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1.
Nat Immunol ; 21(8): 868-879, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32690950

RESUMO

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.


Assuntos
Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/fisiologia , Animais , Retículo Endoplasmático/imunologia , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Proteínas de Membrana/imunologia , Camundongos , Proteínas do Tecido Nervoso/imunologia , Proteínas Nucleares , Transporte Proteico/fisiologia
3.
Immunity ; 54(7): 1478-1493.e6, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34015257

RESUMO

Viral infections during pregnancy are a considerable cause of adverse outcomes and birth defects, and the underlying mechanisms are poorly understood. Among those, cytomegalovirus (CMV) infection stands out as the most common intrauterine infection in humans, putatively causing early pregnancy loss. We employed murine CMV as a model to study the consequences of viral infection on pregnancy outcome and fertility maintenance. Even though pregnant mice successfully controlled CMV infection, we observed highly selective, strong infection of corpus luteum (CL) cells in their ovaries. High infection densities indicated complete failure of immune control in CL cells, resulting in progesterone insufficiency and pregnancy loss. An abundance of gap junctions, absence of vasculature, strong type I interferon (IFN) responses, and interaction of innate immune cells fully protected the ovarian follicles from viral infection. Our work provides fundamental insights into the effect of CMV infection on pregnancy loss and mechanisms protecting fertility.


Assuntos
Corpo Lúteo/imunologia , Infecções por Citomegalovirus/imunologia , Fertilidade/imunologia , Imunidade Inata/imunologia , Animais , Corpo Lúteo/virologia , Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Feminino , Junções Comunicantes/imunologia , Interferon Tipo I/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Gravidez , Progesterona/imunologia
4.
Nature ; 632(8024): 383-389, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39048823

RESUMO

The brain is highly sensitive to damage caused by infection and inflammation1,2. Herpes simplex virus 1 (HSV-1) is a neurotropic virus and the cause of herpes simplex encephalitis3. It is unknown whether neuron-specific antiviral factors control virus replication to prevent infection and excessive inflammatory responses, hence protecting the brain. Here we identify TMEFF1 as an HSV-1 restriction factor using genome-wide CRISPR screening. TMEFF1 is expressed specifically in neurons of the central nervous system and is not regulated by type I interferon, the best-known innate antiviral system controlling virus infections. Depletion of TMEFF1 in stem-cell-derived human neurons led to elevated viral replication and neuronal death following HSV-1 infection. TMEFF1 blocked the HSV-1 replication cycle at the level of viral entry through interactions with nectin-1 and non-muscle myosin heavy chains IIA and IIB, which are core proteins in virus-cell binding and virus-cell fusion, respectively4-6. Notably, Tmeff1-/- mice exhibited increased susceptibility to HSV-1 infection in the brain but not in the periphery. Within the brain, elevated viral load was observed specifically in neurons. Our study identifies TMEFF1 as a neuron-specific restriction factor essential for prevention of HSV-1 replication in the central nervous system.


Assuntos
Fatores de Restrição Antivirais , Encéfalo , Herpes Simples , Herpesvirus Humano 1 , Proteínas de Membrana , Neurônios , Internalização do Vírus , Replicação Viral , Animais , Feminino , Humanos , Masculino , Camundongos , Fatores de Restrição Antivirais/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/virologia , Morte Celular , Sistemas CRISPR-Cas/genética , Herpes Simples/imunologia , Herpes Simples/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/crescimento & desenvolvimento , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Neurônios/virologia , Neurônios/metabolismo , Carga Viral , Nectinas/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Interferon Tipo I , Doenças Neuroinflamatórias/imunologia , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Doenças Neuroinflamatórias/prevenção & controle , Doenças Neuroinflamatórias/virologia
5.
Nature ; 632(8024): 390-400, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39048830

RESUMO

Most cases of herpes simplex virus 1 (HSV-1) encephalitis (HSE) remain unexplained1,2. Here, we report on two unrelated people who had HSE as children and are homozygous for rare deleterious variants of TMEFF1, which encodes a cell membrane protein that is preferentially expressed by brain cortical neurons. TMEFF1 interacts with the cell-surface HSV-1 receptor NECTIN-1, impairing HSV-1 glycoprotein D- and NECTIN-1-mediated fusion of the virus and the cell membrane, blocking viral entry. Genetic TMEFF1 deficiency allows HSV-1 to rapidly enter cortical neurons that are either patient specific or derived from CRISPR-Cas9-engineered human pluripotent stem cells, thereby enhancing HSV-1 translocation to the nucleus and subsequent replication. This cellular phenotype can be rescued by pretreatment with type I interferon (IFN) or the expression of exogenous wild-type TMEFF1. Moreover, ectopic expression of full-length TMEFF1 or its amino-terminal extracellular domain, but not its carboxy-terminal intracellular domain, impairs HSV-1 entry into NECTIN-1-expressing cells other than neurons, increasing their resistance to HSV-1 infection. Human TMEFF1 is therefore a host restriction factor for HSV-1 entry into cortical neurons. Its constitutively high abundance in cortical neurons protects these cells from HSV-1 infection, whereas inherited TMEFF1 deficiency renders them susceptible to this virus and can therefore underlie HSE.


Assuntos
Encéfalo , Encefalite por Herpes Simples , Herpesvirus Humano 1 , Proteínas de Membrana , Internalização do Vírus , Animais , Feminino , Humanos , Masculino , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/virologia , Encefalite por Herpes Simples/virologia , Encefalite por Herpes Simples/metabolismo , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 1/fisiologia , Homozigoto , Interferon Tipo I/metabolismo , Interferon Tipo I/imunologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nectinas/genética , Nectinas/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Neurônios/virologia , Células-Tronco Pluripotentes/citologia , Replicação Viral , Pré-Escolar , Adulto Jovem , Linhagem
6.
Nat Immunol ; 17(2): 150-8, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26595890

RESUMO

Mucosal surfaces are exposed to environmental substances and represent a major portal of entry for microorganisms. The innate immune system is responsible for early defense against infections and it is believed that the interferons (IFNs) constitute the first line of defense against viruses. Here we identify an innate antiviral pathway that works at epithelial surfaces before the IFNs. The pathway is activated independently of known innate sensors of viral infections through a mechanism dependent on viral O-linked glycans, which induce CXCR3 chemokines and stimulate antiviral activity in a manner dependent on neutrophils. This study therefore identifies a previously unknown layer of antiviral defense that exerts its action on epithelial surfaces before the classical IFN response is operative.


Assuntos
Imunidade Inata , Interferons/metabolismo , Mucosa/imunologia , Mucosa/metabolismo , Viroses/imunologia , Viroses/metabolismo , Animais , Linhagem Celular , Quimiocina CXCL10/biossíntese , Modelos Animais de Doenças , Feminino , Expressão Gênica , Glicosilação , Herpes Simples/genética , Herpes Simples/imunologia , Herpes Simples/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 2/imunologia , Humanos , Interferons/genética , Ligantes , Camundongos , Camundongos Knockout , Mucosa/virologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Polissacarídeos/imunologia , Receptores CXCR3/deficiência , Receptores CXCR3/metabolismo , Vagina/imunologia , Vagina/metabolismo , Vagina/virologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Carga Viral , Viroses/virologia
7.
EMBO J ; 42(19): e113118, 2023 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-37646198

RESUMO

Neurotropic viruses, including herpes simplex virus (HSV) types 1 and 2, have the capacity to infect neurons and can cause severe diseases. This is associated with neuronal cell death, which may contribute to morbidity or even mortality if the infection is not controlled. However, the mechanistic details of HSV-induced neuronal cell death remain enigmatic. Here, we report that lytic HSV-2 infection of human neuron-like SH-SY5Y cells and primary human and murine brain cells leads to cell death mediated by gasdermin E (GSDME). HSV-2-induced GSDME-mediated cell death occurs downstream of replication-induced endoplasmic reticulum stress driven by inositol-requiring kinase 1α (IRE1α), leading to activation of caspase-2, cleavage of the pro-apoptotic protein BH3-interacting domain death agonist (BID), and mitochondria-dependent activation of caspase-3. Finally, necrotic neurons released alarmins, which activated inflammatory responses in human iPSC-derived microglia. In conclusion, lytic HSV infection in neurons activates an ER stress-driven pathway to execute GSDME-mediated cell death and promote inflammation.

8.
Trends Immunol ; 45(9): 662-677, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39198098

RESUMO

Herpesviruses are DNA viruses and the cause of diseases ranging from mild skin conditions to severe brain diseases. Mammalian antiviral host defense comprises an array of mechanisms, including restriction factors (RFs), which block specific steps in viral replication cycles. In recent years, knowledge of RFs that contribute to controlling herpesvirus infections has expanded significantly, along with a new understanding of viral evasion mechanisms and disease pathogenesis. By integrating findings from human genetics, murine models, and cellular studies, this review provides a current view of RF control of herpesvirus infections. We also explore the regulation of RF expression, discuss the roles of RFs in diseases, and point towards their growing potential as candidate therapeutic targets.


Assuntos
Infecções por Herpesviridae , Humanos , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Animais , Herpesviridae/imunologia , Herpesviridae/fisiologia , Replicação Viral , Interações Hospedeiro-Patógeno/imunologia , Evasão da Resposta Imune
9.
EMBO J ; 41(10): e109622, 2022 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-35178710

RESUMO

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS-CoV-2 infection is critical for developing treatments for severe COVID-19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID-19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL-6. Using an in vitro stem cell-based human pDC model, we further demonstrate that pDCs, while not supporting SARS-CoV-2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL-6, IL-8, CXCL10) cytokines that protect epithelial cells from de novo SARS-CoV-2 infection. Via targeted deletion of virus-recognition innate immune pathways, we identify TLR7-MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll-like receptor (TLR)2 is responsible for the inflammatory IL-6 response. We further show that SARS-CoV-2 engages the receptor neuropilin-1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL-6 response, suggesting neuropilin-1 as potential therapeutic target for stimulation of TLR7-mediated antiviral protection.


Assuntos
COVID-19 , Células Dendríticas , Receptor 2 Toll-Like , Receptor 7 Toll-Like , COVID-19/imunologia , COVID-19/patologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/patologia , Humanos , Interferon Tipo I/imunologia , Interferon-alfa/imunologia , Interleucina-6/imunologia , Neuropilina-1/imunologia , SARS-CoV-2 , Receptor 2 Toll-Like/imunologia , Receptor 7 Toll-Like/imunologia
10.
EMBO Rep ; 25(9): 3777-3788, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39060455

RESUMO

RNA vaccines elicit protective immunity against SARS-CoV-2, but the use of mRNA as an antiviral immunotherapeutic is unexplored. Here, we investigate the activity of lipidoid nanoparticle (LNP)-formulated mRNA encoding human IFNλ1 (ETH47), which is a critical driver of innate immunity at mucosal surfaces protecting from viral infections. IFNλ1 mRNA administration promotes dose-dependent protein translation, induction of interferon-stimulated genes without relevant signs of unspecific immune stimulation, and dose-dependent inhibition of SARS-CoV-2 replication in vitro. Pulmonary administration of IFNλ1 mRNA in mice results in a potent reduction of virus load, virus-induced body weight loss and significantly increased survival. These data support the development of inhaled administration of IFNλ1 mRNA as a potential prophylactic option for individuals exposed to SARS-CoV-2 or at risk suffering from COVID-19. Based on the broad antiviral activity of IFNλ1 regardless of virus or variant, this approach might also be utilized for other respiratory viral infections or pandemic preparedness.


Assuntos
COVID-19 , Interferon lambda , RNA Mensageiro , SARS-CoV-2 , Animais , Feminino , Humanos , Camundongos , Antivirais , Chlorocebus aethiops , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Imunomodulação , Interferons/metabolismo , Lipossomos , Nanopartículas/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , SARS-CoV-2/imunologia , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Carga Viral , Replicação Viral , Interferon lambda/administração & dosagem , Interferon lambda/genética
11.
J Clin Immunol ; 44(2): 56, 2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-38277122

RESUMO

Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus exclusively infecting humans, causing two distinct pathologies: varicella (chickenpox) upon primary infection and herpes zoster (shingles) following reactivation. In susceptible individuals, VZV can give rise to more severe clinical manifestations, including disseminated infection, pneumonitis, encephalitis, and vasculopathy with stroke. Here, we describe a 3-year-old boy in whom varicella followed a complicated course with thrombocytopenia, hemorrhagic and necrotic lesions, pneumonitis, and intermittent encephalopathy. Hemophagocytic lymphohistiocytosis (HLH) was strongly suspected and as the condition deteriorated, HLH therapy was initiated. Although the clinical condition improved, longstanding hemophagocytosis followed despite therapy. We found that the patient carries a rare monoallelic variant in autocrine motility factor receptor (AMFR), encoding a ubiquitin ligase involved in innate cytosolic DNA sensing and interferon (IFN) production through the cyclic GMP-AMP synthase-stimulator of IFN genes (cGAS-STING) pathway. Peripheral blood mononuclear cells (PBMCs) from the patient exhibited impaired signaling downstream of STING in response dsDNA and 2'3'-cGAMP, agonists of cGAS and STING, respectively, and fibroblasts from the patient showed impaired type I IFN responses and significantly increased VZV replication. Overexpression of the variant AMFR R594C resulted in decreased K27-linked STING ubiquitination compared to WT AMFR. Moreover, ImageStream technology revealed reduced STING trafficking from ER to Golgi in cells expressing the patient AMFR R594C variant. This was supported by a dose-dependent dominant negative effect of expression of the patient AMFR variant as measured by IFN-ß reporter gene assay. Finally, lentiviral transduction with WT AMFR partially reconstituted 2'3'-cGAMP-induced STING-mediated signaling and ISG expression in patient PBMCs. This work links defective AMFR-STING signaling to severe VZV disease and hyperinflammation and suggests a direct role for cGAS-STING in the control of viral infections in humans. In conclusion, we describe a novel genetic etiology of severe VZV disease in childhood, also representing the first inborn error of immunity related to a defect in the cGAS-STING pathway.


Assuntos
Varicela , Herpes Zoster , Interferon Tipo I , Linfo-Histiocitose Hemofagocítica , Pneumonia , Pré-Escolar , Humanos , Herpesvirus Humano 3/genética , Imunidade Inata , Leucócitos Mononucleares/metabolismo , Linfo-Histiocitose Hemofagocítica/diagnóstico , Linfo-Histiocitose Hemofagocítica/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Receptores do Fator Autócrino de Motilidade , Ubiquitina-Proteína Ligases/genética , Masculino
12.
Nat Immunol ; 13(8): 737-43, 2012 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-22706339

RESUMO

The innate immune system senses infection by detecting either evolutionarily conserved molecules essential for the survival of microbes or the abnormal location of molecules. Here we demonstrate the existence of a previously unknown innate detection mechanism induced by fusion between viral envelopes and target cells. Virus-cell fusion specifically stimulated a type I interferon response with expression of interferon-stimulated genes, in vivo recruitment of leukocytes and potentiation of signaling via Toll-like receptor 7 (TLR7) and TLR9. The fusion-dependent response was dependent on the stimulator of interferon genes STING but was independent of DNA, RNA and viral capsid. We suggest that membrane fusion is sensed as a danger signal with potential implications for defense against enveloped viruses and various conditions of giant-cell formation.


Assuntos
Fusão Celular , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/fisiologia , Imunidade Inata , Interferon Tipo I/biossíntese , Fusão de Membrana , Proteínas de Membrana/metabolismo , Animais , Quimiocina CXCL10/metabolismo , Células HEK293 , Células HeLa , Humanos , Leucócitos/imunologia , Leucócitos/metabolismo , Ativação Linfocitária , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Internalização do Vírus
13.
J Med Virol ; 96(6): e29690, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38804180

RESUMO

Autophagy is a degradational pathway with pivotal roles in cellular homeostasis and survival, including protection of neurons in the central nervous system (CNS). The significance of autophagy as antiviral defense mechanism is recognized and some viruses hijack and modulate this process to their advantage in certain cell types. Here, we present data demonstrating that the human neurotropic herpesvirus varicella zoster virus (VZV) induces autophagy in human SH-SY5Y neuronal cells, in which the pathway exerts antiviral activity. Productively VZV-infected SH-SY5Y cells showed increased LC3-I-LC3-II conversion as well as co-localization of the viral glycoprotein E and the autophagy receptor p62. The activation of autophagy was dependent on a functional viral genome. Interestingly, inducers of autophagy reduced viral transcription, whereas inhibition of autophagy increased viral transcript expression. Finally, the genotype of patients with severe ocular and brain VZV infection were analyzed to identify potential autophagy-associated inborn errors of immunity. Two patients expressing genetic variants in the autophagy genes ULK1 and MAP1LC3B2, respectively, were identified. Notably, cells of both patients showed reduced autophagy, alongside enhanced viral replication and death of VZV-infected cells. In conclusion, these results demonstrate a neuro-protective role for autophagy in the context of VZV infection and suggest that failure to mount an autophagy response is a potential predisposing factor for development of severe VZV disease.


Assuntos
Autofagia , Herpesvirus Humano 3 , Neurônios , Humanos , Herpesvirus Humano 3/fisiologia , Herpesvirus Humano 3/patogenicidade , Neurônios/virologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Replicação Viral , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Infecção pelo Vírus da Varicela-Zoster/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Linhagem Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Interações Hospedeiro-Patógeno
14.
EMBO Rep ; 23(12): e55839, 2022 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-36268590

RESUMO

ZBP1 is an interferon-induced cytosolic nucleic acid sensor that facilitates antiviral responses via RIPK3. Although ZBP1-mediated programmed cell death is widely described, whether and how it promotes inflammatory signaling is unclear. Here, we report a ZBP1-induced inflammatory signaling pathway mediated by K63- and M1-linked ubiquitin chains, which depends on RIPK1 and RIPK3 as scaffolds independently of cell death. In human HT29 cells, ZBP1 associated with RIPK1 and RIPK3 as well as ubiquitin ligases cIAP1 and LUBAC. ZBP1-induced K63- and M1-linked ubiquitination of RIPK1 and ZBP1 to promote TAK1- and IKK-mediated inflammatory signaling and cytokine production. Inhibition of caspase activity suppressed ZBP1-induced cell death but enhanced cytokine production in a RIPK1- and RIPK3 kinase activity-dependent manner. Lastly, we provide evidence that ZBP1 signaling contributes to SARS-CoV-2-induced cytokine production. Taken together, we describe a ZBP1-RIPK3-RIPK1-mediated inflammatory signaling pathway relayed by the scaffolding role of RIPKs and regulated by caspases, which may induce inflammation when ZBP1 is activated below the threshold needed to trigger a cell death response.


Assuntos
Morte Celular , Proteínas de Ligação a RNA , Proteína Serina-Treonina Quinases de Interação com Receptores , Humanos , Citocinas , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais , Ubiquitina , Proteínas de Ligação a RNA/genética , Células HT29 , Inflamação
15.
Mol Ther ; 31(11): 3163-3175, 2023 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-37658603

RESUMO

In vivo CRISPR gene therapy holds large clinical potential, but the safety and efficacy remain largely unknown. Here, we injected a single dose of herpes simplex virus 1 (HSV-1)-targeting CRISPR formulation in the cornea of three patients with severe refractory herpetic stromal keratitis (HSK) during corneal transplantation. Our study is an investigator-initiated, open-label, single-arm, non-randomized interventional trial at a single center (NCT04560790). We found neither detectable CRISPR-induced off-target cleavages by GUIDE-seq nor systemic adverse events for 18 months on average in all three patients. The HSV-1 remained undetectable during the study. Our preliminary clinical results suggest that in vivo gene editing targeting the HSV-1 genome holds acceptable safety as a potential therapy for HSK.


Assuntos
Herpesvirus Humano 1 , Ceratite Herpética , Humanos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Ceratite Herpética/terapia , Ceratite Herpética/tratamento farmacológico , Córnea , Herpesvirus Humano 1/genética
16.
Proc Natl Acad Sci U S A ; 118(30)2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34301873

RESUMO

Nanotechnology enables investigations of single biomacromolecules, but technical challenges have limited the application in liquid biopsies, for example, blood plasma. Nonetheless, tools to characterize single molecular species in such samples represent a significant unmet need with the increasing appreciation of the physiological importance of protein structural changes at nanometer scale. Mannose-binding lectin (MBL) is an oligomeric plasma protein and part of the innate immune system through its ability to activate complement. MBL also serves a role as a scavenger for cellular debris, especially DNA. This may link functions of MBL with several inflammatory diseases in which cell-free DNA now appears to play a role, but mechanistic insight has been lacking. By making nanoparticle tracking analysis possible in human plasma, we now show that superoligomeric structures of MBL form nanoparticles with DNA. These oligomers correlate with disease activity in systemic lupus erythematosus patients. With the direct quantification of the hydrodynamic radius, calculations following the principles of Taylor dispersion in the blood stream connect the size of these complexes to endothelial inflammation, which is among the most important morbidities in lupus. Mechanistic insight from an animal model of lupus supported that DNA-stabilized superoligomers stimulate the formation of germinal center B cells and drive loss of immunological tolerance. The formation involves an inverse relationship between the concentration of MBL superoligomers and antibodies to double-stranded DNA. Our approach implicates the structure of DNA-protein nanoparticulates in the pathobiology of autoimmune diseases.


Assuntos
DNA/química , Lúpus Eritematoso Sistêmico/diagnóstico , Nanopartículas/química , Proteínas/química , Adolescente , Adulto , Animais , Linfócitos B , Biomarcadores , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Lectina de Ligação a Manose , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Adulto Jovem
17.
Proc Natl Acad Sci U S A ; 118(50)2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34876524

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2'-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer-spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants.


Assuntos
Aptâmeros de Nucleotídeos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Humanos , Mutação , Testes de Neutralização , Conformação de Ácido Nucleico , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2/fisiologia , Técnica de Seleção de Aptâmeros , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
18.
EMBO J ; 38(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30696688

RESUMO

Cytomegaloviruses (CMVs) are master manipulators of the host immune response. Here, we reveal that the murine CMV (MCMV) protein m152 specifically targets the type I interferon (IFN) response by binding to stimulator of interferon genes (STING), thereby delaying its trafficking to the Golgi compartment from where STING initiates type I IFN signaling. Infection with an MCMV lacking m152 induced elevated type I IFN responses and this leads to reduced viral transcript levels both in vitro and in vivo This effect is ameliorated in the absence of STING Interestingly, while m152 inhibits STING-mediated IRF signaling, it did not affect STING-mediated NF-κB signaling. Analysis of how m152 targets STING translocation reveals that STING activates NF-κB signaling already from the ER prior to its trafficking to the Golgi. Strikingly, this response is important to promote early MCMV replication. Our results show that MCMV has evolved a mechanism to specifically antagonize the STING-mediated antiviral IFN response, while preserving its pro-viral NF-κB response, providing an advantage in the establishment of an infection.


Assuntos
Infecções por Citomegalovirus/imunologia , Interações Hospedeiro-Patógeno/imunologia , Fatores Reguladores de Interferon/metabolismo , Interferon Tipo I/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , NF-kappa B/metabolismo , Proteínas Virais/metabolismo , Animais , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Fatores Reguladores de Interferon/genética , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Muromegalovirus/genética , Muromegalovirus/isolamento & purificação , Muromegalovirus/patogenicidade , NF-kappa B/genética , Ligação Proteica , Proteínas Virais/genética , Replicação Viral
19.
Eur J Immunol ; 52(5): 800-809, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35128644

RESUMO

The patterns of humoral and cellular responses to SARS-CoV-2 were studied in Swedish primary health care workers (n = 156) for 6 months during the Covid-19 pandemic. Serum IgA and IgG to SARS-CoV-2, T-cell proliferation and cytokine secretion, demographic and clinical data, PCR-verified infection, and self-reported symptoms were monitored. The multivariate method OPLS-DA was used to identify immune response patterns coupled to protection from Covid-19. Contracting Covid-19 was associated with SARS-CoV-2-specific neutralizing serum IgG, T cell, IFN-γ, and granzyme B responses to SARS-CoV-2, self-reported typical Covid-19 symptoms, male sex, higher BMI, and hypertension. Not contracting Covid-19 was associated with female sex, IgA-dominated, or no antibody responses to SARS-CoV-2, airborne allergy, and smoking. The IgG-responders had SARS-CoV-2-specific T-cell responses including a cytotoxic CD4+ T-cell population expressing CD25, CD38, CD69, CD194, CD279, CTLA-4, and granzyme B. IgA-responders with no IgG response to SARS-CoV-2 constituted 10% of the study population. The IgA responses were partially neutralizing and only seen in individuals who did not succumb to Covid-19. To conclude, serum IgG-dominated responses correlated with T-cell responses to SARS-CoV-2 and PCR-confirmed Covid-19, whereas IgA-dominated responses correlated with not contracting the infection.


Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Antivirais , Feminino , Granzimas , Humanos , Imunoglobulina A , Imunoglobulina G , Masculino , Pandemias/prevenção & controle , Atenção Primária à Saúde
20.
J Virol ; 96(6): e0131121, 2022 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-35045263

RESUMO

Spread of herpes simplex virus 1 (HSV1) from the periphery to the central nervous system (CNS) can lead to extensive infection and pathological inflammation in the brain, causing herpes simplex encephalitis (HSE). It has been shown that microglia, the CNS-resident macrophages, are involved in early sensing of HSV1 and induction of antiviral responses. In addition, infiltration of peripheral immune cells may contribute to the control of viral infection. In this study, we tested the effect of microglia depletion in a mouse model of HSE. Increased viral titers and increased disease severity were observed in microglia-depleted mice. The effect of microglia depletion was more pronounced in wild-type than in cGas-/- mice, revealing that this immune sensor contributes to the antiviral activity of microglia. Importantly, microglia depletion led to reduced production of type I interferon (IFN), proinflammatory cytokines, and chemokines at early time points after viral entry into the CNS. In line with this, in vitro experiments on murine primary CNS cells demonstrated microglial presence to be essential for IFN RNA induction, and control of HSV1 replication. However, the effect of microglia depletion on the expression of IFNs, and inflammatory cytokines was restricted to the early time point of HSV1 entry into the CNS. There was no major alteration of infiltration of CD45-positive cells in microglia-depleted mice. Collectively, our data demonstrate a key role for microglia in controlling HSV1 replication early after viral entry into the CNS and highlight the importance of a prompt antiviral innate response to reduce the risk of HSE development. IMPORTANCE One of the most devastating and acute neurological conditions is encephalitis, i.e., inflammation of brain tissue. Herpes simplex virus 1 (HSV1) is a highly prevalent pathogen in humans, and the most frequent cause of viral sporadic encephalitis called herpes simplex encephalitis (HSE). HSV1 can infect peripheral neurons and reach the central nervous system (CNS) of humans, where it can be detected by brain resident cells and infiltrating immune cells, leading to protective and damaging immune responses. In this study, we investigated the effects of microglia depletion, the main brain-resident immune cell type. For this purpose, we used a mouse model of HSE. We found that viral levels increased, and disease symptoms worsened in microglia-depleted mice. In addition, mice lacking a major sensor of viral DNA, cGAS, manifested a more pronounced disease than wild-type mice, highlighting the importance of this immune sensor in the activity of microglia. Microglia depletion led to reduced production of many known antiviral factors, most notably type I interferon (IFN). The importance of microglia in the early control of HSV1 spread and the generation of antiviral responses is further demonstrated by experiments on murine mixed glial cell cultures. Interestingly, mice with microglia depletion exhibited an unaltered activation of antiviral responses and recruitment of immune cells from the periphery at later time points of infection, but this did not prevent the development of the disease. Overall, the data highlight the importance of rapid activation of the host defense, with microglia playing a critical role in controlling HSV1 infection, which eventually prevents damage to neurons and brain tissue.


Assuntos
Encefalite por Herpes Simples , Herpesvirus Humano 1 , Imunidade , Interferon Tipo I , Microglia , Internalização do Vírus , Animais , Encéfalo/imunologia , Encéfalo/virologia , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalite por Herpes Simples/imunologia , Encefalite por Herpes Simples/fisiopatologia , Herpesvirus Humano 1/metabolismo , Imunidade/imunologia , Inflamação/patologia , Interferon Tipo I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/imunologia , Microglia/virologia , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo
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