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1.
Immunity ; 52(1): 109-122.e6, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31882361

RESUMO

Recent work suggests that cholesterol metabolism impacts innate immune responses against infection. However, the key enzymes or the natural products and mechanisms involved are not well elucidated. Here, we have shown that upon DNA and RNA viral infection, macrophages reduced 7-dehydrocholesterol reductase (DHCR7) expression. DHCR7 deficiency or treatment with the natural product 7-dehydrocholesterol (7-DHC) could specifically promote phosphorylation of IRF3 (not TBK1) and enhance type I interferon (IFN-I) production in macrophages. We further elucidated that viral infection or 7-DHC treatment enhanced AKT3 expression and activation. AKT3 directly bound and phosphorylated IRF3 at Ser385, together with TBK1-induced phosphorylation of IRF3 Ser386, to achieve IRF3 dimerization. Deletion of DHCR7 and the DHCR7 inhibitors including AY9944 and the chemotherapy drug tamoxifen promoted clearance of Zika virus and multiple viruses in vitro or in vivo. Taken together, we propose that the DHCR7 inhibitors and 7-DHC are potential therapeutics against emerging or highly pathogenic viruses.


Assuntos
Desidrocolesteróis/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/biossíntese , Macrófagos/imunologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Estomatite Vesicular/imunologia , Células A549 , Animais , Linhagem Celular , Colesterol/metabolismo , Ativação Enzimática/imunologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células RAW 264.7 , Interferência de RNA , RNA Interferente Pequeno/genética , Vírus da Estomatite Vesicular Indiana/imunologia
2.
Nat Commun ; 10(1): 3233, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324787

RESUMO

MAVS is essential for antiviral immunity, but the molecular mechanisms responsible for its tight regulation remain poorly understood. Here, we show that NLK inhibits the antiviral immune response during viral infection by targeting MAVS for degradation. NLK depletion promotes virus-induced antiviral cytokine production and decreases viral replication, which is potently rescued by the reintroduction of NLK. Moreover, the depletion of NLK promotes antiviral effects and increases the survival times of mice after infection with VSV. NLK interacts with and phosphorylates MAVS at multiple sites on mitochondria or peroxisomes, thereby inducing the degradation of MAVS and subsequent inactivation of IRF3. Most importantly, a peptide derived from MAVS promotes viral-induced IFN-ß production and antagonizes viral replication in vitro and in vivo. These findings provide direct insights into the molecular mechanisms by which phosphorylation of MAVS regulates its degradation and influences its activation and identify an important peptide target for propagating antiviral responses.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Imunidade Inata/imunologia , Interferon beta/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células HCT116 , Células HEK293 , Humanos , Imunidade Inata/genética , Interferon beta/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/imunologia , Células Vero , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/fisiologia
3.
Nanotechnology ; 30(34): 345502, 2019 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-30865941

RESUMO

The direct method of detecting a virus with extremely low concentration is recommended for the diagnosis of viral disease. In this study, coplanar-gate graphene field-effect transistors (GFETs) were built on flexible polyethylene terephthalate substrates for the attomolar detection of a virus. The GFETs exhibited a very low detection limit of 47.8 aM with relatively low source/drain voltage due to aqueous dielectric media which stabilizes viruses and antibodies for specific bonding. The antibody as a probe molecule was decorated on a graphene surface using 1-pyrenebutanoic acid succinimidyl ester that had previously been immobilized on a graphene surface. The Dirac point voltage shifted downward after dropping the virus solution, due to the electrostatic gating effect of graphene in the antigen (namely, virus)-antibody complex. The virus detection platform used in this study is expected to be beneficial for direct diagnosis in saline environments, since the performances of GFETs were not significantly affected by the presence of Na+ and Cl-. Furthermore, since our flexible and transparent virus sensors can be used in a wearable device, they provide a simple and fast method for diagnosing viruses.


Assuntos
Grafite/química , Imunoensaio/métodos , Plásticos/química , Transistores Eletrônicos , Vírus/isolamento & purificação , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , HIV-1/imunologia , HIV-1/isolamento & purificação , Imunoensaio/instrumentação , Limite de Detecção , Polietilenotereftalatos/química , Eletricidade Estática , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/isolamento & purificação , Vírus/imunologia
4.
Biochem Biophys Res Commun ; 511(2): 287-293, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30795865

RESUMO

Innate immunity is a system that recognizes primarily and excludes pathogenic microorganism. MAVS/IPS-1/Cardif/Visa functions as an adapter protein for RIG-I like receptors (RLRs) and plays a key role in the production of antiviral proteins, interferons (IFNs), for RNA viruses. However, the activation mechanism is not fully understood. Here, we show that BinCARD isoform2 (BinCARD2), carrying CARD domain structure like MAVS, functions in innate immune response. Knockdown of BinCARD2 reduced the RLR ligand-induced expression of IFN-ß mRNA and activation of the IFNB promoter. The activation of the IFNB promoter by overexpression of MAVS or TBK1 was suppressed by silencing of BinCARD2, but no effect on IFNB promoter activation by overexpression of TRIF or constitutive activated IRF-3. Furthermore, we confirmed that BinCARD2 protein associated with MAVS but not TBK1 by immunoprecipitation and colocalized with MAVS. Accordingly, we investigated whether BinCARD2 was involved in MAVS activation and showed that siBinCARD2 did not affect RIG-I/MAVS binding but impaired the MAVS oligomerization. Moreover, we infected A549 cells with vesicular stomatitis virus (VSV) and found that induction of IFN-ß and IL-6 mRNA after VSV infection was decreased by BinCARD2 knockdown. Thus, these data may suggest that BinCARD2 associates with MAVS to positively modulate the oligomerization in the RIG-I like receptors pathway and activates innate immune response.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/imunologia , Imunidade Inata , Interferon beta/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Apoptose , Linhagem Celular , Humanos , Membranas Mitocondriais/imunologia , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia
5.
Mol Cell ; 73(4): 803-814.e6, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30639243

RESUMO

Intron retention (IR) has emerged as an important mechanism of gene expression control, but the factors controlling IR events remain poorly understood. We observed consistent IR in one intron of the Irf7 gene and identified BUD13 as an RNA-binding protein that acts at this intron to increase the amount of successful splicing. Deficiency in BUD13 was associated with increased IR, decreased mature Irf7 transcript and protein levels, and consequently a dampened type I interferon response, which compromised the ability of BUD13-deficient macrophages to withstand vesicular stomatitis virus (VSV) infection. Global analysis of BUD13 knockdown and BUD13 cross-linking to RNA revealed a subset of introns that share many characteristics with the one found in Irf7 and are spliced in a BUD13-dependent manner. Deficiency of BUD13 led to decreased mature transcript from genes containing such introns. Thus, by acting as an antagonist to IR, BUD13 facilitates the expression of genes at which IR occurs.


Assuntos
Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Íntrons , Macrófagos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Estomatite Vesicular/metabolismo , Vírus da Estomatite Vesicular Indiana/patogenicidade , Animais , Sítios de Ligação , Sequência Rica em GC , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Fator Regulador 7 de Interferon/genética , Interferon Tipo I/imunologia , Macrófagos/imunologia , Macrófagos/virologia , Camundongos Endogâmicos C57BL , Ligação Proteica , Sítios de Splice de RNA , Processamento de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Células Vero , Estomatite Vesicular/genética , Estomatite Vesicular/imunologia , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/imunologia
6.
J Virol ; 93(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30541859

RESUMO

Therapeutic vaccines may be an important component of a treatment regimen for curing chronic hepatitis B virus (HBV) infection. We previously demonstrated that recombinant wild-type vesicular stomatitis virus (VSV) expressing the HBV middle surface glycoprotein (MHBs) elicits functional immune responses in mouse models of HBV replication. However, VSV has some undesirable pathogenic properties, and the use of this platform in humans requires further viral attenuation. We therefore generated a highly attenuated VSV that expresses MHBs and contains two attenuating mutations. This vector was evaluated for immunogenicity, pathogenesis, and anti-HBV function in mice. Compared to wild-type VSV, the highly attenuated virus displayed markedly reduced pathogenesis but induced similar MHBs-specific CD8+ T cell and antibody responses. The CD8+ T cell responses elicited by this vector in naive mice prevented HBV replication in animals that were later challenged by hydrodynamic injection or transduction with adeno-associated virus encoding the HBV genome (AAV-HBV). In mice in which persistent HBV replication was first established by AAV-HBV transduction, subsequent immunization with the attenuated VSV induced MHBs-specific CD8+ T cell responses that corresponded with reductions in serum and liver HBV antigens and nucleic acids. HBV control was associated with an increase in the frequency of intrahepatic HBV-specific CD8+ T cells and a transient elevation in serum alanine aminotransferase activity. The ability of VSV to induce a robust multispecific T cell response that controls HBV replication combined with the improved safety profile of the highly attenuated vector suggests that this platform offers a new approach for HBV therapeutic vaccination.IMPORTANCE A curative treatment for chronic hepatitis B must eliminate the virus from the liver, but current antiviral therapies typically fail to do so. Immune-mediated resolution of infection occurs in a small fraction of chronic HBV patients, which suggests the potential efficacy of therapeutic strategies that boost the patient's own immune response to the virus. We modified a safe form of VSV to express an immunogenic HBV protein and evaluated the efficacy of this vector in the prevention and treatment of HBV infection in mouse models. Our results show that this vector elicits HBV-specific immune responses that prevent the establishment of HBV infection and reduce viral proteins in the serum and viral DNA/RNA in the liver of mice with persistent HBV replication. These findings suggest that highly attenuated and safe virus-based vaccine platforms have the potential to be utilized for the development of an effective therapeutic vaccine against chronic HBV infection.


Assuntos
Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/prevenção & controle , Hepatite B Crônica/terapia , Vacinas Atenuadas/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Alanina Transaminase/sangue , Animais , Linfócitos T CD8-Positivos/imunologia , Hepatite B Crônica/imunologia , Imunoterapia/métodos , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vacinas Virais/imunologia , Replicação Viral/imunologia
7.
Viruses ; 10(12)2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30513968

RESUMO

Small Ubiquitin-like MOdifier (SUMO) conjugation to proteins has essential roles in several processes including localization, stability, and function of several players implicated in intrinsic and innate immunity. In human, five paralogs of SUMO are known of which three are ubiquitously expressed (SUMO1, 2, and 3). Infection by rhabdoviruses triggers cellular responses through the activation of pattern recognition receptors, which leads to the production and secretion of interferon. This review will focus on the effects of the stable expression of the different SUMO paralogs or Ubc9 depletion on rhabdoviruses-induced interferon production and interferon signaling pathways as well as on the expression and functions of restriction factors conferring the resistance to rhabdoviruses.


Assuntos
Infecções por Rhabdoviridae/imunologia , Rhabdoviridae/imunologia , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Humanos , Imunidade Inata , Interferons/imunologia , Camundongos , Proteínas de Resistência a Myxovirus/genética , Ligação Proteica , Vírus da Raiva/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/imunologia , Sumoilação , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Vírus da Estomatite Vesicular Indiana/imunologia , eIF-2 Quinase/genética
8.
Cell Host Microbe ; 24(6): 791-803.e6, 2018 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-30543776

RESUMO

Increased glucose metabolism in immune cells not only serves as a hallmark feature of acute inflammation but also profoundly affects disease outcome following bacterial infection and tissue damage. However, the role of individual glucose metabolic pathways during viral infection remains largely unknown. Here we demonstrate an essential function of the hexosamine biosynthesis pathway (HBP)-associated O-linked ß-N-acetylglucosamine (O-GlcNAc) signaling in promoting antiviral innate immunity. Challenge of macrophages with vesicular stomatitis viruses (VSVs) enhances HBP activity and downstream protein O-GlcNAcylation. Human and murine cells deficient of O-GlcNAc transferase, a key enzyme for protein O-GlcNAcylation, show defective antiviral immune responses upon VSV challenge. Mechanistically, O-GlcNAc transferase-mediated O-GlcNAcylation of the signaling adaptor MAVS on serine 366 is required for K63-linked ubiquitination of MAVS and subsequent downstream retinoic-acid inducible gene-like receptor -antiviral signaling activation. Thus, our study identifies a molecular mechanism by which HBP-mediated O-GlcNAcylation regulates MAVS function and highlights the importance of glucose metabolism in antiviral innate immunity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Imunidade Inata/imunologia , N-Acetilglucosaminiltransferases/metabolismo , Infecções por Rhabdoviridae/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Glucose/metabolismo , Células HEK293 , Células HT29 , Hexosaminas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , N-Acetilglucosaminiltransferases/genética , Infecções por Rhabdoviridae/virologia , Células THP-1 , Células Vero
9.
Prev Vet Med ; 160: 68-75, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30389000

RESUMO

The aim of this survey was to estimate the apparent herd-level and animal-level prevalences, as well as to identify risk factors and spatial clustering of vesicular stomatitis virus (VSV) positive herds in the state of Paraíba, semiarid of Brazil. The state was divided into three sampling strata: Sertão, Borborema and Zona da Mata/Agreste. For each sampling stratum, herd-level and animal-level prevalences were estimated by a two-stage sampling survey. First, a pre-established number of herds (primary sampling units) were randomly selected; second, within each herd, a pre-established number of cows aged ≥ 24 months were systematically selected (secondary sampling units). In total, 2279 animals were sampled from 468 herds. Serum samples were submitted to virus neutralization (VN) test for detection of antibodies to VSV using three viral strains: VSIV-3 2013SaoBento/Paraiba E, strain Indiana (VSIV-1) and VSNJV. A herd was considered positive for VSV if it included at least one positive animal in herds of up to 10 females, two positive animals in herds of 11-99 females, and three positive in herds with more than 99 females. The spatial clustering was assessed using the Cuzick-Edwards' k-nearest neighbor method and spatial scan statistics. The apparent herd-level prevalence in the state of Paraíba was 38.5% (95% CI = 35.5-41.6%), 80.6% (95% CI = 73.6-86.2%) in the region of Sertão, 7.0% (95% CI = 3.9-12.2%) in Borborema, and 2.6% (95% CI = 1.0-6.7%) in Agreste/Zona da Mata. The apparent animal-level prevalence was 26.2% (95% CI = 20.6-32.8%) in the state of Paraíba, 48.2% (95% CI = 41.5-54.9%) in Sertão, 6.3% (95% CI = 2.7-14%) in Borborema, and 3.2% 1.9% (95% CI = 0.4-8.4%) in Agreste/Zona da Mata. The risk factors identified were as follows: mixed production (milk/beef) (OR = 4.54), herd size > 23 animals (OR = 3.57), presence of cervids (OR = 15.24), rental of pastures (OR = 3.02), sharing of water sources (OR = 2.57) and presence of horses (OR = 1.69). Two significant clusters of positive herds were detected: the primary cluster covered the Sertão region and the secondary cluster covered part of the Sertão and Borborema regions. Our results suggest high VSV circulation in the bovine population of the state of Paraíba, semiarid region of Brazil, mainly in the Sertão mesoregion, and based on risk factor analysis it was possible to identify important associations that deserve more investigation on causal factors.


Assuntos
Doenças dos Bovinos/epidemiologia , Estomatite Vesicular/epidemiologia , Animais , Anticorpos Antivirais/sangue , Brasil/epidemiologia , Bovinos , Doenças dos Bovinos/virologia , Feminino , Testes de Neutralização/veterinária , Prevalência , Fatores de Risco , Análise Espacial , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular New Jersey/imunologia
10.
Can J Vet Res ; 82(4): 316-321, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30363380

RESUMO

Foot-and-mouth disease (FMD) and vesicular stomatitis (VS) cause such similar clinical signs and lesions that laboratory tests are required to distinguish between infections caused by each virus. Using mouse anti-foot-and-mouth disease virus (FMDV) 3B monoclonal or polyclonal anti-vesicular stomatitis virus-New Jersey (VSV-NJ) antibodies and recombinant FMDV 3ABC or VSV-NJ glycoprotein (G) antigens coated to MagPlex beads, competitive Luminex immunoassays (cLIAs) were developed for FMDV and VSV-NJ, respectively. The cLIAs successfully detected antibodies to FMDV 3ABC and VSV-NJ G in sera from infected animals. The diagnostic sensitivity and specificity were 93% and 98%, respectively for FMDV and 93% and 95.4%, respectively for VSV-NJ. These cLIAs are potential alternatives for competitive enzyme-linked immunosorbent assays (cELISAs) and provide the opportunity for multiplexing to reduce time and the amount of serum required for testing.


Assuntos
Anticorpos Antivirais/sangue , Febre Aftosa/diagnóstico , Imunoensaio/veterinária , Estomatite Vesicular/diagnóstico , Animais , Diagnóstico Diferencial , Febre Aftosa/imunologia , Vírus da Febre Aftosa/imunologia , Sensibilidade e Especificidade , Especificidade da Espécie , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia
11.
J Virol ; 92(23)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30232190

RESUMO

Vesicular stomatitis virus Indiana strain G protein (VSVind.G) is the most commonly used envelope glycoprotein to pseudotype lentiviral vectors (LV) for experimental and clinical applications. Recently, G proteins derived from other vesiculoviruses (VesG), for example, Cocal virus, have been proposed as alternative LV envelopes with possible advantages over VSVind.G. Well-characterized antibodies that recognize VesG will be useful for vesiculovirus research, development of G protein-containing advanced therapy medicinal products (ATMPs), and deployment of VSVind-based vaccine vectors. Here, we show that one commercially available monoclonal antibody, 8G5F11, binds to and neutralizes G proteins from three strains of VSV, as well as Cocal and Maraba viruses, whereas the other commercially available monoclonal anti-VSVind.G antibody, IE9F9, binds to and neutralizes only VSVind.G. Using a combination of G protein chimeras and site-directed mutations, we mapped the binding epitopes of IE9F9 and 8G5F11 on VSVind.G. IE9F9 binds close to the receptor binding site and competes with soluble low-density lipoprotein receptor (LDLR) for binding to VSVind.G, explaining its mechanism of neutralization. In contrast, 8G5F11 binds close to a region known to undergo conformational changes when the G protein moves to its postfusion structure, and we propose that 8G5F11 cross-neutralizes VesGs by inhibiting this.IMPORTANCE VSVind.G is currently regarded as the gold-standard envelope glycoprotein to pseudotype lentiviral vectors. However, recently other G proteins derived from vesiculoviruses have been proposed as alternative envelopes. Here, we investigated two commercially available anti-VSVind.G monoclonal antibodies for their ability to cross-react with other vesiculovirus G proteins, identified the epitopes they recognize, and explored their neutralization activity. We have identified 8G5F11, for the first time, as a cross-neutralizing antibody against several vesiculovirus G proteins. Furthermore, we elucidated the two different neutralization mechanisms employed by these two monoclonal antibodies. Understanding how cross-neutralizing antibodies interact with other G proteins may be of interest in the context of host-pathogen interaction and coevolution, as well as providing the opportunity to modify the G proteins and improve G protein-containing medicinal products and vaccine vectors.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Epitopos/imunologia , Glicoproteínas de Membrana/imunologia , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Antígenos Virais/genética , Antígenos Virais/metabolismo , Reações Cruzadas , Epitopos/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Testes de Neutralização , Filogenia , Homologia de Sequência , Estomatite Vesicular/metabolismo , Estomatite Vesicular/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
12.
Vaccine ; 36(41): 6061-6069, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30219365

RESUMO

The ability to rapidly and accurately determine viral infectivity can help improve the speed of vaccine product development and manufacturing. Current methods to determine infectious viral titers, such as the end-point dilution (50% tissue culture infective dose, TCID50) and plaque assays are slow, labor intensive, and often subjective. In order to accelerate virus quantification, Laser Force Cytology (LFC) was used to monitor vesicular stomatitis virus (VSV) infection in Vero (African green monkey kidney) cells. LFC uses a combination of optical and fluidic forces to interrogate single cells without the use of labels or antibodies. Using a combination of variables measured by the Radiance™ LFC instrument (LumaCyte), an infection metric was developed that correlates well with the viral titer as measured by TCID50 and shortens the timeframe from infection to titer determination from 3 days to 16 h (a 4.5 fold reduction). A correlation was also developed between in-process cellular measurements and the viral titer of collected supernatant, demonstrating the potential for real-time infectivity measurements. Overall, these results demonstrate the utility of LFC as a tool for rapid infectivity measurements throughout the vaccine development process.


Assuntos
Estomatite Vesicular/virologia , Vesiculovirus/isolamento & purificação , Vesiculovirus/patogenicidade , Animais , Anticorpos Antivirais/imunologia , Técnicas Citológicas , Células Vero , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/isolamento & purificação , Vírus da Estomatite Vesicular Indiana/patogenicidade , Vesiculovirus/imunologia
13.
Microbiol Immunol ; 62(9): 585-593, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30160073

RESUMO

MicroRNAs are short, non-coding RNAs that have been shown to regulate a wide range of biological processes, including host antiviral immune responses. In the present study, microRNA-92a (miR-92a) was identified as a negative regulator in macrophage-mediated antiviral responses. Overexpression of miR-92a decreases vesicular stomatitis virus (VSV)-induced production of type-I IFNs and facilitates viral replication in macrophages. The mechanism is that miR-92a directly targets RIG-I and reduces its expression, thereby attenuating VSV-triggered activation of TBK-binding kinase 1 and IRF3, both of which are crucial for initiating transcription of type-I IFN genes. Our results demonstrate for the first time the novel role of miR-92a in suppressing antiviral innate immunity.


Assuntos
Proteína DEAD-box 58/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , MicroRNAs/antagonistas & inibidores , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Antivirais/metabolismo , Citocinas/metabolismo , Proteína DEAD-box 58/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Imunidade Inata/imunologia , Fator Regulador 3 de Interferon/imunologia , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Células RAW 264.7/efeitos dos fármacos , Alinhamento de Sequência , Regulação para Cima/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
14.
Semin Immunol ; 39: 65-72, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30041831

RESUMO

Ebola virus disease is a deadly infection which occurs in sporadic outbreaks. Several vaccine candidates have been developed. The most advanced candidate is the recombinant VSVΔG-ZEBOV-GP vaccine, in which the Vesicular Stomatitis Virus (VSV) envelope glycoprotein is replaced by the Zaire strain Ebola virus (ZEBOV) glycoprotein (GP). This vaccine demonstrated 100% protection in a ring vaccination trial performed in Guinea in 2015, was granted "Breakthrough Therapy Designation" by the FDA and PRIority Medicines (PRIME), and is currently (June 2018) used to support outbreak control in Democratic Republic of Congo. rVSVΔG-ZEBOV-GP elicits a strong and durable antibody response in most vaccinees. This sustained Ebola GP-specific antibody response correlates with an early activation of innate immunity, especially of monocytes and of type-I interferon induced genes. Despite significant progress in the characterization of vaccine-induced immunity, human correlates of protection against Ebolavirus infection have not yet been fully established. A systems biology approach, integrating clinical, immunological, transcriptomic and metabolomic data from pre-clinical and clinical vaccine studies, together with data from disease survivors, will be instrumental to identify Ebola vaccine correlates of protection. The information generated for the rVSVΔG-ZEBOV-GP vaccine may also help identify the correlates of protection of the other Ebola vaccine candidates.


Assuntos
Anticorpos Antivirais/biossíntese , Vacinas contra Ebola/imunologia , Ebolavirus/efeitos dos fármacos , Determinação de Ponto Final/métodos , Doença pelo Vírus Ebola/prevenção & controle , Vacinação , África/epidemiologia , Vacinas contra Ebola/administração & dosagem , Vacinas contra Ebola/genética , Ebolavirus/imunologia , Ebolavirus/patogenicidade , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Imunidade Inata/efeitos dos fármacos , Imunogenicidade da Vacina , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Monócitos/imunologia , Biologia de Sistemas/métodos , Potência de Vacina , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
15.
Nat Commun ; 9(1): 2789, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-30018336

RESUMO

Interferon (IFN)-stimulated genes (ISGs) play crucial roles in the antiviral immune response; however, IFNs also induce negative regulators that attenuate the antiviral response. Here, we show that both viral and bacterial invasion downregulate Nuclear Dbf2-related kinase 1 (NDR1) expression via the type I IFN signaling pathway. NDR1 promotes the virus-induced production of type I IFN, proinflammatory cytokines and ISGs in a kinase-independent manner. NDR1 deficiency also renders mice more susceptible to viral and bacterial infections. Mechanistically, NDR1 enhances STAT1 translation by directly binding to the intergenic region of miR146a, thereby inhibiting miR146a expression and liberating STAT1 from miR146a-mediated translational inhibition. Furthermore, STAT1 binds to the miR146a promoter, thus decreasing its expression. Together, our results suggest that NDR1 promotion of STAT1 translation is an important event for IFN-dependent antiviral immune response, and suggest that NDR1 has an important role in controlling viral infections.


Assuntos
Bronquiolite Viral/genética , Retroalimentação Fisiológica , Herpesvirus Humano 1/genética , MicroRNAs/genética , Proteínas Serina-Treonina Quinases/genética , Fator de Transcrição STAT1/genética , Vírus da Estomatite Vesicular Indiana/genética , Animais , Bronquiolite Viral/imunologia , Bronquiolite Viral/virologia , Estudos de Casos e Controles , Criança , Regulação da Expressão Gênica , Células HEK293 , Herpesvirus Humano 1/imunologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon beta/genética , Interferon beta/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/imunologia , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/imunologia , Células RAW 264.7 , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/imunologia , Fator de Transcrição STAT1/imunologia , Transdução de Sinais , Vírus da Estomatite Vesicular Indiana/imunologia
16.
Nat Commun ; 9(1): 2770, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-30018345

RESUMO

Detection of viral genomes by the innate immune system elicits an antiviral gene program mediated by type I interferons (IFNs). While viral RNA and DNA species induce IFN via separate pathways, the mechanisms by which these pathways are differentially modulated are unknown. Here we show that the positive regulator of IFN in the RNA pathway, TRAF3, has an inhibitory function in the DNA pathway. Loss of TRAF3 coincides with increased expression of the alternative NF-κB-inducing molecule, NIK, which interacts with the DNA pathway adaptor, STING, to enhance IFN induction. Cells lacking NIK display defective IFN activation in the DNA pathway due to impaired STING signaling, and NIK-deficient mice are more susceptible to DNA virus infection. Mechanistically, NIK operates independently from alternative NF-κB signaling components and instead requires autophosphorylation and oligomerization to activate STING. Thus a previously undescribed pathway for NIK exists in activating IFN in the DNA pathway.


Assuntos
DNA Viral/genética , Herpesvirus Humano 1/genética , Interações Hospedeiro-Patógeno , Proteínas Serina-Treonina Quinases/genética , RNA Viral/genética , Fator 3 Associado a Receptor de TNF/genética , Vírus da Estomatite Vesicular Indiana/genética , Células A549 , Animais , DNA Viral/imunologia , Feminino , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Células HEK293 , Herpesvirus Humano 1/imunologia , Humanos , Imunidade Inata , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon beta/genética , Interferon beta/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/imunologia , Proteínas Serina-Treonina Quinases/imunologia , RNA Viral/imunologia , Transdução de Sinais , Células THP-1 , Fator 3 Associado a Receptor de TNF/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia
17.
Vet Microbiol ; 219: 30-39, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29778202

RESUMO

Vesicular stomatitis virus (VSV) can cause serious vesicular lesions in pigs, and the matrix (M) protein is its predominant virulence factor. Dendritic cells (DCs) act as the bridge between innate and adaptive immune responses. However, the susceptibility of porcine DCs to VSV infection and the role of M protein in modulating the function of infected DCs are still poorly defined. Thus, this study aimed to determine the ability of virulent wild-type VSV(wtVSV) and two attenuated M protein variants (VSVΔM51 and VSVMT) to induce maturation of porcine monocyte-derived DCs (MoDCs) in vitro. It was found that both wtVSV and the M protein mutant VSVs could productively replicate in porcine MoDCs. Infection with wtVSV resulted in weak proinflammatory cytokine responses and interfered with DC maturation via downregulation of the costimulatory molecule complex CD80/86. Whilst VSVΔM51 could activate porcine MoDCs, VSVMT, a highly attenuated recombinant VSV with triple mutations in the M protein, induced a potent maturation of MoDCs, as evidenced by efficient cytokine induction, and upregulation of CD80/86 and MHC class II. Overall, our findings reveal that porcine MoDCs are differentially activated by VSV, dependent on the presence of a functional M protein. M protein plays a crucial role in modulating porcine DC-VSV interactions. The data further support the potential use of VSVMT as a vaccine vector for pigs.


Assuntos
Células Dendríticas/virologia , Monócitos/virologia , Vírus da Estomatite Vesicular Indiana/genética , Proteínas da Matriz Viral/farmacologia , Animais , Moléculas de Adesão Celular/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Interleucina-1beta/biossíntese , Interleucina-1beta/imunologia , Monócitos/imunologia , Monócitos/fisiologia , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Proteínas Mutantes/farmacologia , Suínos , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/patogenicidade , Proteínas da Matriz Viral/genética
18.
Immunity ; 48(4): 716-729.e8, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29625895

RESUMO

Protective immunity against pathogens depends on the efficient generation of functionally diverse effector and memory T lymphocytes. However, whether plasticity during effector-to-memory CD8+ T cell differentiation affects memory lineage specification and functional versatility remains unclear. Using genetic fate mapping analysis of highly cytotoxic KLRG1+ effector CD8+ T cells, we demonstrated that KLRG1+ cells receiving intermediate amounts of activating and inflammatory signals downregulated KLRG1 during the contraction phase in a Bach2-dependent manner and differentiated into all memory T cell linages, including CX3CR1int peripheral memory cells and tissue-resident memory cells. "ExKLRG1" memory cells retained high cytotoxic and proliferative capacity distinct from other populations, which contributed to effective anti-influenza and anti-tumor immunity. Our work demonstrates that developmental plasticity of KLRG1+ effector CD8+ T cells is important in promoting functionally versatile memory cells and long-term protective immunity.


Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/imunologia , Ativação Linfocitária/imunologia , Receptores Imunológicos/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Linhagem da Célula/imunologia , Vírus da Influenza A/imunologia , Subunidade p35 da Interleucina-12/imunologia , Listeria monocytogenes/imunologia , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Imunológicos/genética , Vírus da Estomatite Vesicular Indiana/imunologia
20.
J Innate Immun ; 10(2): 131-144, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29306950

RESUMO

BACKGROUND: Oncolytic vesicular stomatitis virus (VSV) can be delivered intravenously to target primary and metastatic lesions, but the interaction between human peripheral blood leukocytes (PBLs) and VSV remains poorly understood. Our study aimed to assess the overall immunological consequences of ex vivo infection of PBLs with VSV. METHODS: Phenotypic analysis of lymphocyte subsets and apoptosis were evaluated with flow cytometry. Caspase 3/7 activity was detected by luminescence assay. Virus release was evaluated in a murine cell line (L929). Gene expression and cytokine/chemokine secretion were assessed by real-time PCR and multiplex assay, respectively. RESULTS: Ex vivo infection of PBLs with VSV elicited upregulated expression of RIG-I, MDA-5, tetherin, IFITM3, and MxA. VSV infection triggered rapid differentiation of blood monocytes into immature dendritic cells as well as their apoptosis, which depended on caspase 3/7 activation. Monocyte differentiation required infectious VSV, but loss of CD14+ cells was also associated with the presence of a cytokine/chemokine milieu produced in response to VSV infection. CONCLUSIONS: Systemic delivery is a major goal in the field of oncolytic viruses. Our results shed further light on immune mechanisms in response to VSV infection and the underlying VSV-PBL interactions bringing hope for improved cancer immunotherapies, particularly those based on intravenous delivery of oncolytic VSV.


Assuntos
Leucócitos Mononucleares/virologia , Vírus Oncolíticos/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Apoptose , Caspases Efetoras/metabolismo , Diferenciação Celular , Linhagem Celular , Citocinas/metabolismo , Células Dendríticas , Fibroblastos/virologia , Voluntários Saudáveis , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Receptores de Lipopolissacarídeos/metabolismo , Camundongos , Replicação Viral
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