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Hepatitis delta virus (HDV) infection represents the most severe form of human viral hepatitis; however, the mechanisms underlying its pathology remain incompletely understood. We recently developed an HDV mouse model by injecting adeno-associated viral vectors (AAV) containing replication-competent HBV and HDV genomes. This model replicates many features of human infection, including liver injury. Notably, the extent of liver damage can be diminished with anti-TNF-α treatment. Here, we found that TNF-α is mainly produced by macrophages. Downstream of the TNF-α receptor (TNFR), the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) serves as a cell fate regulator, playing roles in both cell survival and death pathways. In this study, we explored the function of RIPK1 and other host factors in HDV-induced cell death. We determined that the scaffolding function of RIPK1, and not its kinase activity, offers partial protection against HDV-induced apoptosis. A reduction in RIPK1 expression in hepatocytes through CRISPR-Cas9-mediated gene editing significantly intensifies HDV-induced damage. Contrary to our expectations, the protective effect of RIPK1 was not linked to TNF-α or macrophage activation, as their absence did not alter the extent of damage. Intriguingly, in the absence of RIPK1, macrophages confer a protective role. However, in animals unresponsive to type-I IFNs, RIPK1 downregulation did not exacerbate the damage, suggesting RIPK1's role in shielding hepatocytes from type-I IFN-induced cell death. Interestingly, while the damage extent is similar between IFNα/ßR KO and wild type mice in terms of transaminase elevation, their cell death mechanisms differ. In conclusion, our findings reveal that HDV-induced type-I IFN production is central to inducing hepatocyte death, and RIPK1's scaffolding function offers protective benefits. Thus, type-I IFN together with TNF-α, contribute to HDV-induced liver damage. These insights may guide the development of novel therapeutic strategies to mitigate HDV-induced liver damage and halt disease progression.
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Citocinas , Vírus Delta da Hepatite , Hepatócitos , Proteína Serina-Treonina Quinases de Interação com Receptores , Animais , Camundongos , Hepatócitos/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Citocinas/metabolismo , Vírus Delta da Hepatite/fisiologia , Hepatite D/metabolismo , Morte Celular , Camundongos Endogâmicos C57BL , Apoptose , Camundongos Knockout , Humanos , Fator de Necrose Tumoral alfa/metabolismo , Modelos Animais de DoençasRESUMO
Hepatitis D virus (HDV) infection represents the most severe form of chronic viral hepatitis. We have shown that the delivery of HDV replication-competent genomes to the hepatocytes using adeno-associated virus (AAV-HDV) as gene delivery vehicles offers a unique platform to investigate the molecular aspects of HDV and associated liver damage. For the purpose of this study, we generated HDV genomes modified by site-directed mutagenesis aimed to (i) prevent some post-translational modifications of HDV antigens (HDAgs) such as large-HDAg (L-HDAg) isoprenylation or short-HDAg (S-HDAg) phosphorylation; (ii) alter the localization of HDAgs within the subcellular compartments; and (iii) inhibit the right conformation of the delta ribozyme. First, the different HDV mutants were tested in vitro using plasmid-transfected Huh-7 cells and then in vivo in C57BL/6 mice using AAV vectors. We found that Ser177 phosphorylation and ribozymal activity are essential for HDV replication and HDAg expression. Mutations of the isoprenylation domain prevented the formation of infectious particles and increased cellular toxicity and liver damage. Furthermore, altering HDAg intracellular localization notably decreased viral replication, though liver damage remained unchanged versus normal HDAg distribution. In addition, a mutation in the nuclear export signal impaired the formation of infectious viral particles. These findings contribute valuable insights into the intricate mechanisms of HDV biology and have implications for therapeutic considerations.
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Vírus Delta da Hepatite , RNA Viral , Animais , Camundongos , Antígenos da Hepatite delta/genética , Antígenos da Hepatite delta/metabolismo , RNA Viral/metabolismo , Camundongos Endogâmicos C57BL , Replicação Viral/genética , Processamento de Proteína Pós-Traducional , Fígado/metabolismoRESUMO
The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. In addition, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients.
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Sistemas CRISPR-Cas , Hiperoxalúria Primária , Humanos , Animais , Camundongos , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Edição de Genes , Hiperoxalúria Primária/genética , Hiperoxalúria Primária/terapiaRESUMO
Background: The main objective was to evaluate the efficacy of intranasal photodynamic therapy (PDT) in SARS-CoV-2 mildly symptomatic carriers on decreasing the infectivity period. SARS-CoV-2-specific immune-stimulating effects and safety were also analysed. Methods: We performed a randomized, placebo-controlled, clinical trial in a tertiary hospital (NCT05184205). Patients with a positive SARS-CoV-2 PCR in the last 48 hours were recruited and aleatorily assigned to PDT or placebo. Patients with pneumonia were excluded. Participants and investigators were masked to group assignment. The primary outcome was the reduction in in vitro infectivity of nasopharyngeal samples at days 3 and 7. Additional outcomes included safety assessment and quantification of humoral and T-cell immune-responses. Findings: Patients were recruited between December 2021 and February 2022. Most were previously healthy adults vaccinated against COVID-19 and most carried Omicron variant. 38 patients were assigned to placebo and 37 to PDT. Intranasal PDT reduced infectivity at day 3 post-treatment when compared to placebo with a ß-coefficient of -812.2 (CI95%= -478660 - -1.3, p<0.05) infectivity arbitrary units. The probability of becoming PCR negative (ct>34) at day 7 was higher on the PDT-group, with an OR of 0.15 (CI95%=0.04-0.58). There was a decay in anti-Spike titre and specific SARS-CoV-2 T cell immunity in the placebo group 10 and 20 weeks after infection, but not in the PDT-group. No serious adverse events were reported. Interpretation: Intranasal-PDT is safe in pauci-symptomatic COVID-19 patients, it reduces SARS-CoV-2 infectivity and decelerates the decline SARS-CoV-2 specific immune-responses.
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COVID-19 , SARS-CoV-2 , Adulto , Humanos , Linfócitos T , NarizRESUMO
The enormous societal impact of the ongoing COVID-19 pandemic has been particularly harsh for some social groups, such as the elderly. Recently, it has been suggested that senescent cells could play a central role in pathogenesis by exacerbating the pro-inflammatory immune response against SARS-CoV-2. Therefore, the selective clearance of senescent cells by senolytic drugs may be useful as a therapy to ameliorate the symptoms of COVID-19 in some cases. Using the established COVID-19 murine model K18-hACE2, we demonstrated that a combination of the senolytics dasatinib and quercetin (D/Q) significantly reduced SARS-CoV-2-related mortality, delayed its onset, and reduced the number of other clinical symptoms. The increase in senescent markers that we detected in the lungs in response to SARS-CoV-2 may be related to the post-COVID-19 sequelae described to date. These results place senescent cells as central targets for the treatment of COVID-19, and make D/Q a new and promising therapeutic tool.
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COVID-19 , Quercetina , Camundongos , Humanos , Animais , Quercetina/farmacologia , Quercetina/uso terapêutico , Dasatinibe/farmacologia , Dasatinibe/uso terapêutico , SARS-CoV-2 , Senescência Celular , Senoterapia , PandemiasRESUMO
SARS-CoV-2 is responsible for the COVID-19 pandemic, which has caused almost 570 million infections and over six million deaths worldwide. To help curb its spread, solutions using ultraviolet light (UV) for quick virus inactivation inside buildings without human intervention could be very useful to reduce chances of contagion. The UV dose must be sufficient to inactivate the virus considering the different materials in the room, but it should not be too high, not to degrade the environment. In the present study, we have analyzed the ability of a 254 nm wavelength UV-C lamp to inactivate dried samples of SARS-CoV-2 exposed at a distance of two meters, simulating a full-scale scenario. Our results showed that virus inactivation was extremely efficient in most tested materials, which included plastic, metal, wood, and textile, with a UV-C exposure of only 42 s (equivalent to 10 mJ/cm2). However, porous materials like medium density fibreboard, were hard to decontaminate, indicating that they should be avoided in hospital rooms and public places.
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Identification of relevant epitopes is crucial for the development of subunit peptide vaccines inducing neutralizing and cellular immunity against SARS-CoV-2. Our aim was the characterization of epitopes in the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein to generate a peptide vaccine. Epitope mapping using a panel of 10 amino acid overlapped 15-mer peptides covering region 401-515 from RBD did not identify linear epitopes when tested with sera from infected individuals or from RBD-immunized mice. However, immunization of mice with these 15-mer peptides identified four peptides located at region 446-480 that induced antibodies recognizing the peptides and RBD/S1 proteins. Immunization with peptide 446-480 from S protein formulated with Freund's adjuvant or with CpG oligodeoxinucleotide/Alum induced polyepitopic antibody responses in BALB/c and C56BL/6J mice, recognizing RBD (titres of 3 × 104-3 × 105, depending on the adjuvant) and displaying neutralizing capacity (80-95% inhibition capacity; p < 0.05) against SARS-CoV-2. Murine CD4 and CD8T-cell epitopes were identified in region 446-480 and vaccination experiments using HLA transgenic mice suggested the presence of multiple human T-cell epitopes. Antibodies induced by peptide 446-480 showed broad recognition of S proteins and S-derived peptides belonging to SARS-CoV-2 variants of concern. Importantly, vaccination with peptide 446-480 or with a cyclic version of peptide 446-488 containing a disulphide bridge between cysteines 480 and 488, protected humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2 (62.5 and 75% of protection; p < 0.01 and p < 0.001, respectively). This region could be the basis for a peptide vaccine or other vaccine platforms against Covid-19.
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Anticorpos Neutralizantes/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Imunidade Celular , Imunidade Humoral , SARS-CoV-2/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Vacinas contra COVID-19/normas , Reações Cruzadas/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B , Epitopos de Linfócito T/imunologia , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Hepatitis delta virus (HDV) infection causes the most severe form of viral hepatitis, but little is known about the molecular mechanisms involved. We have recently developed an HDV mouse model based on the delivery of HDV replication-competent genomes using adeno-associated vectors (AAV), which developed a liver pathology very similar to the human disease and allowed us to perform mechanistic studies. We have generated different AAV-HDV mutants to eliminate the expression of HDV antigens (HDAgs), and we have characterized them both in vitro and in vivo. We confirmed that S-HDAg is essential for HDV replication and cannot be replaced by L-HDAg or host cellular proteins, and that L-HDAg is essential to produce the HDV infectious particle and inhibits its replication. We have also found that lack of L-HDAg resulted in the increase of S-HDAg expression levels and the exacerbation of liver damage, which was associated with an increment in liver inflammation but did not require T cells. Interestingly, early expression of L-HDAg significantly ameliorated the liver damage induced by the mutant expressing only S-HDAg. In summary, the use of AAV-HDV represents a very attractive platform to interrogate in vivo the role of viral components in the HDV life cycle and to better understand the mechanism of HDV-induced liver pathology.
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Dependovirus/genética , Vetores Genéticos/genética , Hepatite D/virologia , Vírus Delta da Hepatite/fisiologia , Replicação Viral , Animais , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Engenharia Genética , Hepatite D/patologia , Humanos , Técnicas In Vitro , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Camundongos , MutaçãoRESUMO
Antiviral agents with different mechanisms of action could induce synergistic effects against SARS-CoV-2 infection. Some reports suggest the therapeutic potential of the heme oxygenase-1 (HO-1) enzyme against virus infection. Given that hemin is a natural inducer of the HO-1 gene, the aim of this study was to develop an in vitro assay to analyze the antiviral potency of hemin against SARS-CoV-2 infection. A SARS-CoV-2 infectivity assay was conducted in Vero-E6 and Calu-3 epithelial cell lines. The antiviral effect of hemin, and chloroquine as a control, against SARS-CoV-2 virus infection was quantified by RT-qPCR using specific oligonucleotides for the N gene. Chloroquine induced a marked reduction of viral genome copies in kidney epithelial Vero-E6 cells but not in lung cancer Calu-3 cells. Hemin administration to the culture medium induced a high induction in the expression of the HO-1 gene that was stronger in Vero-E6 macaque-derived cells than in the human Calu-3 cell line. However, hemin treatment did not modify SARS-CoV-2 replication, as measured by viral genome quantification 48 h post-infection for Vero-E6 and 72 h post-infection for the Calu-3 lineages. In conclusion, although exposure to hemin induced strong HO-1 up-regulation, this effect was unable to inhibit or delay the progression of SARS-CoV-2 infection in two epithelial cell lines susceptible to infection.
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Antivirais/farmacologia , Heme Oxigenase-1/metabolismo , Hemina/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , COVID-19 , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Cloroquina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND & AIMS: HDV infection induces the most severe form of human viral hepatitis. However, the specific reasons for the severity of the disease remain unknown. Recently, we developed an HDV replication mouse model in which, for the first time, liver damage was detected. METHODS: HDV and HBV replication-competent genomes and HDV antigens were delivered to mouse hepatocytes using adeno-associated vectors (AAVs). Aminotransferase elevation, liver histopathology, and hepatocyte death were evaluated and the immune infiltrate was characterized. Liver transcriptomic analysis was performed. Mice deficient for different cellular and molecular components of the immune system, as well as depletion and inhibition studies, were employed to elucidate the causes of HDV-mediated liver damage. RESULTS: AAV-mediated HBV/HDV coinfection caused hepatocyte necrosis and apoptosis. Activated T lymphocytes, natural killer cells, and proinflammatory macrophages accounted for the majority of the inflammatory infiltrate. However, depletion studies and the use of different knockout mice indicated that neither T cells, natural killer cells nor macrophages were necessary for HDV-induced liver damage. Transcriptomic analysis revealed a strong activation of type I and II interferon (IFN) and tumor necrosis factor (TNF)-α pathways in HBV/HDV-coinfected mice. While the absence of IFN signaling had no effect, the use of a TNF-α antagonist resulted in a significant reduction of HDV-associated liver injury. Furthermore, hepatic expression of HDAg resulted in the induction of severe liver damage, which was T cell- and TNF-α-independent. CONCLUSIONS: Both host (TNF-α) and viral (HDV antigens) factors play a relevant role in HDV-induced liver damage. Importantly, pharmacological inhibition of TNF-α may offer an attractive strategy to aid control of HDV-induced acute liver damage. LAY SUMMARY: Chronic hepatitis delta constitutes the most severe form of viral hepatitis. There is limited data on the mechanism involved in hepatitis delta virus (HDV)-induced liver pathology. Our data indicate that a cytokine (TNF-α) and HDV antigens play a relevant role in HDV-induced liver damage.
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Adeno-associated viruses (AAV) have become one of the most promising tools for gene transfer in clinics. Among all the serotypes, AAV9 has been described as the most efficient for cardiac transduction. In order to achieve optimal therapeutic delivery in heart disease, we have explored AAV9 transduction efficiency in an infarcted heart using different routes of administration and promoters, including a cardiac-specific one. AAV9 vectors carrying luciferase or green fluorescence protein under the control of the ubiquitous elongation-factor-1-alpha or the cardiac-specific troponin-T (TnT) promoters were administered by intramyocardial or intravenous injection, either in healthy or myocardial-infarcted mice. The transduction efficacy and specificity, the time-course expression, and the safety of each vector were tested. High transgene expression levels were found in the heart, but not in the liver, of mice receiving AAV-TnT, which was significantly higher after intramyocardial injection regardless of ischemia-induction. On the contrary, high hepatic transgene expression levels were detected with the elongation-factor-1-alpha-promoter, independently of the administration route and heart damage. Moreover, tissue-specific green fluorescence protein expression was found in cardiomyocytes with the TnT vector, whereas minimal cardiac expression was detected with the ubiquitous one. Interestingly, we found that myocardial infarction greatly increased the transcriptional activity of AAV genomes. Our findings show that the use of cardiac promoters allows for specific and stable cardiac gene expression, which is optimal and robust when intramyocardially injected. Furthermore, our data indicate that the pathological status of the tissue can alter the transcriptional activity of AAV genomes, an aspect that should be carefully evaluated for clinical applications.
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Dependovirus/genética , Isquemia Miocárdica/patologia , Animais , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos , Genoma Viral , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Coração/fisiologia , Humanos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miócitos Cardíacos/citologia , Fator 1 de Elongação de Peptídeos/metabolismo , Regiões Promotoras Genéticas , Distribuição Tecidual , Transdução Genética , Transgenes , Troponina T/metabolismoRESUMO
CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1-/- mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites.
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Oxirredutases do Álcool/antagonistas & inibidores , Sistemas CRISPR-Cas , Terapia Genética/métodos , Hiperoxalúria Primária/terapia , Oxalatos/urina , Oxirredutases do Álcool/genética , Animais , Modelos Animais de Doenças , Edição de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Nefrocalcinose/prevenção & controleRESUMO
The innate immune system provides a primary line of defense against pathogens. Stimulator of IFN genes (STING), encoded by the TMEM173 gene, is a critical protein involved in IFN-ß induction in response to infection by different pathogens. In this study, we describe the expression of three different alternative-spliced human (h) TMEM173 mRNAs producing STING truncated isoforms 1, 2, and 3 in addition to the full-length wild-type (wt) hSTING. All of the truncated isoforms lack exon 7 and share the N-terminal transmembrane region with wt hSTING. Overexpression of the three STING truncated isoforms failed to induce IFN-ß, and they acted as selective pathway inhibitors of wt hSTING even in combination with upstream inducer cyclic-di-GMP-AMP synthase. Truncated isoforms alter the stability of wt hSTING, reducing protein t 1/2 to some extent by the induction of proteasome-dependent degradation. Knocking down expression of truncated isoforms increased production of IFN-ß by THP1 monocytes in response to intracellular cytosolic DNA or HSV-1 infection. At early stages of infection, viruses like HSV-1 or vesicular stomatitis virus reduced the ratio of full-length wt hSTING/truncated STING isoforms, suggesting the skewing of alternative splicing of STING toward truncated forms as a tactic to evade antiviral responses. Finally, in silico analysis revealed that the human intron-exon gene architecture of TMEM173 (splice sites included) is preserved in other mammal species, predominantly primates, stressing the relevance of alternative splicing in regulating STING antiviral biology.
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Proteínas de Membrana/imunologia , Replicação Viral/imunologia , Processamento Alternativo/imunologia , Animais , Chlorocebus aethiops , Simulação por Computador , Células HEK293 , Células HeLa , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Humanos , Imunidade Inata , Interferon beta/imunologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Monócitos/imunologia , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Viroses/genética , Viroses/imunologia , Replicação Viral/genéticaRESUMO
BACKGROUND & AIMS: Studying hepatitis delta virus (HDV) and developing new treatments is hampered by the limited availability of small animal models. Herein, a description of a robust mouse model of HDV infection that mimics several important characteristics of the human disease is presented. METHODS: HDV and hepatitis B virus (HBV) replication competent genomes were delivered to the mouse liver using adeno-associated viruses (AAV; AAV-HDV and AAV-HBV). Viral load, antigen expression and genomes were quantified at different time points after AAV injection. Furthermore, liver pathology, genome editing, and the activation of the innate immune response were evaluated. RESULTS: AAV-HDV infection initiated HDV replication in mouse hepatocytes. Genome editing was confirmed by the presence of small and large HDV antigens and sequencing. Viral replication was detected for 45days, even after the AAV-HDV vector had almost disappeared. In the presence of HBV, HDV infectious particles were detected in serum. Furthermore, as observed in patients, co-infection was associated with the reduction of HBV antigen expression and the onset of liver damage that included the alteration of genes involved in the development of liver pathologies. HDV replication induced a sustained type I interferon response, which was significantly reduced in immunodeficient mice and almost absent in mitochondrial antiviral signaling protein (MAVS)-deficient mice. CONCLUSION: The animal model described here reproduces important characteristics of human HDV infection and provides a valuable tool for characterizing the viral infection and for developing new treatments. Furthermore, MAVS was identified as a main player in HDV detection and adaptive immunity was found to be involved in the amplification of the innate immune response. Lay summary: Co-infection with hepatitis B and D virus (HBV and HDV, respectively) often causes a more severe disease condition than HBV alone. Gaining more insight into HDV and developing new treatments is hampered by limited availability of adequate immune competent small animal models and new ones are needed. Here, a mouse model of HDV infection is described, which mimics several important characteristics of the human disease, such as the initiation and maintenance of replication in murine hepatocytes, genome editing and, in the presence of HBV, generation of infectious particles. Lastly, the involvement of an adaptive immunity and the intracellular signaling molecule MAVS in mounting a strong and lasting innate response was shown. Thus, our model serves as a useful tool for the investigation of HDV biology and new treatments.
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Proteínas Adaptadoras de Transdução de Sinal/imunologia , Hepatite D/imunologia , Interferon beta/biossíntese , Imunidade Adaptativa , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Coinfecção/imunologia , Coinfecção/patologia , Coinfecção/virologia , Dependovirus/genética , Modelos Animais de Doenças , Genoma Viral , Hepatite B/complicações , Hepatite B/imunologia , Hepatite B/virologia , Antígenos da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite D/complicações , Hepatite D/virologia , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/imunologia , Vírus Delta da Hepatite/fisiologia , Antígenos da Hepatite delta/metabolismo , Humanos , Imunidade Inata , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Imunológicos , Transdução de Sinais/imunologia , Replicação ViralRESUMO
Interleukin-15 (IL-15) is a cell growth-factor that regulates lymphocyte function and homeostasis. Its strong immunostimulatory activity coupled with an apparent lack of toxicity makes IL-15 an exciting candidate for cancer therapy, somehow limited by its short half-life in circulation. To increase IL-15 bioavailability we constructed a recombinant adeno-associated vector expressing murine IL-15 (AAV-mIL15) in the liver. Mice injected with AAV-mIL15 showed sustained and vector dose-dependent levels of IL-15/IL-15Rα complexes in serum, production of IFN-γ and activation of CD8+ T-cells and macrophages. The antitumoral efficacy of AAV-mIL15 was tested in a mouse model of metastatic colorectal cancer established by injection of MC38 cells. AAV-mIL15 treatment slightly inhibits MC38 tumor-growth and significantly increases the survival of mice. However, mIL-15 sustained expression was associated with development of side effects like hepatosplenomegaly, liver damage and the development of haematological stress, which results in the expansion of hematopoietic precursors in the bone marrow. To elucidate the mechanism, we treated IFN-γ receptor-, RAG1-, CD1d- and µMT-deficient mice and performed adoptive transfer of bone marrow cells from WT mice to RAG1-defcient mice. We demonstrated that the side effects of murine IL-15 administration were mainly mediated by IFN-γ-producing T-cells. CONCLUSIONS: IL-15 induces the activation and survival of effector immune cells that are necessary for its antitumoral activity; but, long-term exposure to IL-15 is associated with the development of important side effects mainly mediated by IFN-γ-producing T-cells. Strategies to modulate T-cell activation should be combined with IL-15 administration to reduce secondary adverse events while maintaining its antitumoral effect.
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Interferon gama/metabolismo , Interleucina-15/farmacologia , Linfócitos T/metabolismo , Transferência Adotiva , Animais , Antígenos CD1d/metabolismo , Células da Medula Óssea/citologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Neoplasias Colorretais/terapia , Dependovirus , Progressão da Doença , Feminino , Regulação da Expressão Gênica , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos , Proteínas de Homeodomínio/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Fígado/metabolismo , Ativação Linfocitária , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
UNLABELLED: In chronic hepatitis B (CHB), failure to control hepatitis B virus (HBV) is associated with T cell dysfunction. HBV transgenic mice mirror many features of the human disease, including T cell unresponsiveness, and thus represent an appropriate model in which to test novel therapeutic strategies. To date, the tolerant state of CD8(+) T cells in these animals could be altered only by strong immunogens or by immunization with HBV antigen-pulsed dendritic cells; however, the effectors induced were unable to suppress viral gene expression or replication. Because of the known stimulatory properties of alpha interferon (IFN-α) and interleukin-15 (IL-15), this study explored the therapeutic potential of liver-directed gene transfer of these cytokines in a murine model of CHB using adeno-associated virus (AAV) delivery. This combination not only resulted in a reduction in the viral load in the liver and the induction of an antibody response but also gave rise to functional and specific CD8(+) immunity. Furthermore, when splenic and intrahepatic lymphocytes from IFN-α- and IL-15-treated animals were transferred to new HBV carriers, partial antiviral immunity was achieved. In contrast to previous observations made using either cytokine alone, markedly attenuated PD-L1 induction in hepatic tissue was observed upon coadministration. An initial study with CHB patient samples also gave promising results. Hence, we demonstrated synergy between two stimulating cytokines, IL-15 and IFN-α, which, given together, constitute a potent approach to significantly enhance the CD8(+) T cell response in a state of immune hyporesponsiveness. Such an approach may be useful for treating chronic viral infections and neoplastic conditions. IMPORTANCE: With 350 million people affected worldwide and 600,000 annual deaths due to HBV-induced liver cirrhosis and/or hepatocellular carcinoma, chronic hepatitis B (CHB) is a major health problem. However, current treatment options are costly and not very effective and/or need to be administered for life. The unprecedented efficacy of the strategy described in our paper may offer an alternative and is relevant for a broad spectrum of readers because of its clear translational importance to other chronic viral infections in which a hyporesponsive antigen-specific T cell repertoire prevents clearance of the pathogen.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Interferon-alfa/administração & dosagem , Interleucina-15/administração & dosagem , Adenoviridae/genética , Animais , Modelos Animais de Doenças , Portadores de Fármacos , Terapia Genética , Anticorpos Anti-Hepatite B/sangue , Interferon-alfa/genética , Interleucina-15/genética , Fígado/virologia , Camundongos Transgênicos , Resultado do Tratamento , Carga ViralRESUMO
RIG-I-like receptors (RLRs) are cellular sensor proteins that detect certain RNA species produced during viral infections. RLRs activate a signaling cascade that results in the production of IFN-ß as well as several other cytokines with antiviral and proinflammatory activities. We explored the potential of different constructs based on RLRs to induce the IFN-ß pathway and create an antiviral state in type I IFN-unresponsive models. A chimeric construct composed of RIG-I 2CARD and the first 200 amino acids of MAVS (2CARD-MAVS200) showed an enhanced ability to induce IFN-ß when compared to other stimulatory constructs. Furthermore, this human chimeric construct showed a superior ability to activate IFN-ß expression in cells from various species. This construct was found to overcome the restrictions of blocking IFN-ß induction or signaling by a number of viral IFN-antagonist proteins. Additionally, the antiviral activity of this chimera was demonstrated in influenza virus and HBV infection mouse models using adeno-associated virus (AAV) vectors as a delivery vehicle. We propose that AAV vectors expressing 2CARD-MAVS200 chimeric protein can reconstitute IFN-ß induction and recover a partial antiviral state in different models that do not respond to recombinant IFN-ß treatment.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , RNA Helicases DEAD-box/metabolismo , Vírus da Hepatite B/imunologia , Hepatite B Crônica/terapia , Vírus da Influenza A/imunologia , Interferon beta/metabolismo , Infecções por Orthomyxoviridae/terapia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Células HEK293 , Hepatite B Crônica/imunologia , Humanos , Imunidade Inata , Interferon beta/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infecções por Orthomyxoviridae/imunologia , Fragmentos de Peptídeos/genética , Receptores Imunológicos , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais/genéticaRESUMO
Interferon-α is a potent antiviral agent and a vigorous adjuvant in the induction of T-cell responses but its use is limited by hematologic toxicity. Interferon-α alters hematopoietic stem cell dormancy and impairs myelocytic and erythrocytic/megakaryocytic differentiation from hematopoietic progenitors. However, the effect of chronic interferon-α exposure on hematopoietic precursors has still not been well characterized. Here, we transduced the liver of mice with an adenoassociated vector encoding interferon-α to achieve sustained high serum levels of the cytokine. The bone marrow of these animals showed diminished long-term and short-term hematopoietic stem cells, reduction of multipotent progenitor cells, and marked decrease of B cells, but significant increase in the proportion of CD8(+) and CD4(+)CD8(+) T cells. Upon adoptive transfer to RAG(-/-) mice, bone marrow cells from interferon-α-treated animals generated CD4(+) and CD8(+) T cells while CD19(+), CD11b(+) and NK1.1(+) lineages failed to develop. These effects are associated with the transcriptional downregulation of transcription factors involved in B-cell differentiation and modulation of key factors for T-cell development. Thus, sustained interferon-α exposure causes hematopoietic stem cells exhaustion and drives common lymphoid progenitors towards T-cell generation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Interferon-alfa/farmacologia , Linfopoese/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Imunofenotipagem , Interferon-alfa/genética , Contagem de Leucócitos , Leucócitos/citologia , Leucócitos/metabolismo , Linfopoese/genética , Masculino , Camundongos , Camundongos Knockout , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND & AIMS: Adenoviral (Ad) vectors are currently one of the most efficient tools for in vivo gene transfer to the liver. However, anti-Ad immune responses limit the safety and efficacy of these vectors. The initial inflammatory reaction is a concern in terms of toxicity, and it favours the development of cellular and humoral responses leading to short transgene persistence and inefficient vector re-administrations. Therefore, safe and simple ways to interfere with these processes are needed. Study ways to deplete specific immune cell populations and their impact on liver-directed gene transfer. METHODS: First-generation Ad vectors encoding reporter genes (luciferase or ß-galactosidase) were injected intravenously into Balb/c mice. Kupffer cells and splenic macrophages were depleted by intravenous administration of clodronate liposomes. B lymphocytes, CD4(+) , CD8(+) T lymphocytes or NK cells were depleted by intraperitoneal injection of anti-M plus anti-D, anti-CD4, anti-CD8 or anti-asialo-GM1 antibodies respectively. Long-term evolution of luciferase expression in the liver was monitored by bioluminescence imaging. RESULTS: The anti-CD4 monoclonal antibody impaired cellular and humoral immune responses, leading to efficient vector re-administration. Clodronate liposomes had no impact on humoral responses but caused a 100-1000 fold increase in liver transduction, stabilized transgene expression, reduced the concentration of inflammatory cytokines, and inhibited lymphocyte activation. CONCLUSIONS: Transient CD4(+) T-cell depletion using antibodies is a clinically feasible procedure that allows efficient Ad redosing. Systemic administration of clodronate liposomes may further increase the safety and efficacy of vectors.