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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.
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Vesículas Extracelulares , Infecções por Flaviviridae , Flaviviridae , Humanos , Infecções por Flaviviridae/tratamento farmacológico , Evasão da Resposta Imune , Inflamação/terapiaRESUMO
Proteasome inhibitors are used in the therapy of several cancers, and clinical trials are underway for their use in the treatment of glioblastoma (GBM). However, GBM becomes resistant to chemotherapy relatively rapidly. Recently, the overexpression of ribonucleotide reductase (RNR) genes was found to mediate therapy resistance in GBM. The use of combinations of chemotherapeutic agents is considered a promising direction in cancer therapy. The present work aimed to evaluate the efficacy of the combination of proteasome and RNR inhibitors in yeast and GBM cell models. We have shown that impaired proteasome function results in increased levels of RNR subunits and increased enzyme activity in yeast. Co-administration of the proteasome inhibitor bortezomib and the RNR inhibitor hydroxyurea was found to significantly reduce the growth rate of S. cerevisiae yeast. Accordingly, the combination of bortezomib and another RNR inhibitor gemcitabine reduced the survival of DBTRG-05MG compared to the HEK293 cell line. Thus, yeast can be used as a simple model to evaluate the efficacy of combinations of proteasome and RNR inhibitors.
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Glioblastoma , Saccharomyces cerevisiae , Humanos , Complexo de Endopeptidases do Proteassoma , Glioblastoma/tratamento farmacológico , Bortezomib/farmacologia , Células HEK293RESUMO
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.
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Vírus da Encefalite Transmitidos por Carrapatos , Encefalite Transmitida por Carrapatos , Proteínas não Estruturais Virais , Animais , Camundongos , Vírus da Encefalite Transmitidos por Carrapatos/classificação , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Encefalite Transmitida por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/virologia , Interleucina-10/genética , Zoonoses , Proteínas não Estruturais Virais/metabolismoRESUMO
Currently, many DNA vaccines against infectious diseases are in clinical trials; however, their efficacy needs to be improved. The potency of DNA immunogen can be optimized by targeting technologies. In the current study, to increase the efficacy of NS1 encoded by plasmid, proteasome targeting was applied. NS1 variants with or without translocation sequence and with ornithine decarboxylase as a signal of proteasomal degradation were tested for expression, localization, protein turnover, proteasomal degradation and protection properties. Deletion of translocation signal abrogated presentation of NS1 on the cell surface and increased proteasomal processing of NS1. Fusion with ornithine decarboxylase led to an increase of protein turnover and the proteasome degradation rate of NS1. Immunization with NS1 variants with increased proteasome processing protected mice from viral challenge only partially; however, the survival time of infected mice was prolonged in these groups. These data can give a presupposition for formulation of specific immune therapy for infected individuals.
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Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Proteólise , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismo , Vacinas Virais/imunologia , Animais , Camundongos , Análise de Sobrevida , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
BACKGROUND: Infection with tick-borne encephalitis virus (TBEV) causes pathological changes in the central nervous system. However, the possible redox alterations in the infected cells that can contribute to the virus pathogenicity remain unknown. OBJECTIVE: In the current study we explored the ability of TBEV nonstructural protein 1 (NS1) to induce oxidative stress and activate antioxidant defense via the nuclear factor (erythroid-derived-2)-like 2/antioxidant response element (Nrf2/ARE) pathway. METHODS: HEK 293T cells were transfected with plasmid encoding NS1 protein, and the production of reactive oxygen species (ROS) was measured using oxidation-sensitive dyes, the activation of the ARE promoter was estimated using a reporter plasmid, and the expression of phase II detoxifying enzymes was quantified by measuring their mRNA levels using RT-qPCR. RESULTS: A high level of ROS production was detected in cells transfected with NS1-expressing plasmid. In addition, this protein activated the promoter with an ARE and upregulated the transcription of ARE-dependent genes that encode phase II enzymes. CONCLUSION: TBEV NS1 protein both triggers ROS production and activates a defense Nrf2/ARE pathway. These data suggest that a role of redox-mediated processes in TBEV-induced damage of the central nervous system should also be explored. These data can contribute to a better understanding of TBEV pathogenicity, further improvement of TBE treatment, and the development of vaccine candidates against this infection.
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Elementos de Resposta Antioxidante , Vírus da Encefalite Transmitidos por Carrapatos/química , Vírus da Encefalite Transmitidos por Carrapatos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Transdução de Sinais , Proteínas não Estruturais Virais/fisiologia , Vírus da Encefalite Transmitidos por Carrapatos/genética , Células HEK293 , Células HeLa , Humanos , Fator 2 Relacionado a NF-E2/genética , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Transfecção , Proteínas não Estruturais Virais/genéticaRESUMO
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.
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Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Vírus Delta da Hepatite/imunologia , Antígenos da Hepatite delta/imunologia , Animais , Anticorpos Antivirais/sangue , Linhagem Celular , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Imunofluorescência , Vírus Delta da Hepatite/genética , Antígenos da Hepatite delta/biossíntese , Humanos , RNA Viral/genética , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The efficient cell-mediated immune response clears cells expressing deoxyribonucleic acid (DNA) immunogens, but there are no methods to monitor this in vivo. We hypothesized that immune-mediated clearance can be monitored in vivo if DNA immunogens are coexpressed with reporter(s). To test this, we designed genes encoding human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) fused via its N- or C-terminus to 30-amino acid-long Gly-Ala-repeat of Epstein-Barr virus nuclear antigen 1 or via the N-terminus to the transport signal of invariant chain/Ii or inserted between the cytoplasmic and luminal domains of lysosome-associated membrane protein I (LAMP). DNA immunogens mixed with luciferase gene were injected into BALB/c mice with subsequent electroporation. Reporter expression seen as luminescence was monitored by in vivo imaging. When luminescence faded, mice were sacrificed, and their splenocytes were stimulated with RT-derived antigens. Fading of luminescence correlated with the RT-specific secretion of interferon-γ and interleukin-2. Both immune and in vivo imaging techniques concordantly demonstrated an enhanced immunogenicity of RT-LAMP and of the N-terminal Gly-Ala-RT fusion genes. In vivo imaging performed as an animal-sparing method to estimate the overall performance of DNA immunogens, predicting it early in the experiment. So far, in vivo imaging cannot be a substitute for conventional immune assays, but it is supplementary to them. Further experiments are needed to identify which arms of cellular immune response in vivo imaging monitors best.
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Imagem Óptica , Vacinas Sintéticas/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Epitopos/imunologia , Feminino , Células HEK293 , Transcriptase Reversa do HIV/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Linfócitos T/imunologia , Vacinas de DNA/imunologia , Vacinas Sintéticas/genéticaRESUMO
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-κB and cGAS-STING signaling, and activation of inflammasomes.
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Flaviviridae , Flaviviridae/metabolismo , Humanos , Imunidade Inata , Inflamassomos , Inflamação , Transdução de SinaisRESUMO
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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[This corrects the article DOI: 10.1371/journal.pone.0197902.].
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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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Telomerase reverse transcriptase (TERT) is a classic tumor-associated antigen overexpressed in majority of tumors. Several TERT-based cancer vaccines are currently in clinical trials, but immune correlates of their antitumor activity remain largely unknown. Here, we characterized fine specificity and lytic potential of immune response against rat TERT in mice. BALB/c mice were primed with plasmids encoding expression-optimized hemagglutinin-tagged or nontagged TERT or empty vector and boosted with same DNA mixed with plasmid encoding firefly luciferase (Luc DNA). Injections were followed by electroporation. Photon emission from booster sites was assessed by in vivo bioluminescent imaging. Two weeks post boost, mice were sacrificed and assessed for IFN-γ, interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α) production by T-cells upon their stimulation with TERT peptides and for anti-TERT antibodies. All TERT DNA-immunized mice developed cellular and antibody response against epitopes at the N-terminus and reverse transcriptase domain (rtTERT) of TERT. Photon emission from mice boosted with TERT/TERT-HA+Luc DNA was 100 times lower than from vector+Luc DNA-boosted controls. Bioluminescence loss correlated with percent of IFN-γ/IL-2/TNF-α producing CD8+ and CD4+ T-cells specific to rtTERT, indicating immune clearance of TERT/Luc-coexpressing cells. We made murine adenocarcinoma 4T1luc2 cells to express rtTERT by lentiviral transduction. Expression of rtTERT significantly reduced the capacity of 4T1luc2 to form tumors and metastasize in mice, while not affecting in vitro growth. Mice which rejected the tumors developed T-cell response against rtTERT and low/no response to the autoepitope of TERT. This advances rtTERT as key component of TERT-based therapeutic vaccines against cancer.
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BACKGROUND: Hepatitis C core protein is an attractive target for HCV vaccine aimed to exterminate HCV infected cells. However, although highly immunogenic in natural infection, core appears to have low immunogenicity in experimental settings. We aimed to design an HCV vaccine prototype based on core, and devise immunization regimens that would lead to potent anti-core immune responses which circumvent the immunogenicity limitations earlier observed. METHODS: Plasmids encoding core with no translation initiation signal (pCMVcore); with Kozak sequence (pCMVcoreKozak); and with HCV IRES (pCMVcoreIRES) were designed and expressed in a variety of eukaryotic cells. Polyproteins corresponding to HCV 1b amino acids (aa) 1-98 and 1-173 were expressed in E. coli. C57BL/6 mice were immunized with four 25-microg doses of pCMVcoreKozak, or pCMV (I). BALB/c mice were immunized with 100 microg of either pCMVcore, or pCMVcoreKozak, or pCMVcoreIRES, or empty pCMV (II). Lastly, BALB/c mice were immunized with 20 microg of core aa 1-98 in prime and boost, or with 100 microg of pCMVcoreKozak in prime and 20 microg of core aa 1-98 in boost (III). Antibody response, [3H]-T-incorporation, and cytokine secretion by core/core peptide-stimulated splenocytes were assessed after each immunization. RESULTS: Plasmids differed in core-expression capacity: mouse fibroblasts transfected with pCMVcore, pCMVcoreIRES and pCMVcoreKozak expressed 0.22 +/- 0.18, 0.83 +/- 0.5, and 13 +/- 5 ng core per cell, respectively. Single immunization with highly expressing pCMVcoreKozak induced specific IFN-gamma and IL-2, and weak antibody response. Single immunization with plasmids directing low levels of core expression induced similar levels of cytokines, strong T-cell proliferation (pCMVcoreIRES), and antibodies in titer 103(pCMVcore). Boosting with pCMVcoreKozak induced low antibody response, core-specific T-cell proliferation and IFN-gamma secretion that subsided after the 3rd plasmid injection. The latter also led to a decrease in specific IL-2 secretion. The best was the heterologous pCMVcoreKozak prime/protein boost regiment that generated mixed Th1/Th2-cellular response with core-specific antibodies in titer >or= 3 x 10(3). CONCLUSION: Thus, administration of highly expressed HCV core gene, as one large dose or repeated injections of smaller doses, may suppress core-specific immune response. Instead, the latter is induced by a heterologous DNA prime/protein boost regiment that circumvents the negative effects of intracellular core expression.
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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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Estresse Oxidativo , Vacinas de DNA/imunologia , Proteínas do Core Viral/imunologia , Sequência de Aminoácidos , Animais , Feminino , Células HEK293 , Humanos , Imunidade Celular , Imunização , Interferon gama/biossíntese , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Mutantes/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas do Core Viral/químicaRESUMO
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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Adenocarcinoma/virologia , Neoplasias da Mama/virologia , Infecções por HIV/metabolismo , Transcriptase Reversa do HIV/metabolismo , HIV-1/metabolismo , Neoplasias Mamárias Experimentais/virologia , Proteína 1 Relacionada a Twist/metabolismo , Animais , Carcinogênese , Processos de Crescimento Celular , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Infecções por HIV/patologia , Transcriptase Reversa do HIV/genética , HIV-1/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutação/genética , Metástase Neoplásica , Espécies Reativas de Oxigênio/metabolismo , Proteína 1 Relacionada a Twist/genética , Regulação para CimaRESUMO
Optimization of DNA vaccine delivery improves the potency of the immune response and is crucial to clinical success. Here, we inquired how such optimization impacts the magnitude of the response, its specificity and type. BALB/c mice were DNA-immunized with two model immunogens, HIV-1 protease and reverse transcriptase by intramuscular or intradermal injections with electroporation. DNA immunogens were co-delivered with DNA encoding luciferase. Delivery and expression were monitored by in vivo bioluminescence imaging (BLI). The endpoint immune responses were assessed by IFN-γ/IL-2 FluoroSpot, multiparametric flow cytometry and antibody ELISA. Expression and immunogenicity were compared in relation to the delivery route. Regardless of the route, protease generated mainly IFN-γ, and reverse transcriptase, IL-2 and antibody response. BLI of mice immunized with protease- or reverse transcriptase/reporter plasmid mixtures, demonstrated significant loss of luminescence over time. The rate of decline of luminescence strongly correlated with the magnitude of immunogen-specific response, and depended on the immunogenicity profile and the immunization route. In vitro and in vivo BLI-based assays demonstrated that intradermal delivery strongly improved the immunogenicity of protease, and to a lesser extent, of reverse transcriptase. Immune response polarization and epitope hierarchy were not affected. Thus, by changing delivery/immunogen expression sites, it is possible to modulate the magnitude, but not the type or fine specificity of the induced immune response.
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Imunização , Vacinas de DNA/imunologia , Animais , Anticorpos Antivirais/imunologia , Citocinas/metabolismo , Epitopos/imunologia , Feminino , Expressão Gênica , Protease de HIV/metabolismo , Espaço Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Músculos/imunologia , Pele/imunologia , Vacinas de DNA/genéticaRESUMO
Envelope protein E2 of human hepatitis C virus (HCV) is an attractive component of a prototype HCV vaccine. Delivered by DNA immunogens, E2 evokes specific immune response of Th1-type, failing to induce either considerable antibody production, or T-helper cell proliferation. We aimed at modulating the immunogenic performance of E2 gene by changing the mode of protein expression in eukaryotic cells. Plasmids were constructed encoding full-length E2 and nonstructural protein 1 (p7) fused to either 13 or 38 C-terminal amino acids (aa) of HCV E1 that contain second hydrophobic segment of E1 stop-transfer signal, or a complete E1 stop-transfer signal with duplicated second hydrophobic segment. Injected into BALB/c mice, E2/p7 genes induced potent antibody and T-helper cell response targeted against hypervariable region 1, aa 472-586 of E2, and a novel epitope at aa 774-796 of p7. Profile of cytokines secreted by proliferating mouse splenocytes stimulated in vitro with E2- and p7-derived peptides, indicated mixed Th1/Th2 type of immune response. Thus, the full-length E2 and p7 genes supplied in one cassette were both immunogenic. E2/p7 containing a complete E1 stop-transfer signal with prolonged membrane spanning domain was superior to the shorter E2/p7 version in terms of both antibody and cellular immunogenicity. Optimal performance of HCV E2 could thus be achieved without the aid of external/heterologous signals by easing, through modification of the E2 signal sequence, the release of E2 from the rough ER while retaining full-length E2 and p7 sequences. This finding may help to improve the Th2 performance of HCV envelope genes as prototype vaccines.
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Genes Virais , Hepacivirus/imunologia , Sinais Direcionadores de Proteínas/fisiologia , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/imunologia , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular Transformada , Transformação Celular Viral , Chlorocebus aethiops , Escherichia coli/genética , Variação Genética , Células HeLa , Hepacivirus/genética , Humanos , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plasmídeos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas Virais/imunologiaRESUMO
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. 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.
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Vacinas contra a AIDS/imunologia , Transcriptase Reversa do HIV/imunologia , Transcriptase Reversa do HIV/metabolismo , Imunogenicidade da Vacina , Estresse Oxidativo , Sinais Direcionadores de Proteínas/genética , Vacinas de DNA/imunologia , Animais , Linhagem Celular , Feminino , Granzimas/genética , Anticorpos Anti-HIV/sangue , Transcriptase Reversa do HIV/genética , HIV-1/enzimologia , HIV-1/imunologia , Humanos , Interferon gama , Interleucina-2 , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Vacinas de DNA/administração & dosagem , Vacinas de DNA/efeitos adversosRESUMO
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.
Assuntos
Infecções por HIV/virologia , HIV-1/patogenicidade , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Fármacos Anti-HIV/efeitos adversos , Relação CD4-CD8 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , HIV-1/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Oxidantes/uso terapêutico , Oxirredução , Transdução de Sinais , Resultado do Tratamento , Replicação ViralRESUMO
Mutations in reverse transcriptase (RT) confer high levels of HIV resistance to drugs. However, while conferring drug resistance, they can lower viral replication capacity (fitness). The molecular mechanisms behind remain largely unknown. The aim of the study was to characterize the effect of drug-resistance mutations on HIV RT expression. Genes encoding AZT-resistant RTs with single or combined mutations D67N, K70R, T215F, and K219Q, and RTs derived from drug-resistant HIV-1 strains were designed and expressed in a variety of eukaryotic cells. Expression in transiently transfected cells was assessed by Western blotting and immunofluorescent staining with RT-specific antibodies. To compare the levels of expression, mutated RT genes were microinjected into the nucleus of the oocytes of Xenopus laevis. Expression of RT was quantified by sandwich ELISA. Relative stability of RTs was assessed by pulse-chase experiments. Xenopus oocytes microinjected with the genes expressed 2-50 pg of RT mutants per cell. The level of RT expression decreased with accumulation of drug-resistance mutations. Pulse-chase experiments demonstrated that poor expression of DR-RTs was due to proteolytic instability. Instability could be attributed to additional cleavage sites predicted to appear in the vicinity of resistance mutations. Accumulation of drug-resistance mutations appears to affect the level of eukaryotic expression of HIV-1 RT by inducing proteolytic instability. Low RT levels might be one of the determinants of impaired replication fitness of drug-resistant HIV-1 strains.