RESUMO
The human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, possibly due to the properties of the immature neonatal pulmonary immune system. Using the newborn lamb, a classical model of human lung development and a translational model of RSV infection, we aimed to explore the role of cell-mediated immunity in RSV disease during early life. Remarkably, in healthy conditions, the developing T cell compartment of the neonatal lung showed major differences to that seen in the mature adult lung. The most striking observation being a high baseline frequency of bronchoalveolar IL-4-producing CD4+ and CD8+ T cells, which declined progressively over developmental age. RSV infection exacerbated this pro-type 2 environment in the bronchoalveolar space, rather than inducing a type 2 response per se. Moreover, regulatory T cell suppressive functions occurred very early to dampen this pro-type 2 environment, rather than shutting them down afterwards, while γδ T cells dropped and failed to produce IL-17. Importantly, RSV disease severity was related to the magnitude of those unconventional bronchoalveolar T cell responses. These findings provide novel insights in the mechanisms of RSV immunopathogenesis in early life, and constitute a major step for the understanding of RSV disease severity.
Assuntos
Pulmão/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções Respiratórias/imunologia , Linfócitos T/patologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/imunologia , Células Cultivadas , Pré-Escolar , Modelos Animais de Doenças , Progressão da Doença , Humanos , Pulmão/crescimento & desenvolvimento , Pulmão/patologia , Pulmão/virologia , Infecções por Vírus Respiratório Sincicial/congênito , Infecções por Vírus Respiratório Sincicial/patologia , Infecções Respiratórias/congênito , Infecções Respiratórias/patologia , Ovinos/crescimento & desenvolvimento , Ovinos/imunologia , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
Lung-resident (LR) mesenchymal stem and stromal cells (MSCs) are key elements of the alveolar niche and fundamental regulators of homeostasis and regeneration. We interrogated their function during virus-induced lung injury using the highly prevalent respiratory syncytial virus (RSV) which causes severe outcomes in infants. We applied complementary approaches with primary pediatric LR-MSCs and a state-of-the-art model of human RSV infection in lamb. Remarkably, RSV-infection of pediatric LR-MSCs led to a robust activation, characterized by a strong antiviral and pro-inflammatory phenotype combined with mediators related to T cell function. In line with this, following in vivo infection, RSV invades and activates LR-MSCs, resulting in the expansion of the pulmonary MSC pool. Moreover, the global transcriptional response of LR-MSCs appears to follow RSV disease, switching from an early antiviral signature to repair mechanisms including differentiation, tissue remodeling, and angiogenesis. These findings demonstrate the involvement of LR-MSCs during virus-mediated acute lung injury and may have therapeutic implications.
Assuntos
Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/virologia , Pulmão/imunologia , Células-Tronco Mesenquimais/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Animais , Humanos , Pulmão/citologia , Pulmão/metabolismo , Células-Tronco Mesenquimais/metabolismo , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sincicial Respiratório Humano/imunologia , OvinosRESUMO
Approved vaccines are effective against severe COVID-19, but broader immunity is needed against new variants and transmission. Therefore, we developed genome-modified live-attenuated vaccines (LAV) by recoding the SARS-CoV-2 genome, including 'one-to-stop' (OTS) codons, disabling Nsp1 translational repression and removing ORF6, 7ab and 8 to boost host immune responses, as well as the spike polybasic cleavage site to optimize the safety profile. The resulting OTS-modified SARS-CoV-2 LAVs, designated as OTS-206 and OTS-228, are genetically stable and can be intranasally administered, while being adjustable and sustainable regarding the level of attenuation. OTS-228 exhibits an optimal safety profile in preclinical animal models, with no side effects or detectable transmission. A single-dose vaccination induces a sterilizing immunity in vivo against homologous WT SARS-CoV-2 challenge infection and a broad protection against Omicron BA.2, BA.5 and XBB.1.5, with reduced transmission. Finally, this promising LAV approach could be applicable to other emerging viruses.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Genoma Viral , SARS-CoV-2 , Vacinas Atenuadas , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/genética , Vacinas Atenuadas/administração & dosagem , SARS-CoV-2/genética , SARS-CoV-2/imunologia , COVID-19/prevenção & controle , COVID-19/transmissão , COVID-19/imunologia , COVID-19/virologia , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/genética , Animais , Genoma Viral/genética , Humanos , Camundongos , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Feminino , Chlorocebus aethiops , Modelos Animais de Doenças , Células Vero , Anticorpos Neutralizantes/imunologiaRESUMO
We present a protocol to generate an advanced ex vivo model of human placenta. We use a vibrating tissue slicer to obtain precision-cut slices representative of the entire thickness of human placenta. This approach delivers standardized cultures with a preserved microstructure and cellular composition comparable to the native tissue. We applied this system to study SARS-CoV-2 infection at the maternal-fetal interface. Moreover, this system can be used to investigate the basic functions of the human placenta in health and disease. For complete details on the use and execution of this protocol, please refer to Fahmi et al. (2021).
Assuntos
COVID-19 , SARS-CoV-2 , Feminino , Humanos , Placenta , GravidezRESUMO
Here, we present a protocol to analyze the T cell profiles of the neonatal ovine lung during respiratory syncytial virus (RSV) infection. The protocol delivers standardized multiparameter flow cytometry (FCM) analysis of CD4+, CD8+, regulatory, and γδ T cells isolated from lung, lymph nodes, and bronchoalveolar lavages (BALs). We detail the preparation of RSV and transtracheal inoculation of newborn lambs. We then describe tissue isolation and preparation of cell suspensions, followed by FCM acquisition to identify different T cell subsets. For complete details on the use and execution of this protocol, please refer to Démoulins et al. (2021).
Assuntos
Infecções por Vírus Respiratório Sincicial , Animais , Ovinos , Infecções por Vírus Respiratório Sincicial/patologia , Citometria de Fluxo , Vírus Sinciciais Respiratórios , Pulmão/patologia , Subpopulações de Linfócitos TRESUMO
The deacetylase HDAC6 has tandem catalytic domains and a zinc finger domain (ZnF) binding ubiquitin (Ub). While the catalytic domain has an antiviral effect, the ZnF facilitates influenza A virus (IAV) infection and cellular stress responses. By recruiting Ub via the ZnF, HDAC6 promotes the formation of aggresomes and stress granules (SGs), dynamic structures associated with pathologies such as neurodegeneration. IAV subverts the aggresome/HDAC6 pathway to facilitate capsid uncoating during early infection. To target this pathway, we generate designed ankyrin repeat proteins (DARPins) binding the ZnF; one of these prevents interaction with Ub in vitro and in cells. Crystallographic analysis shows that it blocks the ZnF pocket where Ub engages. Conditional expression of this DARPin reversibly impairs infection by IAV and Zika virus; moreover, SGs and aggresomes are downregulated. These results validate the HDAC6 ZnF as an attractive target for drug discovery.
Assuntos
Vírus da Influenza A , Influenza Humana , Infecção por Zika virus , Zika virus , Desacetilase 6 de Histona/metabolismo , Humanos , Vírus da Influenza A/metabolismo , Ubiquitina/metabolismo , Zika virus/metabolismoRESUMO
The ongoing SARS-CoV-2 pandemic continues to lead to high morbidity and mortality. During pregnancy, severe maternal and neonatal outcomes and placental pathological changes have been described. We evaluate SARS-CoV-2 infection at the maternal-fetal interface using precision-cut slices (PCSs) of human placenta. Remarkably, exposure of placenta PCSs to SARS-CoV-2 leads to a full replication cycle with infectious virus release. Moreover, the susceptibility of placental tissue to SARS-CoV-2 replication relates to the expression levels of ACE2. Viral proteins and/or viral RNA are detected in syncytiotrophoblasts, cytotrophoblasts, villous stroma, and possibly Hofbauer cells. While SARS-CoV-2 infection of placenta PCSs does not cause a detectable cytotoxicity or a pro-inflammatory cytokine response, an upregulation of one order of magnitude of interferon type III transcripts is measured. In conclusion, our data demonstrate the capacity of SARS-CoV-2 to infect and propagate in human placenta and constitute a basis for further investigation of SARS-CoV-2 biology at the maternal-fetal interface.
Assuntos
Placenta/virologia , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/transmissão , COVID-19/virologia , Vilosidades Coriônicas/virologia , Feminino , Humanos , Transmissão Vertical de Doenças Infecciosas , Interferons/metabolismo , Placenta/citologia , Placenta/metabolismo , Gravidez , RNA Viral/metabolismo , Trofoblastos/citologia , Trofoblastos/virologia , Proteínas Virais/metabolismo , Liberação de Vírus , Replicação Viral , Interferon lambdaRESUMO
INTRODUCTION: Rhinovirus (RV) infection is a major cause of cystic fibrosis (CF) lung morbidity with limited therapeutic options. Various diseases involving chronic inflammatory response and infection are associated with endoplasmic reticulum (ER) stress and subsequent activation of the unfolded protein response (UPR), an adaptive response to maintain cellular homeostasis. Recent evidence suggests impaired ER stress response in CF airway epithelial cells, this might be a reason for recurrent viral infection in CF. Therefore, assuming that ER stress inducing drugs have antiviral properties, we evaluated the activation of the UPR by selected ER stress inducers as an approach to control virus replication in the CF bronchial epithelium. METHODS: We assessed the levels of UPR markers, namely the glucose-regulated protein 78 (Grp78) and the C/EBP homologous protein (CHOP), in primary CF and control bronchial epithelial cells and in a CF and control bronchial epithelial cell line before and after infection with RV. The cells were also pretreated with ER stress-inducing drugs and RV replication and shedding was measured by quantitative RT-PCR and by a TCID50 assay, respectively. Cell death was assessed by a lactate dehydrogenate (LDH) activity test in supernatants. RESULTS: We observed a significantly impaired induction of Grp78 and CHOP in CF compare to control cells following RV infection. The ER stress response could be significantly induced in CF cells by pharmacological ER stress inducers Brefeldin A, Tunicamycin, and Thapsigargin. The chemical induction of the UPR pathway prior to RV infection of CF and control cells reduced viral replication and shedding by up to two orders of magnitude and protected cells from RV-induced cell death. CONCLUSION: RV infection causes an impaired activation of the UPR in CF cells. Rescue of the ER stress response by chemical ER stress inducers reduced significantly RV replication in CF cells. Thus, pharmacological modulation of the UPR might represent a strategy to control respiratory virus replication in the CF bronchial epithelium.
Assuntos
Antivirais/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Rhinovirus/efeitos dos fármacos , Resposta a Proteínas não Dobradas , Replicação Viral/efeitos dos fármacos , Brônquios/citologia , Brônquios/virologia , Estudos de Casos e Controles , Células Cultivadas , Criança , Fibrose Cística/complicações , Chaperona BiP do Retículo Endoplasmático , Células Epiteliais/virologia , Humanos , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , Rhinovirus/fisiologia , Transdução de SinaisRESUMO
Flaviviruses replicate in a wide variety of species and have a broad cellular tropism. They are isolated from various body fluids, and Zika virus (ZIKV), Japanese encephalitis virus (JEV), and West Nile virus (WNV) RNAs have been detected in nasopharyngeal swabs. Consequently, we evaluated the cellular tropism and host responses upon ZIKV, JEV, WNV, and Usutu virus (USUV) infection using a relevant model of the human upper respiratory tract epithelium based on primary human nasal epithelial cells (NECs) cultured at the air-liquid interface. NECs were susceptible to all the viruses tested, and confocal analysis showed evidence of infection of ciliated and non-ciliated cells. Each flavivirus productively infected NECs, leading to apical and basolateral live virus shedding with particularly high basal release for JEV and WNV. As demonstrated by a paracellular permeability assay, the integrity of the epithelium was not affected by flavivirus infection, suggesting an active release of live virus through the basolateral surface. Also, we detected a significant secretion of interferon type III and the pro-inflammatory cytokine IP-10/CXCL10 upon infection with JEV. Taken together, our data suggest that the human upper respiratory tract epithelium is a target for flaviviruses and could potentially play a role in the spread of infection to other body compartments through basolateral virus release. Undoubtedly, further work is required to evaluate the risks and define the adapted measures to protect individuals exposed to flavivirus-contaminated body fluids.