DNA immunization site determines the level of gene expression and the magnitude, but not the type of the induced immune response

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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

Funding Information: Funding: Experiments were supported by the grants of the Russian Science Fund 15-15-30039, Russian Fund for Basic Research 20-04-01034, Latvian Science Fund LZP 2018-2-03-08, and EU-ROPARTNER project “Strengthening and spreading international partnership activities of the Faculty of Biology and Environmental Protection of University of Lodz, Poland, for interdisciplinary research and innovation”. Mobility and method acquisition were supported by Swedish institute PI project 19806/2016TP, and Horizon 2020 project VACTRAIN#692293. MI and BW were supported by Horizon 2020 grant EAVI contract N68113. Funding Information: Experiments were supported by the grants of the Russian Science Fund 15-15-30039, Russian Fund for Basic Research 20-04-01034, Latvian Science Fund LZP 2018-2-03-08, and EU-ROPARTNER project ?Strengthening and spreading international partnership activities of the Faculty of Biology and Environmental Protection of University of Lodz, Poland, for interdisciplinary research and innovation?. Mobility and method acquisition were supported by Swedish institute PI project 19806/2016TP, and Horizon 2020 project VACTRAIN#692293. MI and BW were supported by Horizon 2020 grant EAVI contract N68113. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.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.publishersversionPeer reviewe

HIV-1 Reverse Transcriptase Promotes Tumor Growth and Metastasis Formation via ROS-Dependent Upregulation of Twist

Funding Information: https://orcid.org/0000-0002-6160-2203 Bayurova Ekaterina [email protected] 1 2 Jansons Juris [email protected] 3 4 Skrastina Dace [email protected] 3 4 https://orcid.org/0000-0002-4980-9754 Smirnova Olga [email protected] 5 Mezale Dzeina [email protected] 3 Kostyusheva Anastasia [email protected] 6 Kostyushev Dmitry [email protected] 6 Petkov Stefan [email protected] 7 Podschwadt Philip [email protected] 7 https://orcid.org/0000-0003-0365-570X Valuev-Elliston Vladimir [email protected] 5 Sasinovich Sviataslau [email protected] 7 https://orcid.org/0000-0003-2278-4451 Korolev Sergey [email protected] 8 Warholm Per [email protected] 9 https://orcid.org/0000-0002-2260-6551 Latanova Anastasia [email protected] 1 5 https://orcid.org/0000-0003-2183-0858 Starodubova Elizaveta [email protected] 1 5 https://orcid.org/0000-0001-8506-2339 Tukhvatulin Amir [email protected] 1 Latyshev Oleg [email protected] 1 Selimov Renat [email protected] 10 Metalnikov Pavel [email protected] 10 Komarov Alexander [email protected] 10 https://orcid.org/0000-0002-3673-4714 Ivanova Olga [email protected] 5 Gorodnicheva Tatiana [email protected] 11 https://orcid.org/0000-0002-7443-6961 Kochetkov Sergey [email protected] 5 Gottikh Marina [email protected] 8 Strumfa Ilze [email protected] 3 https://orcid.org/0000-0002-5659-9679 Ivanov Alexander [email protected] 5 Gordeychuk Ilya [email protected] 1 2 12 https://orcid.org/0000-0001-9382-2254 Isaguliants Maria [email protected] 1 2 3 7 García-Rivas Gerardo 1 NF Gamaleya Research Center of Epidemiology and Microbiology Moscow Russia 2 Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences Moscow Russia chumakovs.ru 3 Department of Pathology Riga Stradins University Riga Latvia rsu.lv 4 Latvian Biomedical Research and Study Centre Riga Latvia lu.lv 5 Engelhardt Institute of Molecular Biology Russian Academy of Sciences Moscow Russia ras.ru 6 National Medical Research Center for Tuberculosis and Infectious Diseases Moscow Russia 7 Department of Microbiology Tumor and Cell Biology Karolinska Institutet Stockholm Sweden ki.se 8 Chemistry Department and Belozersky Institute of Physico-Chemical Biology Lomonosov Moscow State University Moscow Russia msu.ru 9 Science for Life Laboratory Stockholm University Stockholm Sweden su.se 10 Russian State Center for Quality and Standardization of Veterinary Drugs and Feed (VGNKI) Moscow Russia 11 Evrogen Moscow Russia 12 Sechenov First Moscow State Medical University Moscow Russia mma.ru 2019 2 12 2019 2019 08 05 2019 01 11 2019 05 11 2019 2 12 2019 2019 Copyright © 2019 Ekaterina Bayurova et al. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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. Ministry of Science and Higher Education of the Russian Federation 075-15-2019-1660 Latvian Science Council LZP-2018/2-0308 EU VACTRAIN Russian Foundation for Basic Research 17-00-00085 17_04_00583 17_54_30002 Publisher Copyright: © 2019 Ekaterina Bayurova et al.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.publishersversionPeer reviewe

Extracellular Vesicles in <i>Flaviviridae</i> Pathogenesis: Their Roles in Viral Transmission, Immune Evasion, and Inflammation

The members of the Flaviviridae family are becoming an emerging threat for public health, causing an increasing number of infections each year and requiring effective treatment. The consequences of these infections can be severe and include liver inflammation with subsequent carcinogenesis, endothelial damage with hemorrhage, neuroinflammation, and, in some cases, death. The mechanisms of Flaviviridae pathogenesis are being actively investigated, but there are still many gaps in their understanding. Extracellular vesicles may play important roles in these mechanisms, and, therefore, this topic deserves detailed research. Recent data have revealed the involvement of extracellular vesicles in steps of Flaviviridae pathogenesis such as transmission, immune evasion, and inflammation, which is critical for disease establishment. This review covers recent papers on the roles of extracellular vesicles in the pathogenesis of Flaviviridae and includes examples of clinical applications of the accumulated data

Flaviviridae Nonstructural Proteins: The Role in Molecular Mechanisms of Triggering Inflammation

Members of the Flaviviridae family are posing a significant threat to human health worldwide. Many flaviviruses are capable of inducing severe inflammation in humans. Flaviviridae nonstructural proteins, apart from their canonical roles in viral replication, have noncanonical functions strongly affecting antiviral innate immunity. Among these functions, antagonism of type I IFN is the most investigated; meanwhile, more data are accumulated on their role in the other pathways of innate response. This review systematizes the last known data on the role of Flaviviridae nonstructural proteins in molecular mechanisms of triggering inflammation, with an emphasis on their interactions with TLRs and RLRs, interference with NF-&kappa;B and cGAS-STING signaling, and activation of inflammasomes

Activation of Early Proinflammatory Responses by TBEV NS1 Varies between the Strains of Various Subtypes

Tick-borne encephalitis (TBE) is an emerging zoonosis that may cause long-term neurological sequelae or even death. Thus, there is a growing interest in understanding the factors of TBE pathogenesis. Viral genetic determinants may greatly affect the severity and consequences of TBE. In this study, nonstructural protein 1 (NS1) of the tick-borne encephalitis virus (TBEV) was tested as such a determinant. NS1s of three strains with similar neuroinvasiveness belonging to the European, Siberian and Far-Eastern subtypes of TBEV were studied. Transfection of mouse cells with plasmids encoding NS1 of the three TBEV subtypes led to different levels of NS1 protein accumulation in and secretion from the cells. NS1s of TBEV were able to trigger cytokine production either in isolated mouse splenocytes or in mice after delivery of NS1 encoding plasmids. The profile and dynamics of TNF-α, IL-6, IL-10 and IFN-γ differed between the strains. These results demonstrated the involvement of TBEV NS1 in triggering an immune response and indicated the diversity of NS1 as one of the genetic factors of TBEV pathogenicity

Correction: DNA immunization site determines the level of gene expression and the magnitude, but not the type of the induced immune response.

[This corrects the article DOI: 10.1371/journal.pone.0197902.]

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

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

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

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-&gamma;/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

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

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-gamma/IL-2, RT1.14opt-in induced granzyme B, and RT1.14oil induced perforin production. Perforin secretion correlated with coproduction of IFN-gamma and IL-2 (R = 0,97). Both DNA immunogens induced strong anti-RT antibody response. Ld peptide 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.Funding Agencies|Russian Foundation of Basic Research [14-04-01817]; Russian Science Foundation [15-15-30039]; EU Twinning Project VACTRAIN [692293]; Swedish Institute PI [19806_2016]</p