RESUMO
Influenza A virus (IAV) and SARS-CoV-2 (COVID-19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1-dependent targeting of LC3 to single-membrane, non-autophagosome compartments - referred to as non-canonical autophagy - protects mice from lethal IAV infection. Mice with systemic loss of non-canonical autophagy are exquisitely sensitive to low-pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non-canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non-canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces.
Assuntos
Proteínas Relacionadas à Autofagia/genética , Vírus da Influenza A/patogenicidade , Proteínas Associadas aos Microtúbulos/metabolismo , Infecções por Orthomyxoviridae/genética , Deleção de Sequência , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Animais , Autofagia , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/metabolismo , Embrião de Galinha , Citocinas/metabolismo , Cães , Células Madin Darby de Rim Canino , Camundongos , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/mortalidade , Domínios Proteicos , Replicação ViralRESUMO
Human metapneumovirus (hMPV) is a leading cause of morbidity and hospitalization among children worldwide, however, no vaccines or therapeutics are currently available for hMPV disease prevention and treatment. The hMPV fusion (F) protein is the sole target of neutralizing antibodies. To map the immunodominant epitopes on the hMPV F protein, we isolated a panel of human monoclonal antibodies (mAbs), and the mAbs were assessed for binding avidity, neutralization potency, and epitope specificity. We found the majority of the mAbs target diverse epitopes on the hMPV F protein, and we discovered multiple mAb binding approaches for antigenic site III. The most potent mAb, MPV467, which had picomolar potency, was examined in prophylactic and therapeutic mouse challenge studies, and MPV467 limited virus replication in mouse lungs when administered 24 h before or 72 h after viral infection. We determined the structure of MPV467 in complex with the hMPV F protein using cryo-electron microscopy to a resolution of 3.3 Å, which revealed a complex novel prefusion-specific epitope overlapping antigenic sites II and V on a single protomer. Overall, our data reveal insights into the immunodominant antigenic epitopes on the hMPV F protein, identify a mAb therapy for hMPV F disease prevention and treatment, and provide the discovery of a prefusion-specific epitope on the hMPV F protein.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Antígenos Virais , Metapneumovirus , Infecções por Paramyxoviridae , Proteínas Virais de Fusão , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/química , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/uso terapêutico , Antígenos Virais/química , Antígenos Virais/imunologia , Microscopia Crioeletrônica , Epitopos/imunologia , Humanos , Metapneumovirus/imunologia , Camundongos , Infecções por Paramyxoviridae/prevenção & controle , Prevenção Primária , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologiaRESUMO
Dual oxidase 1 (DUOX1) is an NADPH oxidase that is highly expre-ssed in respiratory epithelial cells and produces H2O2 in the airway lumen. While a line of prior in vitro observations suggested that DUOX1 works in partnership with an airway peroxidase, lactoperoxidase (LPO), to produce antimicrobial hypothiocyanite (OSCN-) in the airways, the in vivo role of DUOX1 in mammalian organisms has remained unproven to date. Here, we show that Duox1 promotes antiviral innate immunity in vivo. Upon influenza airway challenge, Duox1-/- mice have enhanced mortality, morbidity, and impaired lung viral clearance. Duox1 increases the airway levels of several cytokines (IL-1ß, IL-2, CCL1, CCL3, CCL11, CCL19, CCL20, CCL27, CXCL5, and CXCL11), contributes to innate immune cell recruitment, and affects epithelial apoptosis in the airways. In primary human tracheobronchial epithelial cells, OSCN- is generated by LPO using DUOX1-derived H2O2 and inactivates several influenza strains in vitro. We also show that OSCN- diminishes influenza replication and viral RNA synthesis in infected host cells that is inhibited by the H2O2 scavenger catalase. Binding of the influenza virus to host cells and viral entry are both reduced by OSCN- in an H2O2-dependent manner in vitro. OSCN- does not affect the neuraminidase activity or morphology of the influenza virus. Overall, this antiviral function of Duox1 identifies an in vivo role of this gene, defines the steps in the infection cycle targeted by OSCN-, and proposes that boosting this mechanism in vivo can have therapeutic potential in treating viral infections.
Assuntos
Antivirais/imunologia , Oxidases Duais/metabolismo , Imunidade Inata , Animais , Apoptose , Brônquios/patologia , Brônquios/virologia , Citocinas/metabolismo , Modelos Animais de Doenças , Células Epiteliais/patologia , Humanos , Peróxido de Hidrogênio/metabolismo , Influenza Humana/imunologia , Influenza Humana/patologia , Influenza Humana/virologia , Lactoperoxidase/metabolismo , Camundongos , Neuraminidase/química , Neuraminidase/metabolismo , Orthomyxoviridae/fisiologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Proteólise , RNA Viral/metabolismo , Tiocianatos , Proteínas Virais/química , Proteínas Virais/metabolismo , Inativação de Vírus , Internalização do Vírus , Replicação ViralRESUMO
Influenza virus causes epidemics and sporadic pandemics resulting in morbidity, mortality, and economic losses. Influenza viruses require host genes to replicate. RNA interference (RNAi) screens can identify host genes coopted by influenza virus for replication. Targeting these proinfluenza genes can provide therapeutic strategies to reduce virus replication. Nineteen proinfluenza G-protein-coupled receptor (GPCR) and 13 proinfluenza ion channel genes were identified in human lung (A549) cells by use of small interfering RNAs (siRNAs). These proinfluenza genes were authenticated by testing influenza virus A/WSN/33-, A/CA/04/09-, and B/Yamagata/16/1988-infected A549 cells, resulting in the validation of 16 proinfluenza GPCR and 5 proinfluenza ion channel genes. These findings showed that several GPCR and ion channel genes are needed for the production of infectious influenza virus. These data provide potential targets for the development of host-directed therapeutic strategies to impede the influenza virus productive cycle so as to limit infection.IMPORTANCE Influenza epidemics result in morbidity and mortality each year. Vaccines are the most effective preventive measure but require annual reformulation, since a mismatch of vaccine strains can result in vaccine failure. Antiviral measures are desirable particularly when vaccines fail. In this study, we used RNAi screening to identify several GPCR and ion channel genes needed for influenza virus replication. Understanding the host genes usurped by influenza virus during viral replication can help identify host genes that can be targeted for drug repurposing or for the development of antiviral drugs. The targeting of host genes is refractory to drug resistance generated by viral mutations, as well as providing a platform for the development of broad-spectrum antiviral drugs.
Assuntos
Interações entre Hospedeiro e Microrganismos , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza B/fisiologia , Influenza Humana/virologia , Canais Iônicos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células A549 , Animais , Cães , Humanos , Células Madin Darby de Rim Canino , Replicação ViralRESUMO
Swine influenza virus (SIV) can cause respiratory illness in swine. Swine contribute to influenza virus reassortment, as avian, human, and/or swine influenza viruses can infect swine and reassort, and new viruses can emerge. Thus, it is important to determine the host antiviral responses that affect SIV replication. In this study, we examined the innate antiviral cytokine response to SIV by swine respiratory epithelial cells, focusing on the expression of interferon (IFN) and interferon-stimulated genes (ISGs). Both primary and transformed swine nasal and tracheal respiratory epithelial cells were examined following infection with field isolates. The results show that IFN and ISG expression is maximal at 12 h postinfection (hpi) and is dependent on cell type and virus genotype. IMPORTANCE Swine are considered intermediate hosts that have facilitated influenza virus reassortment events that have given rise pandemics or genetically related viruses have become established in swine. In this study, we examine the innate antiviral response to swine influenza virus in primary and immortalized swine nasal and tracheal epithelial cells, and show virus strain- and host cell type-dependent differential expression of key interferons and interferon-stimulated genes.
Assuntos
Citocinas/metabolismo , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N2/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Mucosa Respiratória/imunologia , Animais , Linhagem Celular , Citocinas/imunologia , Cães , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno/imunologia , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N2/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Interferons/imunologia , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/imunologia , Mucosa Respiratória/citologia , Suínos , Replicação Viral/fisiologiaRESUMO
Human metapneumovirus (hMPV) is an important cause of acute viral respiratory infection. As the only target of neutralizing antibodies, the hMPV fusion (F) protein has been a major focus for vaccine development and targeting by drugs and monoclonal antibodies (MAbs). While X-ray structures of trimeric prefusion and postfusion hMPV F proteins from genotype A, and monomeric prefusion hMPV F protein from genotype B have been determined, structural data for the postfusion conformation for genotype B is lacking. We determined the crystal structure of this protein and compared the structural differences of postfusion hMPV F between hMPV A and B genotypes. We also assessed the receptor binding properties of the hMPV F protein to heparin and heparan sulfate (HS). A library of HS oligomers was used to verify the HS binding activity of hMPV F, and several compounds showed binding to predominantly prefusion hMPV F, but had limited binding to postfusion hMPV F. Furthermore, MAbs to antigenic sites III and the 66-87 intratrimeric epitope block heparin binding. In addition, we evaluated the efficacy of postfusion hMPV B2 F protein as a vaccine candidate in BALB/c mice. Mice immunized with hMPV B2 postfusion F protein showed a balanced Th1/Th2 immune response and generated neutralizing antibodies against both subgroup A2 and B2 hMPV strains, which protected the mice from hMPV challenge. Antibody competition analysis revealed the antibodies generated by immunization target two known antigenic sites (III and IV) on the hMPV F protein. Overall, this study provides new characteristics of the hMPV F protein, which may be informative for vaccine and therapy development. IMPORTANCE Human metapneumovirus (hMPV) is an important cause of viral respiratory disease. In this paper, we report the X-ray crystal structure of the hMPV fusion (F) protein in the postfusion conformation from genotype B. We also assessed binding of the hMPV F protein to heparin and heparan sulfate, a previously reported receptor for the hMPV F protein. Furthermore, we determined the immunogenicity and protective efficacy of postfusion hMPV B2 F protein, which is the first study using a homogenous conformation of the protein. Antibodies generated in response to vaccination give a balanced Th1/Th2 response and target two previously discovered neutralizing epitopes.
Assuntos
Anticorpos Antivirais/imunologia , Epitopos/imunologia , Heparina/metabolismo , Metapneumovirus/imunologia , Infecções por Paramyxoviridae/imunologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Feminino , Heparina/análogos & derivados , Humanos , Imunização , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Paramyxoviridae/metabolismo , Infecções por Paramyxoviridae/virologia , Ligação Proteica , Conformação Proteica , Proteoglicanas/metabolismo , Células Th1/imunologia , Células Th2/imunologia , Proteínas Virais de Fusão/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pbio.1002580.].
RESUMO
A rapid, portable, and cost-effective method to detect the infection of SARS-CoV-2 is fundamental toward mitigating the current COVID-19 pandemic. Herein, a human angiotensin-converting enzyme 2 protein (ACE2) functionalized silver nanotriangle (AgNT) array localized surface plasmon resonance (LSPR) sensor is developed for rapid coronavirus detection, which is validated by SARS-CoV-2 spike RBD protein and CoV NL63 virus with high sensitivity and specificity. A linear shift of the LSPR wavelength versus the logarithm of the concentration of the spike RBD protein and CoV NL63 is observed. The limits of detection for the spike RBD protein, CoV NL63 in buffer and untreated saliva are determined to be 0.83 pM, 391 PFU/mL, and 625 PFU/mL, respectively, while the detection time is found to be less than 20 min. Thus, the AgNT array optical sensor could serve as a potential rapid point-of-care COVID-19 diagnostic platform.
RESUMO
Influenza virus causes seasonal epidemics and sporadic pandemics resulting in morbidity, mortality, and economic losses worldwide. Understanding how to regulate influenza virus replication is important for developing vaccine and therapeutic strategies. Identifying microRNAs (miRs) that affect host genes used by influenza virus for replication can support an antiviral strategy. In this study, G-protein coupled receptor (GPCR) and ion channel (IC) host genes in human alveolar epithelial (A549) cells used by influenza virus for replication (Orr-Burks et al., 2021) were examined as miR target genes following A/CA/04/09- or B/Yamagata/16/1988 replication. Thirty-three miRs were predicted to target GPCR or IC genes and their miR mimics were evaluated for their ability to decrease influenza virus replication. Paired miR inhibitors were used as an ancillary measure to confirm or not the antiviral effects of a miR mimic. Fifteen miRs lowered influenza virus replication and four miRs were found to reduce replication irrespective of virus strain and type differences. These findings provide evidence for novel miR disease intervention strategies for influenza viruses.
Assuntos
Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Canais Iônicos/metabolismo , MicroRNAs/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Replicação Viral , Células A549 , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/prevenção & controle , Canais Iônicos/genética , MicroRNAs/genética , Receptores Acoplados a Proteínas G/genéticaRESUMO
Mumps virus (MuV) caused the most viral meningitis before mass immunization. Unfortunately, MuV has reemerged in the United States in the past several years. MuV is a member of the genus Rubulavirus, in the family Paramyxoviridae, and has a nonsegmented negative-strand RNA genome. The viral RNA-dependent RNA polymerase (vRdRp) of MuV consists of the large protein (L) and the phosphoprotein (P), while the nucleocapsid protein (NP) encapsulates the viral RNA genome. These proteins make up the replication and transcription machinery of MuV. The P protein is phosphorylated by host kinases, and its phosphorylation is important for its function. In this study, we performed a large-scale small interfering RNA (siRNA) screen targeting host kinases that regulated MuV replication. The human kinase ribosomal protein S6 kinase beta-1 (RPS6KB1) was shown to play a role in MuV replication and transcription. We have validated the role of RPS6KB1 in regulating MuV using siRNA knockdown, an inhibitor, and RPS6KB1 knockout cells. We found that MuV grows better in cells lacking RPS6KB1, indicating that it downregulates viral growth. Furthermore, we detected an interaction between the MuV P protein and RPS6KB1, suggesting that RPS6KB1 directly regulates MuV replication and transcription.IMPORTANCE Mumps virus is an important human pathogen. In recent years, MuV has reemerged in the United State, with outbreaks occurring in young adults who have been vaccinated. Our work provides insight into a previously unknown mumps virus-host interaction. RPS6KB1 negatively regulates MuV replication, likely through its interaction with the P protein. Understanding virus-host interactions can lead to novel antiviral drugs and enhanced vaccine production.
Assuntos
Genoma Viral , Vírus da Caxumba/genética , Proteínas do Nucleocapsídeo/genética , Fosfoproteínas/genética , RNA Polimerase Dependente de RNA/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Virais/genética , Animais , Chlorocebus aethiops , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Vírus da Caxumba/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Células Vero , Proteínas Virais/metabolismo , Replicação ViralRESUMO
PURPOSE: SARS-CoV-2 infection is associated with substantial mortality and high morbidity. This study tested the effect of angiotensin II type I receptor blocker, losartan, on SARS-CoV-2 replication and inhibition of the papain-like protease of the virus. METHODS: The dose-dependent inhibitory effect of losartan, in concentrations from 1µM to 100µM as determined by quantitative cell analysis combining fluorescence microscopy, image processing, and cellular measurements (Cellomics analysis) on SARS-CoV-2 replication was investigated in Vero E6 cells. The impact of losartan on deubiquitination and deISGylation of SARS-CoV-2 papain-like protease (PLpro) were also evaluated. Results: Losartan reduced PLpro cleavage of tetraUbiquitin to diUbiquitin. It was less effective in inhibiting PLpro's cleavage of ISG15-AMC than Ubiquitin-AMC. To determine if losartan inhibited SARS-CoV-2 replication, losartan treatment of SARS-CoV-2 infected Vero E6 was examined. Losartan treatment one hour prior to SARS-CoV-2 infection reduced levels of SARS-CoV-2 nuclear protein, an indicator of virus replication, by 80% and treatment one-hour post-infection decreased viral replication by 70%. CONCLUSION: Losartan was not an effective inhibitor of deubiquitinase or deISGylase activity of the PLpro but affected the SARS-CoV-2 replication of Vero E6 cells in vitro. As losartan has a favorable safety profile and is currently available it has features necessary for efficacious drug repurposing and treatment of COVID-19.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Antivirais/farmacologia , Losartan/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Chlorocebus aethiops , Biologia Computacional , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Enzimas Desubiquitinantes/antagonistas & inibidores , Enzimas Desubiquitinantes/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Ubiquitina/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
Human metapneumovirus (hMPV) is a leading cause of viral lower respiratory tract infection in children. The sole target of neutralizing antibodies targeting hMPV is the fusion (F) protein, a class I viral fusion protein mediating virus-cell membrane fusion. There have been several monoclonal antibodies (mAbs) isolated that neutralize hMPV; however, determining the antigenic sites on the hMPV F protein mediating such neutralizing antibody generation would assist efforts for effective vaccine design. In this report, the isolation and characterization of four new human mAbs, termed MPV196, MPV201, MPV314, and MPV364, are described. Among the four mAbs, MPV364 was found to be the most potent neutralizing mAb in vitro Binding studies with monomeric and trimeric hMPV F revealed that MPV364 had the weakest binding affinity for monomeric hMPV F compared to the other three mAbs, yet binding experiments with trimeric hMPV F showed limited differences in binding affinity, suggesting that MPV364 targets an antigenic site incorporating two protomers. Epitope binning studies showed that MPV364 targets antigenic site III on the hMPV F protein and competes for binding with previously discovered mAbs MPE8 and 25P13, both of which cross-react with the respiratory syncytial virus (RSV) F protein. However, MPV364 does not cross-react with the RSV F protein, and the competition profile suggests that it binds to the hMPV F protein in a binding pose slightly shifted from mAbs MPE8 and 25P13. MPV364 was further assessed in vivo and was shown to substantially reduce viral replication in the lungs of BALB/c mice. Overall, these data reveal a new binding region near antigenic site III of the hMPV F protein that elicits potent neutralizing hMPV F-specific mAbs and provide a new panel of neutralizing mAbs that are candidates for therapeutic development.IMPORTANCE Recent progress in understanding the human immune response to respiratory syncytial virus has paved the way for new vaccine antigens and therapeutics to prevent and treat disease. Progress toward understanding the immune response to human metapneumovirus (hMPV) has lagged behind, although hMPV is a leading cause of lower respiratory tract infection in children. In this report, we advanced the field by isolating a panel of human mAbs to the hMPV F protein. One potent neutralizing mAb, MPV364, targets antigenic site III on the hMPV F protein and incorporates two protomers into its epitope yet is unique from previously discovered site III mAbs, as it does not cross-react with the RSV F protein. We further examined MPV364 in vivo and found that it limits viral replication in BALB/c mice. Altogether, these data provide new mAb candidates for therapeutic development and provide insights into hMPV vaccine development.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Epitopos de Linfócito B/imunologia , Metapneumovirus/imunologia , Proteínas Virais de Fusão/imunologia , Sítios de Ligação , Mapeamento de Epitopos , Humanos , Ligação ProteicaRESUMO
Respiratory syncytial virus (RSV) is a leading cause of hospitalization of infants and young children, causing considerable respiratory disease and repeat infections that may lead to chronic respiratory conditions such as asthma, wheezing, and bronchitis. RSV causes â¼34 million new episodes of lower respiratory tract illness (LRTI) in children younger than 5 years of age, with >3 million hospitalizations due to severe RSV-associated LRTI. The standard of care is limited to symptomatic relief as there are no approved vaccines and few effective antiviral drugs; thus, a safe and efficacious RSV therapeutic is needed. Therapeutic targeting of host proteins hijacked by RSV to facilitate replication is a promising antiviral strategy as targeting the host reduces the likelihood of developing drug resistance. The nuclear export of the RSV M protein, mediated by the nuclear export protein exportin 1 (XPO1), is crucial for RSV assembly and budding. Inhibition of RSV M protein export by leptomycin B correlated with reduced RSV replication in vitro In this study, we evaluated the anti-RSV efficacy of Verdinexor (KPT-335), a small molecule designed to reversibly inhibit XPO1-mediated nuclear export. KPT-335 inhibited XPO1-mediated transport and reduced RSV replication in vitro KPT-335 was effective against RSV A and B strains and reduced viral replication following prophylactic or therapeutic administration. Inhibition of RSV replication by KPT-335 was due to a combined effect of reduced XPO1 expression, disruption of the nuclear export of RSV M protein, and inactivation of the NF-κB signaling pathway.IMPORTANCE RSV is an important cause of LRTI in infants and young children for which there are no suitable antiviral drugs offered. We evaluated the efficacy of KPT-335 as an anti-RSV drug and show that KPT-335 inhibits XPO1-mediated nuclear export, leading to nuclear accumulation of RSV M protein and reduction in RSV levels. KPT-335 treatment also resulted in inhibition of proinflammatory pathways, which has important implications for its effectiveness in vivo.
Assuntos
Acrilamidas/farmacologia , Hidrazinas/farmacologia , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Células A549 , Acrilamidas/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Glicoproteínas/imunologia , Humanos , Hidrazinas/metabolismo , Carioferinas/efeitos dos fármacos , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Células Vero , Proteína Exportina 1RESUMO
Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection (LRTI) annually affecting >2 million children in the United States <5 years old. In the elderly (>65 years old), RSV results in â¼175,000 hospitalizations annually in the United States with a worldwide incidence of â¼34 million. There is no approved RSV vaccine, and treatments are limited. Recently, a phase 3 trial in the elderly using a recombinant RSV F protein vaccine failed to meet its efficacy objectives, namely, prevention of moderate-to-severe RSV-associated LRTI and reduced incidence of acute respiratory disease. Moreover, a recent phase 3 trial evaluating suptavumab (REGN2222), an antibody to RSV F protein, did not meet its primary endpoint of preventing medically attended RSV infections in preterm infants. Despite these setbacks, numerous efforts targeting the RSV F protein with vaccines, antibodies, and small molecules continue based on the commercial success of a monoclonal antibody (MAb) against the RSV F protein (palivizumab). As the understanding of RSV biology has improved, the other major coat protein, the RSV G protein, has reemerged as an alternative target reflecting progress in understanding its roles in infecting bronchial epithelial cells and in altering the host immune response. In mouse models, a high-affinity, strain-independent human MAb to the RSV G protein has shown potent direct antiviral activity combined with the alleviation of virus-induced immune system effects that contribute to disease pathology. This MAb, being prepared for clinical trials, provides a qualitatively new approach to managing RSV for populations not eligible for prophylaxis with palivizumab.
Assuntos
Palivizumab/farmacologia , Infecções por Vírus Respiratório Sincicial/terapia , Vírus Sincicial Respiratório Humano , Proteínas Virais de Fusão/antagonistas & inibidores , Animais , Ensaios Clínicos como Assunto , Ensaios Clínicos Fase III como Assunto , Humanos , Camundongos , Infecções por Vírus Respiratório Sincicial/imunologia , Proteínas Virais/antagonistas & inibidoresRESUMO
Analysis of host gene expression profiles following viral infections of target cells/tissues can reveal crucial insights into the host: virus interaction and enables the development of novel therapeutics and prophylactics. Regions of the host genome that do not code for protein, encode structural, and functional non-coding RNAs that are important not only in regulation of host gene expression but also may impact viral replication. This review summarizes the role of host non-coding RNAs during replication of multiple respiratory viruses with a focus on Respiratory Syncytial Virus (RSV), an important pediatric pathogen. This review highlights the current state of knowledge and understanding regarding the function(s) of ncRNAs for respiratory viral infection and host immunity in general.
Assuntos
Interações Hospedeiro-Patógeno , RNA não Traduzido/genética , Infecções por Vírus Respiratório Sincicial/genética , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Vírus Sincicial Respiratório Humano/imunologia , Replicação Viral , Humanos , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/patogenicidadeRESUMO
The extracellular matrix (ECM) provides physical scaffolding for cellular constituents and initiates biochemical and biomechanical cues that are required for physiological activity of living tissues. The ECM enzyme ADAMTS5, a member of the ADAMTS (A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs) protein family, cleaves large proteoglycans such as aggrecan, leading to the destruction of cartilage and osteoarthritis. However, its contribution to viral pathogenesis and immunity is currently undefined. Here, we use a combination of in vitro and in vivo models to show that ADAMTS5 enzymatic activity plays a key role in the development of influenza-specific immunity. Influenza virus infection of Adamts5-/- mice resulted in delayed virus clearance, compromised T cell migration and immunity and accumulation of versican, an ADAMTS5 proteoglycan substrate. Our research emphasises the importance of ADAMTS5 expression in the control of influenza virus infection and highlights the potential for development of ADAMTS5-based therapeutic strategies to reduce morbidity and mortality.
Assuntos
Proteína ADAMTS5/fisiologia , Imunidade Celular/fisiologia , Orthomyxoviridae/imunologia , Linfócitos T/imunologia , Proteína ADAMTS5/genética , Animais , Imunofenotipagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Versicanas/metabolismo , Redução de PesoRESUMO
Although antibodies that effectively neutralize a broad set of influenza viruses exist in the human antibody repertoire, they are rare. We used a single-cell screening technology to identify rare monoclonal antibodies (MAbs) that recognized a broad set of influenza B viruses (IBV). The screen yielded 23 MAbs with diverse germ line origins that recognized hemagglutinins (HAs) derived from influenza strains of both the Yamagata and Victoria lineages of IBV. Of the 23 MAbs, 3 exhibited low expression in a transient-transfection system, 4 were neutralizers that bound to the HA head region, 11 were stalk-binding nonneutralizers, and 5 were stalk-binding neutralizers, with 4 of these 5 having unique antibody sequences. Of these four unique stalk-binding neutralizing MAbs, all were broadly reactive and neutralizing against a panel of multiple strains spanning both IBV lineages as well as highly effective in treating lethal IBV infections in mice at both 24 and 72 h postinfection. The MAbs in this group were thermostable and bound different epitopes in the highly conserved HA stalk region. These characteristics suggest that these MAbs are suitable for consideration as candidates for clinical studies to address their effectiveness in the treatment of IBV-infected patients.
Assuntos
Anticorpos Monoclonais/imunologia , Vírus da Influenza B/patogenicidade , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Animais , Anticorpos Antivirais/imunologia , Epitopos/química , Epitopos/imunologia , Feminino , Hemaglutininas/química , Hemaglutininas/imunologia , Humanos , Vírus da Influenza B/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de NeutralizaçãoRESUMO
Seasonal human influenza virus continues to cause morbidity and mortality annually, and highly pathogenic avian influenza (HPAI) viruses along with other emerging influenza viruses continue to pose pandemic threats. Vaccination is considered the most effective measure for controlling influenza; however, current strategies rely on a precise vaccine match with currently circulating virus strains for efficacy, requiring constant surveillance and regular development of matched vaccines. Current vaccines focus on eliciting specific antibody responses against the hemagglutinin (HA) surface glycoprotein; however, the diversity of HAs across species and antigenic drift of circulating strains enable the evasion of virus-inhibiting antibody responses, resulting in vaccine failure. The neuraminidase (NA) surface glycoprotein, while diverse, has a conserved enzymatic site and presents an appealing target for priming broadly effective antibody responses. Here we show that vaccination with parainfluenza virus 5 (PIV5), a promising live viral vector expressing NA from avian (H5N1) or pandemic (H1N1) influenza virus, elicited NA-specific antibody and T cell responses, which conferred protection against homologous and heterologous influenza virus challenges. Vaccination with PIV5-N1 NA provided cross-protection against challenge with a heterosubtypic (H3N2) virus. Experiments using antibody transfer indicate that antibodies to NA have an important role in protection. These findings indicate that PIV5 expressing NA may be effective as a broadly protective vaccine against seasonal influenza and emerging pandemic threats.IMPORTANCE Seasonal influenza viruses cause considerable morbidity and mortality annually, while emerging viruses pose potential pandemic threats. Currently licensed influenza virus vaccines rely on the antigenic match of hemagglutinin (HA) for vaccine strain selection, and most vaccines rely on HA inhibition titers to determine efficacy, despite the growing awareness of the contribution of neuraminidase (NA) to influenza virus vaccine efficacy. Although NA is immunologically subdominant to HA, and clinical studies have shown variable NA responses to vaccination, in this study, we show that vaccination with a parainfluenza virus 5 recombinant vaccine candidate expressing NA (PIV5-NA) from a pandemic influenza (pdmH1N1) virus or highly pathogenic avian influenza (H5N1) virus elicits robust, cross-reactive protection from influenza virus infection in two animal models. New vaccination strategies incorporating NA, including PIV5-NA, could improve seasonal influenza virus vaccine efficacy and provide protection against emerging influenza viruses.
Assuntos
Proteção Cruzada , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Neuraminidase/imunologia , Vírus da Parainfluenza 5/genética , Animais , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/sangue , Reações Cruzadas , Vetores Genéticos/administração & dosagem , Humanos , Imunização Passiva , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/patogenicidade , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Vacinas contra Influenza/genética , Influenza Humana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Pandemias/prevenção & controle , Vacinas Sintéticas/imunologiaRESUMO
Influenza A(H1N1) viruses entered the U.S. swine population following the 1918 pandemic and remained genetically stable for roughly 80 years. In 1998, there was an outbreak of influenza-like illness among swine that was caused by A(H3N2) viruses containing the triple reassortant internal gene (TRIG) cassette. Following the TRIG cassette emergence, numerous reassortant viruses were isolated in nature, suggesting that the TRIG virus had an enhanced ability to reassort compared to the classical swine virus. The present study was designed to quantify the relative reassortment capacities of classical and TRIG swine viruses. Reverse genetic viruses were generated from the classical H1N1 virus A/swine/MN/37866/1999 (MN/99), the TRIG virus A/swine/NC/18161/2002 (NC/02), and a seasonal human H3N2 virus, A/TX/6/1996 (TX/96), to measure in vitro reassortment and growth potentials. After coinfection with NC/02 or MN/99 plus TX/96, H1/H3 double-positive cells were identified. Delayed TX/96 infection was fully excluded by both swine viruses. We then analyzed reassortant H3 viruses. Seventy-seven of 81 (95.1%) TX/96-NC/02 reassortants contained at least one polymerase gene segment from NC/02, whereas only 34 of 61 (55.7%) MN/99-TX/96 reassortants contained at least one polymerase gene segment from MN/99. Additionally, 38 of 81 (46.9%) NC/02-TX/96 reassortants contained all NC/02 polymerase gene segments, while none of the MN/99-TX/96 reassortants contained all MN/99 polymerase genes. There were 21 H3 reassortants between MN/99 and TX/96, compared to only 17 H3 reassortants between NC/02 and TX/96. Overall, the results indicate that there are no distinct differences in the ability of the TRIG to reassort with a human virus compared to the classical swine virus. IMPORTANCE: There appear to be no differences in the abilities of classical swine and TRIG swine viruses to exclude a second virus, suggesting that under the right circumstances both viruses have similar opportunities to reassort. The increased percentage of TRIG polymerase gene segments in reassortant H3 viruses indicates that these viruses may be more compatible with gene segments from other viruses; however, this needs to be investigated further. Nevertheless, the classical swine virus also showed the ability to reassort, suggesting that factors other than reassortment capacity alone are responsible for the different epidemiologies of TRIG and classical swine viruses. The post-TRIG diversity was likely driven by increased intensive farming practices rather than virologic properties. Our results indicate that host ecology can be a significant factor in viral evolution.
Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Infecções por Orthomyxoviridae/virologia , Doenças dos Suínos/virologia , Animais , Surtos de Doenças , Suínos , Estados UnidosRESUMO
UNLABELLED: Vaccine manufacturing costs prevent a significant portion of the world's population from accessing protection from vaccine-preventable diseases. To enhance vaccine production at reduced costs, a genome-wide RNA interference (RNAi) screen was performed to identify gene knockdown events that enhanced poliovirus replication. Primary screen hits were validated in a Vero vaccine manufacturing cell line using attenuated and wild-type poliovirus strains. Multiple single and dual gene silencing events increased poliovirus titers >20-fold and >50-fold, respectively. Host gene knockdown events did not affect virus antigenicity, and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-mediated knockout of the top candidates dramatically improved viral vaccine strain production. Interestingly, silencing of several genes that enhanced poliovirus replication also enhanced replication of enterovirus 71, a clinically relevant virus to which vaccines are being targeted. The discovery that host gene modulation can markedly increase virus vaccine production dramatically alters mammalian cell-based vaccine manufacturing possibilities and should facilitate polio eradication using the inactivated poliovirus vaccine. IMPORTANCE: Using a genome-wide RNAi screen, a collection of host virus resistance genes was identified that, upon silencing, increased poliovirus and enterovirus 71 production by from 10-fold to >50-fold in a Vero vaccine manufacturing cell line. This report provides novel insights into enterovirus-host interactions and describes an approach to developing the next generation of vaccine manufacturing through engineered vaccine cell lines. The results show that specific gene silencing and knockout events can enhance viral titers of both attenuated (Sabin strain) and wild-type polioviruses, a finding that should greatly facilitate global implementation of inactivated polio vaccine as well as further reduce costs for live-attenuated oral polio vaccines. This work describes a platform-enabling technology applicable to most vaccine-preventable diseases.