Browsing by Author "Petkov, Stefan"

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    Cellular immune response induced by dna immunization of mice with drug resistant integrases of hiv-1 clade a offers partial protection against growth and metastatic activity of integrase-expressing adenocarcinoma cells
    (2021-06) Isaguliants, Maria; Krotova, Olga; Petkov, Stefan; Jansons, Juris; Bayurova, Ekaterina; Mezale, Dzeina; Fridrihsone, Ilze; Kilpelainen, Athina; Podschwadt, Philip; Agapkina, Yulia; Smirnova, Olga; Kostic, Linda; Saleem, Mina; Latyshev, Oleg; Eliseeva, Olesja; Malkova, Anastasia; Gorodnicheva, Tatiana; Wahren, Britta; Gordeychuk, Ilya; Starodubova, Elizaveta; Latanova, Anastasia; Research Department
    Therapeutic DNA-vaccination against drug-resistant HIV-1 may hinder emergence and spread of drug-resistant HIV-1, allowing for longer successful antiretroviral treatment (ART) up-to relief of ART. We designed DNA-vaccines against drug-resistant HIV-1 based on consensus clade A integrase (IN) resistant to raltegravir: IN_in_r1 (L74M/E92Q/V151I/N155H/G163R) or IN_in_r2 (E138K/G140S/Q148K) carrying D64V abrogating IN activity. INs, overexpressed in mammalian cells from synthetic genes, were assessed for stability, route of proteolytic degradation, and ability to induce oxidative stress. Both were found safe in immunotoxicity tests in mice, with no inherent carcinogenicity: their expression did not enhance tumorigenic or metastatic potential of adenocarcinoma 4T1 cells. DNA-immunization of mice with INs induced potent multicytokine T-cell response mainly against aa 209–239, and moderate IgG response cross-recognizing diverse IN variants. DNA-immunization with IN_in_r1 protected 60% of mice from challenge with 4Tlluc2 cells expressing non-mutated IN, while DNA-immunization with IN_in_r2 protected only 20% of mice, although tumor cells expressed IN matching the immunogen. Tumor size inversely correlated with IN-specific IFN-γ/IL-2 T-cell response. IN-expressing tumors displayed compromised metastatic activity restricted to lungs with reduced metastases size. Protective potential of IN immunogens relied on their immunogenicity for CD8+ T-cells, dependent on proteasomal processing and low level of oxidative stress.
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    DNA immunization site determines the level of gene expression and the magnitude, but not the type of the induced immune response
    (2018-06-04) Petkov, Stefan; Starodubova, Elizaveta; Latanova, Anastasia; Kilpelainen, Athina; Latyshev, Oleg; Svirskis, Simons; Wahren, Britta; Chiodi, Francesca; Gordeychuk, Ilya; Isaguliants, Maria; Rīga Stradiņš University
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    Fusion to Flaviviral Leader Peptide Targets HIV-1 Reverse Transcriptase for Secretion and Reduces Its Enzymatic Activity and Ability to Induce Oxidative Stress but Has No Major Effects on Its Immunogenic Performance in DNA-Immunized Mice
    (2017-06-22) Latanova, Anastasia; Petkov, Stefan; Kuzmenko, Yulia; Kilpelaïnen, Athina; Ivanov, Alexander; Smirnova, Olga; Krotova, Olga; Korolev, Sergey; Hinkula, Jorma; Karpov, Vadim; Isaguliants, Maria; Starodubova, Elizaveta; Rīga Stradiņš University
    Reverse transcriptase (RT) is a key enzyme in viral replication and susceptibility to ART and a crucial target of immunotherapy against drug-resistant HIV-1. RT induces oxidative stress which undermines the attempts to make it immunogenic. We hypothesized that artificial secretion may reduce the stress and make RT more immunogenic. Inactivated multidrug-resistant RT (RT1.14opt-in) was N-terminally fused to the signal providing secretion of NS1 protein of TBEV (Ld) generating optimized inactivated Ld-carrying enzyme RT1.14oil. Promotion of secretion prohibited proteasomal degradation increasing the half-life and content of RT1.14oil in cells and cell culture medium, drastically reduced the residual polymerase activity, and downmodulated oxidative stress. BALB/c mice were DNA-immunized with RT1.14opt-in or parental RT1.14oil by intradermal injections with electroporation. Fluorospot and ELISA tests revealed that RT1.14opt-in and RT1.14oil induced IFN-γ/IL-2, RT1.14opt-in induced granzyme B, and RT1.14oil induced perforin production. Perforin secretion correlated with coproduction of IFN-γ and IL-2 (R=0,97). Both DNA immunogens induced strong anti-RT antibody response. Ldpeptide was not immunogenic. Thus, Ld-driven secretion inferred little change to RT performance in DNA immunization. Positive outcome was the abrogation of polymerase activity increasing safety of RT-based DNA vaccines. Identification of the molecular determinants of low cellular immunogenicity of RT requires further studies.
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    HIV-1 Protease as DNA Immunogen against Drug Resistance in HIV-1 Infection : DNA Immunization with Drug Resistant HIV-1 Protease Protects Mice from Challenge with Protease-Expressing Cells
    (2023-01) Petkov, Stefan; Kilpeläinen, Athina; Bayurova, Ekaterina; Latanova, Anastasia; Mezale, Dzeina; Fridrihsone, Ilse; Starodubova, Elizaveta; Jansons, Juris; Dudorova, Alesja; Gordeychuk, Ilya; Wahren, Britta; Isaguliants, Maria; Research Department
    DNA immunization with HIV-1 protease (PR) is advanced for immunotherapy of HIV-1 infection to reduce the number of infected cells producing drug-resistant virus. A consensus PR of the HIV-1 FSU_A strain was designed, expression-optimized, inactivated (D25N), and supplemented with drug resistance (DR) mutations M46I, I54V, and V82A common for FSU_A. PR variants with D25N/M46I/I54V (PR_Ai2mut) and with D25N/M46I/I54V/V82A (PR_Ai3mut) were cloned into the DNA vaccine vector pVAX1, and PR_Ai3mut, into a lentiviral vector for the transduction of murine mammary adenocarcinoma cells expressing luciferase 4T1luc2. BALB/c mice were DNA-immunized by intradermal injections of PR_Ai, PR_Ai2mut, PR_Ai3mut, vector pVAX1, or PBS with electroporation. All PR variants induced specific CD8+ T-cell responses revealed after splenocyte stimulation with PR-derived peptides. Splenocytes of mice DNA-immunized with PR_Ai and PR_Ai2mut were not activated by peptides carrying V82A, whereas splenocytes of PR_Ai3mut-immunized mice recognized both peptides with and without V82A mutation. Mutations M46I and I54V were immunologically silent. In the challenge study, DNA immunization with PR_Ai3mut protected mice from the outgrowth of subcutaneously implanted adenocarcinoma 4T1luc2 cells expressing PR_Ai3mut; a tumor was formed only in 1/10 implantation sites and no metastases were detected. Immunizations with other PR variants were not protective; all mice formed tumors and multiple metastasis in the lungs, liver, and spleen. CD8+ cells of PR_Ai3mut DNA-immunized mice exhibited strong IFN-γ/IL-2 responses against PR peptides, while the splenocytes of mice in other groups were nonresponsive. Thus, immunization with a DNA plasmid encoding inactive HIV-1 protease with DR mutations suppressed the growth and metastatic activity of tumor cells expressing PR identical to the one encoded by the immunogen. This demonstrates the capacity of T-cell response induced by DNA immunization to recognize single DR mutations, and supports the concept of the development of immunotherapies against drug resistance in HIV-1 infection. It also suggests that HIV-1-infected patients developing drug resistance may have a reduced natural immune response against DR HIV-1 mutations causing an immune escape.
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    HIV-1 Reverse Transcriptase Promotes Tumor Growth and Metastasis Formation via ROS-Dependent Upregulation of Twist
    (2019-12-02) Bayurova, Ekaterina; Jansons, Juris; Skrastina, Dace; Smirnova, Olga; Mezale, Dzeina; Kostyusheva, Anastasia; Kostyushev, Dmitry; Petkov, Stefan; Podschwadt, Philip; Valuev-Elliston, Vladimir; Sasinovich, Sviataslau; Korolev, Sergey; Warholm, Per; Latanova, Anastasia; Starodubova, Elizaveta; Tukhvatulin, Amir; Latyshev, Oleg; Selimov, Renat; Metalnikov, Pavel; Komarov, Alexander; Ivanova, Olga; Gorodnicheva, Tatiana; Kochetkov, Sergey; Gottikh, Marina; Strumfa, Ilze; Ivanov, Alexander; Gordeychuk, Ilya; Isaguliants, Maria; Department of Pathology; Rīga Stradiņš University
    HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of "non-AIDS-defining" malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression of Vimentin and Twist. These properties, particularly, the expression of Twist, correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A and Twist. No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression.
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    Reciprocal inhibition of immunogenic performance in mice of two potent dna immunogens targeting hcv-related liver cancer
    (2021-05) Jansons, Juris; Skrastina, Dace; Kurlanda, Alisa; Petkov, Stefan; Avdoshina, Darya; Kuzmenko, Yulia; Krotova, Olga; Trofimova, Olga; Gordeychuk, Ilya; Sominskaya, Irina; Isaguliants, Maria; Institute of Microbiology and Virology
    Chronic HCV infection and associated liver cancer impose a heavy burden on the healthcare system. Direct acting antivirals eliminate HCV, unless it is drug resistant, and partially reverse liver disease, but they cannot cure HCV-related cancer. A possible remedy could be a multi-component immunotherapeutic vaccine targeting both HCV-infected and malignant cells, but also those not infected with HCV. To meet this need we developed a two-component DNA vaccine based on the highly conserved core protein of HCV to target HCV-infected cells, and a renowned tumor-associated antigen telomerase reverse transcriptase (TERT) based on the rat TERT, to target malignant cells. Their synthetic genes were expression-optimized, and HCV core was truncated after aa 152 (Core152opt) to delete the domain interfering with immunogenicity. Core152opt and TERT DNA were highly immunogenic in BALB/c mice, inducing IFN-γ/IL-2/TNF-α response of CD4+ and CD8+ T cells. Additionally, DNA-immunization with TERT enhanced cellular immune response against luciferase encoded by a co-delivered plasmid (Luc DNA). However, DNA-immunization with Core152opt and TERT mix resulted in abrogation of immune response against both components. A loss of bioluminescence signal after co-delivery of TERT and Luc DNA into mice indicated that TERT affects the in vivo expression of luciferase directed by the immediate early cytomegalovirus and interferon-β promoters. Panel of mutant TERT variants was created and tested for their expression effects. TERT with deleted N-terminal nucleoli localization signal and mutations abrogating telomerase activity still suppressed the IFN-β driven Luc expression, while the inactivated reverse transcriptase domain of TERT and its analogue, enzymatically active HIV-1 reverse transcriptase, exerted only weak suppressive effects, implying that suppression relied on the presence of the full-length/nearly full-length TERT, but not its enzymatic activity. The effect(s) could be due to interference of the ectopically expressed xenogeneic rat TERT with biogenesis of mRNA, ribosomes and protein translation in murine cells, affecting the expression of immunogens. HCV core can aggravate this effect, leading to early apoptosis of co-expressing cells, preventing the induction of immune response.