They were also successfully used to characterize the expression of S-HDAg in the eukaryotic cells by immunofluorescent staining/confocal microscopy.