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1.
ACS Appl Mater Interfaces ; 13(1): 88-96, 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33382581

RESUMO

Polymer-protein conjugates are a class of biohybrids with unique properties that are highly useful in biomedicine ranging from protein therapeutics to biomedical imaging; however, it remains a considerable challenge to conjugate polymers to proteins in a site-specific, mild, and efficient way to form polymer-protein conjugates with uniform structures and properties and optimal functions. Herein we report pyridine-2,6-dicarboxaldehyde (PDA)-enabled N-terminal modification of proteins with polymerization initiators for in situ growth of poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA) conjugates uniquely at the N-termini of a range of natural and recombinant proteins in a mild and efficient fashion. The formed POEGMA-protein conjugates showed highly retained in vitro bioactivity as compared with free proteins. Notably, the in vitro bioactivity of a POEGMA-interferon α (IFN) conjugate synthesized by this new chemistry is 8.1-fold higher than that of PEGASYS that is a commercially available and Food and Drug Administration (FDA) approved PEGylated IFN. The circulation half-life of the conjugate is similar to that of PEGASYS but is 46.2 times longer than that of free IFN. Consequently, the conjugate exhibits considerably improved antiviral bioactivity over free IFN and even PEGASYS in a mouse model. These results indicate that the PDA-enabled N-terminal grafting-from method is applicable to a number of proteins whose active sites are far away from the N-terminus for the synthesis of N-terminal polymer-protein conjugates with high yield, well-retained activity, and considerably improved pharmacology for biomedical applications.


Assuntos
Aldeídos/química , Antivirais/farmacologia , Indicadores e Reagentes/química , Interferon-alfa/farmacologia , Polietilenoglicóis/farmacologia , Piridinas/química , 2',5'-Oligoadenilato Sintetase/metabolismo , Sequência de Aminoácidos , Animais , Antivirais/química , Antivirais/farmacocinética , Meia-Vida , Interferon-alfa/química , Interferon-alfa/farmacocinética , Camundongos , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Polimerização
2.
J Virol ; 94(23)2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-32938761

RESUMO

SARS-CoV-2, a novel coronavirus (CoV) that causes COVID-19, has recently emerged causing an ongoing outbreak of viral pneumonia around the world. While distinct from SARS-CoV, both group 2B CoVs share similar genome organization, origins to bat CoVs, and an arsenal of immune antagonists. In this report, we evaluate type I interferon (IFN-I) sensitivity of SARS-CoV-2 relative to the original SARS-CoV. Our results indicate that while SARS-CoV-2 maintains similar viral replication to SARS-CoV, the novel CoV is much more sensitive to IFN-I. In Vero E6 and in Calu3 cells, SARS-CoV-2 is substantially attenuated in the context of IFN-I pretreatment, whereas SARS-CoV is not. In line with these findings, SARS-CoV-2 fails to counteract phosphorylation of STAT1 and expression of ISG proteins, while SARS-CoV is able to suppress both. Comparing SARS-CoV-2 and influenza A virus in human airway epithelial cultures, we observe the absence of IFN-I stimulation by SARS-CoV-2 alone but detect the failure to counteract STAT1 phosphorylation upon IFN-I pretreatment, resulting in near ablation of SARS-CoV-2 infection. Next, we evaluated IFN-I treatment postinfection and found that SARS-CoV-2 was sensitive even after establishing infection. Finally, we examined homology between SARS-CoV and SARS-CoV-2 in viral proteins shown to be interferon antagonists. The absence of an equivalent open reading frame 3b (ORF3b) and genetic differences versus ORF6 suggest that the two key IFN-I antagonists may not maintain equivalent function in SARS-CoV-2. Together, the results identify key differences in susceptibility to IFN-I responses between SARS-CoV and SARS-CoV-2 that may help inform disease progression, treatment options, and animal model development.IMPORTANCE With the ongoing outbreak of COVID-19, differences between SARS-CoV-2 and the original SARS-CoV could be leveraged to inform disease progression and eventual treatment options. In addition, these findings could have key implications for animal model development as well as further research into how SARS-CoV-2 modulates the type I IFN response early during infection.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Interferon Tipo I/farmacologia , Interferon-alfa/farmacologia , Vírus da SARS/efeitos dos fármacos , Animais , Antivirais/antagonistas & inibidores , Antivirais/metabolismo , Betacoronavirus/imunologia , Betacoronavirus/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Interferon Tipo I/antagonistas & inibidores , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Interferon-alfa/antagonistas & inibidores , Interferon-alfa/imunologia , Interferon-alfa/metabolismo , Fosforilação , Proteínas Recombinantes/farmacologia , Vírus da SARS/imunologia , Vírus da SARS/fisiologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Células Vero , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
3.
Rev Med Suisse ; 16(704): 1538-1543, 2020 Sep 02.
Artigo em Francês | MEDLINE | ID: mdl-32880108

RESUMO

Hepatitis D virus causes chronic hepatitis D. The virus is defective, meaning it requires simultaneous presence of hepatitis B virus within the hepatocytes to complete its viral cycle. Globally, 15 to 20 millions people are estimated to be chronically co-infected by hepatitis B and D viruses. Current therapy remains limited to pegylated interferon alfa, which has an unsatisfactory success rate, several contraindications and many side effects. Drugs directly targeting the hepatitis D virus life cycle are being developed with promising results. These drugs target viral entry into hepatocytes, virion assembly or secretion from infected hepatocytes. This article provides an overview of the newly developed therapies and their efficacy.


Assuntos
Antivirais/farmacologia , Antivirais/uso terapêutico , Hepatite B Crônica/tratamento farmacológico , Hepatite D Crônica/tratamento farmacológico , Vírus Delta da Hepatite/efeitos dos fármacos , Hepatite B Crônica/virologia , Hepatite D Crônica/virologia , Humanos , Interferon-alfa/farmacologia , Interferon-alfa/uso terapêutico
4.
PLoS Pathog ; 16(9): e1008773, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32881988

RESUMO

Japanese encephalitis virus (JEV) genotype I (GI) replicates more efficiently than genotype III (GIII) in birds, and this difference is considered to be one of the reasons for the JEV genotype shift. In this study, we utilized duck embryo fibroblasts and domestic ducklings as in vitro and in vivo models of a JEV amplifying avian host to identify the viral determinants of the differing replication efficiency between the GI and GIII strains in birds. GI strains induced significantly lower levels of interferon (IFN)-α and ß production than GIII strains, an effect orrelated with the enhanced replication efficiency of GI strains over GIII strains. By using a series of chimeric viruses with exchange of viral structural and non-structural (NS) proteins, we identified NS5 as the viral determinant of the differences in IFN-α and ß induction and replication efficiency between the GI and III strains. NS5 inhibited IFN-α and ß production induced by poly(I:C) stimulation and harbored 11 amino acid variations, of which the NS5-V372A and NS5-H386Y variations were identified to co-contribute to the differences in IFN-α and ß induction and replication efficiency between the strains. The NS5-V372A and NS5-H386Y variations resulted in alterations in the number of hydrogen bonds formed with neighboring residues, which were associated with the different ability of the GI and GIII strains to inhibit IFN-α and ß production. Our findings indicated that the NS5-V372A and NS5-H386Y variations enabled GI strains to inhibit IFN-α and ß production more efficiently than GIII strains for antagonism of the IFN-I mediated antiviral response, thereby leading to the replication and host adaption advantages of GI strains over GIII strains in birds. These findings provide new insight into the molecular basis of the JEV genotype shift.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/imunologia , Encefalite Japonesa/imunologia , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Mutação , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Animais , Antivirais/farmacologia , Patos , Vírus da Encefalite Japonesa (Espécie)/efeitos dos fármacos , Vírus da Encefalite Japonesa (Espécie)/genética , Encefalite Japonesa/tratamento farmacológico , Encefalite Japonesa/virologia , Interações Hospedeiro-Patógeno , Camundongos , Ligação Proteica , Suínos , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
5.
Nature ; 586(7830): 560-566, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32854108

RESUMO

Coronaviruses are prone to transmission to new host species, as recently demonstrated by the spread to humans of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic1. Small animal models that recapitulate SARS-CoV-2 disease are needed urgently for rapid evaluation of medical countermeasures2,3. SARS-CoV-2 cannot infect wild-type laboratory mice owing to inefficient interactions between the viral spike protein and the mouse orthologue of the human receptor, angiotensin-converting enzyme 2 (ACE2)4. Here we used reverse genetics5 to remodel the interaction between SARS-CoV-2 spike protein and mouse ACE2 and designed mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA), a recombinant virus that can use mouse ACE2 for entry into cells. SARS-CoV-2 MA was able to replicate in the upper and lower airways of both young adult and aged BALB/c mice. SARS-CoV-2 MA caused more severe disease in aged mice, and exhibited more clinically relevant phenotypes than those seen in Hfh4-ACE2 transgenic mice, which express human ACE2 under the control of the Hfh4 (also known as Foxj1) promoter. We demonstrate the utility of this model using vaccine-challenge studies in immune-competent mice with native expression of mouse ACE2. Finally, we show that the clinical candidate interferon-λ1a (IFN-λ1a) potently inhibits SARS-CoV-2 replication in primary human airway epithelial cells in vitro-both prophylactic and therapeutic administration of IFN-λ1a diminished SARS-CoV-2 replication in mice. In summary, the mouse-adapted SARS-CoV-2 MA model demonstrates age-related disease pathogenesis and supports the clinical use of pegylated IFN-λ1a as a treatment for human COVID-196.


Assuntos
Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Modelos Animais de Doenças , Interferons/farmacologia , Interferons/uso terapêutico , Interleucinas/farmacologia , Interleucinas/uso terapêutico , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Vacinas Virais/imunologia , Envelhecimento/imunologia , Animais , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Interferon-alfa/administração & dosagem , Interferon-alfa/farmacologia , Interferon-alfa/uso terapêutico , Interferons/administração & dosagem , Interleucinas/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Modelos Moleculares , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/genética , Pneumonia Viral/imunologia , Receptores Virais/genética , Receptores Virais/metabolismo
6.
PLoS One ; 15(7): e0228302, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628668

RESUMO

Programmed death ligand 1 (PD-L1) has been recently shown to be a major obstacle to antiviral immunity by binding to its receptor programmed death 1 (PD-1) on specific IFN-γ producing T cells in chronic hepatitis B. Currently, IFN-α is widely used to treat hepatitis B virus (HBV) infection, but its antiviral effect vary greatly and the mechanism is not totally clear. We found that IFN-α/γ induced a marked increase of PD-L1 expression in hepatocytes. Signal and activators of transcription (Stat1) was then identified as a major transcription factor involved in IFN-α/γ-mediated PD-L1 elevation both in vitro and in mice. Blockage of the PD-L1/PD-1 interaction by a specific mAb greatly enhanced HBV-specific T cell activity by the gp96 adjuvanted therapeutic vaccine, and promoted HBV clearance in HBV transgenic mice. Our results demonstrate the IFN-α/γ-Stat1-PD-L1 axis plays an important role in mediating T cell hyporesponsiveness and inactivating liver-infiltrating T cells in the hepatic microenvironment. These data raise further potential interest in enhancing the anti-HBV efficacy of IFN-α and therapeutic vaccines.


Assuntos
Antígeno B7-H1/metabolismo , Vírus da Hepatite B/imunologia , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Fator de Transcrição STAT1/metabolismo , Linfócitos T/imunologia , Regulação para Cima/efeitos dos fármacos , Animais , Anticorpos Monoclonais/uso terapêutico , Antígeno B7-H1/química , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Sítios de Ligação , Linhagem Celular , Hepatite B/tratamento farmacológico , Hepatite B/veterinária , Antígenos de Superfície da Hepatite B/sangue , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição STAT1/química , Linfócitos T/metabolismo
7.
J Virol ; 94(17)2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32581092

RESUMO

Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of an infected hepatocyte and serves as the template for the transcription of viral mRNAs. It had been demonstrated by others and us that interferon alpha (IFN-α) treatment of hepatocytes induced a prolonged suppression of human and duck hepatitis B virus cccDNA transcription, which is associated with the reduction of cccDNA-associated histone modifications specifying active transcription (H3K9ac or H3K27ac), but not the histone modifications marking constitutive (H3K9me3) or facultative (H3K27me3) heterochromatin formation. In our efforts to identify IFN-induced cellular proteins that mediate the suppression of cccDNA transcription by the cytokine, we found that downregulating the expression of signal transducer and activator of transcription 1 (STAT1), structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1), or promyelocytic leukemia (PML) protein increased basal level of cccDNA transcription activity and partially attenuated IFN-α suppression of cccDNA transcription. In contrast, ectopic expression of STAT1, SMCHD1, or PML significantly reduced cccDNA transcription activity. SMCHD1 is a noncanonical SMC family protein and implicated in epigenetic silencing of gene expression. PML is a component of nuclear domain 10 (ND10) and is involved in suppressing the replication of many DNA viruses. Mechanistic analyses demonstrated that STAT1, SMCHD1, and PML were recruited to cccDNA minichromosomes and phenocopied the IFN-α-induced posttranslational modifications of cccDNA-associated histones. We thus conclude that STAT1, SMCHD1, and PML may partly mediate the suppressive effect of IFN-α on hepadnaviral cccDNA transcription.IMPORTANCE Pegylated IFN-α is the only therapeutic regimen that can induce a functional cure of chronic hepatitis B in a small, but significant, fraction of treated patients. Understanding the mechanisms underlying the antiviral functions of IFN-α in hepadnaviral infection may reveal molecular targets for development of novel antiviral agents to improve the therapeutic efficacy of IFN-α. By a loss-of-function genetic screening of individual IFN-stimulated genes (ISGs) on hepadnaviral mRNAs transcribed from cccDNA, we found that downregulating the expression of STAT1, SMCHD1, or PML significantly increased the level of viral RNAs without altering the level of cccDNA. Mechanistic analyses indicated that those cellular proteins are recruited to cccDNA minichromosomes and induce the posttranslational modifications of cccDNA-associated histones similar to those induced by IFN-α treatment. We have thus identified three IFN-α-induced cellular proteins that suppress cccDNA transcription and may partly mediate IFN-α silencing of hepadnaviral cccDNA transcription.


Assuntos
DNA Circular/metabolismo , Hepadnaviridae/efeitos dos fármacos , Hepadnaviridae/genética , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Linhagem Celular , Galinhas , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , DNA Viral/genética , Epigênese Genética , Infecções por Hepadnaviridae/virologia , Vírus da Hepatite B do Pato/efeitos dos fármacos , Vírus da Hepatite B , Hepatite B Crônica/virologia , Hepatócitos/virologia , Código das Histonas , Histonas/metabolismo , Humanos , Interferon-alfa/genética , Proteína da Leucemia Promielocítica/metabolismo , Processamento de Proteína Pós-Traducional , RNA Viral , Fator de Transcrição STAT1/metabolismo , Transcrição Genética , Replicação Viral
8.
Int J Nanomedicine ; 15: 3669-3680, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32547021

RESUMO

Introduction: The exhaustion and poor homing of activated lymphocytes are critical obstacles in adoptive cell immunotherapy for solid tumors. In order to effectively deliver immune cells into tumors, we encapsulated interferon-α2b (IFN-α2b) into macroporous hydrogels as an enhancement factor and utilized low-dose irradiation (LDI) as a tumoral attractor of T cells. Methods: Hydroxypropyl cellulose hydrogels were prepared by irradiation techniques, and the cross-sectional microstructure was characterized by scanning electron microscopy. The synergistic antitumor mechanism of combination of IFN-α2b and CIK cells was evaluated by detecting the expression of activation marker CD69 on CIK cell surface and IFN-γ production by CIK cells. The in vivo antitumor activity of IFN-α2b-incorporated hydroxypropyl cellulose hydrogels combined with CIK and radiation was evaluated in an MKN-45 xenografted nude mice model. Results: The bioactivity of IFN-α2b was well maintained in ultraviolet-reactive, rapidly cross-linkable hydroxypropyl cellulose hydrogels. In vitro studies demonstrated IFN-α2b-activated T cells, as evidenced by upregulating early activation marker CD69 and secretion inflammatory cytokine IFN-γ. In vivo real-time image showed our hydrogels kept a higher amount of drug delivery at the tumor site for a long time compared with free drug injection. Low-dose irradiation promoted T cell accumulation and infiltration in subcutaneous tumors. Combination of IFN-α2b-loaded hydrogels (Gel-IFN) with T cells and LDI exhibited higher efficacy to eradicate human gastric cancer xenograted tumors with less proliferating cells and more necrotic regions compared with IFN-α2b or T cells alone. Discussion: HPC hydrogels kept the activity of IFN-α2b and stably release of IFN-α2b to stimulate T cells for a long time. At the same time, low-dose radiation recruits T cells into tumors. This innovative integration mode of IFN-α2b-loaded hydrogels and radiotherapy offers a potent strategy to improve the therapeutic outcome of T cell therapy.


Assuntos
Antineoplásicos/uso terapêutico , Reagentes para Ligações Cruzadas/química , Hidrogéis/química , Interferon-alfa/uso terapêutico , Luz , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/radioterapia , Linfócitos T/imunologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Celulose/análogos & derivados , Celulose/química , Relação Dose-Resposta à Radiação , Elétrons , Humanos , Interferon-alfa/farmacologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Linfócitos T/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Science ; 369(6504): 712-717, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32527928

RESUMO

Excessive cytokine signaling frequently exacerbates lung tissue damage during respiratory viral infection. Type I (IFN-α and IFN-ß) and III (IFN-λ) interferons are host-produced antiviral cytokines. Prolonged IFN-α and IFN-ß responses can lead to harmful proinflammatory effects, whereas IFN-λ mainly signals in epithelia, thereby inducing localized antiviral immunity. In this work, we show that IFN signaling interferes with lung repair during influenza recovery in mice, with IFN-λ driving these effects most potently. IFN-induced protein p53 directly reduces epithelial proliferation and differentiation, which increases disease severity and susceptibility to bacterial superinfections. Thus, excessive or prolonged IFN production aggravates viral infection by impairing lung epithelial regeneration. Timing and duration are therefore critical parameters of endogenous IFN action and should be considered carefully for IFN therapeutic strategies against viral infections such as influenza and coronavirus disease 2019 (COVID-19).


Assuntos
Células Epiteliais Alveolares/patologia , Citocinas/metabolismo , Interferon Tipo I/metabolismo , Interferons/metabolismo , Pulmão/patologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Células Epiteliais Alveolares/imunologia , Animais , Apoptose , Líquido da Lavagem Broncoalveolar/imunologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Citocinas/administração & dosagem , Citocinas/imunologia , Feminino , Vírus da Influenza A Subtipo H3N2 , Interferon Tipo I/administração & dosagem , Interferon Tipo I/farmacologia , Interferon-alfa/administração & dosagem , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Interferon beta/administração & dosagem , Interferon beta/metabolismo , Interferon beta/farmacologia , Interferons/administração & dosagem , Interferons/farmacologia , Masculino , Camundongos , Infecções por Orthomyxoviridae/metabolismo , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
10.
Nat Commun ; 11(1): 2584, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444635

RESUMO

Interferon-α (IFNα), a type I interferon, is expressed in the islets of type 1 diabetic individuals, and its expression and signaling are regulated by T1D genetic risk variants and viral infections associated with T1D. We presently characterize human beta cell responses to IFNα by combining ATAC-seq, RNA-seq and proteomics assays. The initial response to IFNα is characterized by chromatin remodeling, followed by changes in transcriptional and translational regulation. IFNα induces changes in alternative splicing (AS) and first exon usage, increasing the diversity of transcripts expressed by the beta cells. This, combined with changes observed on protein modification/degradation, ER stress and MHC class I, may expand antigens presented by beta cells to the immune system. Beta cells also up-regulate the checkpoint proteins PDL1 and HLA-E that may exert a protective role against the autoimmune assault. Data mining of the present multi-omics analysis identifies two compound classes that antagonize IFNα effects on human beta cells.


Assuntos
Processamento Alternativo , Células Secretoras de Insulina/fisiologia , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Processamento Alternativo/efeitos dos fármacos , Células Cultivadas , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Mineração de Dados , Diabetes Mellitus Tipo 1/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Mapas de Interação de Proteínas , Proteômica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição
11.
Antiviral Res ; 179: 104811, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32360182

RESUMO

There is an urgent need to identify antivirals to curtail the COVID-19 pandemic. Herein, we report the sensitivity of SARS-CoV-2 to recombinant human interferons α and ß (IFNα/ß). Treatment with IFN-α or IFN-ß at a concentration of 50 international units (IU) per milliliter reduces viral titers by 3.4 log or over 4 log, respectively, in Vero cells. The EC50 of IFN-α and IFN-ß treatment is 1.35 IU/ml and 0.76 IU/ml, respectively, in Vero cells. These results suggest that SARS-CoV-2 is more sensitive than many other human pathogenic viruses, including SARS-CoV. Overall, our results demonstrate the potential efficacy of human Type I IFN in suppressing SARS-CoV-2 infection, a finding which could inform future treatment options for COVID-19.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Interferon Tipo I/farmacologia , Pneumonia Viral/tratamento farmacológico , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Animais , Chlorocebus aethiops , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Humanos , Imunidade Inata , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Proteínas Recombinantes/farmacologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/virologia , Células Vero , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
12.
Nat Rev Drug Discov ; 19(3): 149-150, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32127666
13.
Vet Microbiol ; 242: 108591, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32122595

RESUMO

Pigeon circovirus (PiCV) is the most diagnosed virus in pigeons (Columba livia) and have been studied and reported globally. PiCV infections can lead to immunosuppression and pigeons infected with PiCV can result to lymphocyte apoptosis and atrophy of immune organs. Young pigeon disease syndrome (YPDS) is a complex disease and believed that PiCV could be one of the agents leading to this syndrome. An effective treatment regimen is needed to control the spread of PiCV in pigeons. In this study pigeon interferon alpha (PiIFN-α) was cloned and expressed and its antiviral effects were tested against fowl adenovirus type 4 (FAdV-4) in vitro and PiCV in vivo. No detectable levels of FAdV-4 viral genome in LMH cells stimulated with 300 µg/mL PiIFN-α were found. Additionally, PiIFN-α was stable at different temperature and pH for 4 h, and no reduction in antiviral activity was observed in untreated and treated cells. In pigeons naturally and experimentally infected by PiCV, no detectable levels of PiCV virus titers were found after treatment with PiIFN-α. Cytokine and ISG expression levels in liver and spleen samples were detected and IFN-γ and Mx1 genes were dominantly up-regulated following PiIFN-α treatment (p < 0.05). This study demonstrated that PiCV can be inhibited by administration of PiIFN-α and PiFN-α can be used as a therapeutic approach to prevent the spread of PiCV in pigeons.


Assuntos
Doenças das Aves/virologia , Infecções por Circoviridae/veterinária , Circovirus/fisiologia , Citocinas/imunologia , Interferon-alfa/farmacologia , Replicação Viral/imunologia , Animais , Doenças das Aves/imunologia , Linhagem Celular , Infecções por Circoviridae/imunologia , Circovirus/genética , Circovirus/imunologia , Columbidae/imunologia , Columbidae/virologia , Escherichia coli/genética , Feminino , Genoma Viral , Concentração de Íons de Hidrogênio , Fígado/imunologia , Fígado/virologia , Masculino , Estabilidade Proteica , Baço/imunologia , Baço/virologia , Temperatura , Carga Viral/imunologia , Replicação Viral/efeitos dos fármacos
14.
BMC Biotechnol ; 20(1): 16, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32169063

RESUMO

BACKGROUND: The type I human interferon (IFN) family consists of a group of cytokines with a multiplicity of biological activities, including antiviral, antitumor, and immunomodulatory effects. However, because the half-life of IFN is short, its clinical application is limited. Increasing the yield and biological activity of IFN while extending its half-life is currently the focus of IFN research. RESULTS: Two novel long-acting recombinant human IFN-α2b (rhIFN-α2b) proteins were designed in which the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin ß su bunit and N-linked glycosylation sequences were linked to rhIFN-α2b. They were designated IFN-1CTPON (fused at the C-terminus of rhIFN-α2b) and IFN-2CTPON (fused at both the C-terminus and N-terminus of rhIFN-α2b). Monoclonal CHO cell strains stably and efficiently expressing the IFNs were successfully selected with methotrexate (MTX), and the highest expression levels were 1468 mg/l and 1196 mg/l for IFN-1CTPON and IFN-2CTPON, respectively. The proteins were purified with affinity chromatography and molecular sieve chromatography. IFN-1CTPON and IFN-2CTPON showed antiviral and antiproliferative activities in vitro. Notably, the half-life of IFN-1CTPON and IFN-2CTPON in vivo were three-fold and two-fold longer than that of commercially available rhIFN-α2b. CONCLUSIONS: CHO cell strains stably expressing long-acting rhIFN-α2b were screened. The purified IFN-CTPON protein has biological activity and an extended half-life, and therefore potential applications.


Assuntos
Antineoplásicos/farmacologia , Antivirais/farmacologia , Gonadotropina Coriônica Humana Subunidade beta/genética , Interferon-alfa/genética , Proteínas Recombinantes de Fusão/farmacologia , Animais , Células CHO , Proliferação de Células/efeitos dos fármacos , Gonadotropina Coriônica Humana Subunidade beta/química , Cromatografia de Afinidade , Cricetulus , Preparações de Ação Retardada , Glicosilação , Meia-Vida , Células HeLa , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/metabolismo
15.
J Immunother Cancer ; 8(1)2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32152220

RESUMO

BACKGROUND: Interferon-α (IFN-α) plays a pivotal role in host antitumor immunity, and the evasion of IFN-α signaling pathway can lead to IFN-α resistance during the treatment of cancer. Although the interplay between IFN-α and tumor cells has been extensively investigated in differentiated tumor cells, much less attention has been directed to tumor-repopulating cells (TRCs). METHODS: Three-dimentional soft fibrin matrix was used to select and grow highly malignant and tumorigenic melanoma TRCs. The regulation of integrin ß3 (ITGB3)-c-SRC-STAT signaling pathway in melanoma TRCs was investigated both in vitro and in vivo. The relevant mRNA and protein expression levels were analyzed by qRT-PCR and western blot analysis. Immunoprecipitation and chromatin immunoprecipitation (ChIP) followed by qPCR (ChIP-qPCR) assays were performed to detect protein-protein and protein-DNA interactions. The clinical impacts of retinoic acid inducible gene-I (RIG-I) were assessed in melanoma datasets obtained from The Cancer Genome Atlas and Gene Expression Omnibus profiles. RESULTS: IFN-α-induced apoptosis was decreased in melanoma TRCs. Compared with conventional flask-cultured cells, IFN-α-mediated STAT1 activation was diminished in melanoma TRCs. Decreased expression of RIG-I in melanoma TRCs led to diminished activation of STAT1 via enhancing the interaction between Src homology region 2 domain-containing phosphatase-1 and STAT1. In addition, low expression levels of RIG-I correlated with poor prognosis in patients with melanoma. STAT3 was highly phosphorylated in TRCs and knockdown of STAT3 reversed the downregulation of RIG-I in TRCs. Knockdown of STAT3 resulted in STAT1 activation and increased expression of the pro-apoptosis genes in IFN-α-treated TRCs. Combined treatment of STAT3 inhibitor and IFN-α increased the apoptosis rate of TRCs. Disruption of ITGB3/c-SRC/STAT3 signaling pathway significantly elevated the efficiency of IFN-α-induced apoptosis of TRCs. CONCLUSIONS: In melanoma TRCs, ITGB3-c-SRC-STAT3 pathway caused RIG-I repression and then affect STAT1 activation to cause resistance to IFN-α-induced apoptosis. RIG-I is a prognostic marker in patients with melanoma. Combination of STAT3 inhibitor and IFN-α could enhance the efficacy of melanoma treatment. Our findings may provide a new concept of combinatorial treatment for future immunotherapy.


Assuntos
Proteína DEAD-box 58/metabolismo , Integrina beta3/metabolismo , Interferon-alfa/farmacologia , Melanoma Experimental/tratamento farmacológico , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proteína DEAD-box 58/antagonistas & inibidores , Proteína DEAD-box 58/genética , Regulação para Baixo , Feminino , Células Hep G2 , Humanos , Fatores Imunológicos/farmacologia , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/patologia , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Prognóstico , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Taxa de Sobrevida
16.
Int J Mol Sci ; 21(4)2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32098104

RESUMO

Nervous necrosis virus (NNV) results in high mortality rates of infected marine fish worldwide. Interferons (IFNs) are cytokines in vertebrates that suppress viral replication and regulate immune responses. Heterologous overexpression of fish IFN in bacteria could be problematic because of protein solubility and loss of function due to protein misfolding. In this study, a protein model of the IFN-α of Epinephelus septemfasciatus was built based on comparative modeling. In addition, PelB and SacB signal peptides were fused to the N-terminus of E. septemfasciatus IFN-α for overexpression of soluble, secreted IFN in Escherichia coli (E-IFN) and Bacillus subtilis (B-IFN). Cytotoxicity tests indicated that neither recombinant grouper IFN-α were cytotoxic to a grouper head kidney cell line (GK). The GK cells stimulated with E-IFN and B-IFN exhibited elevated expression of antiviral Mx genes when compared with the control group. The NNV challenge experiments demonstrated that GK cells pretreated or co-treated with E-IFN and B-IFN individually had three times the cell survival rates of untreated cells, indicating the cytoprotective ability of our recombinant IFNs. These data provide a protocol for the production of soluble, secreted, and functional grouper IFN of high purity, which may be applied to aquaculture fisheries for antiviral infection.


Assuntos
Bacillus subtilis , Escherichia coli , Proteínas de Peixes , Interferon-alfa , Perciformes/genética , Animais , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Linhagem Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Peixes/biossíntese , Proteínas de Peixes/genética , Proteínas de Peixes/farmacologia , Interferon-alfa/biossíntese , Interferon-alfa/genética , Interferon-alfa/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
17.
Sci Rep ; 10(1): 3229, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32094377

RESUMO

Diabetes mellitus (DM) patients are at an increased risk of complications following influenza-virus infection, seasonal vaccination (SV) is recommended. However, SV with trivalent influenza vaccine (TIV) can induce antibody and type-I interferon (IFN) responses, and the effect of anti-DM treatment on these responses is incompletely understood. We evaluated the antibody response and IFN-α expression in individuals with and without type 2 DM (T2DM) following SV, and examined the effects on anti-DM treatment. TIV elicited sero-protection in all groups, but antibody persistency was <8 months, except for the antibody response to B-antigens in non-DM. T2DM impaired the IgG avidity index, and T2DM showed a significantly decreased response against H1N1 and H3N2, in addition to delaying and reducing haemagglutination-inhibition persistency against influenza B-antigens in DM groups treated with metformin (Met-DM) or glibenclamide (GB-DM). Following TIV, the Met-DM and GB-DM groups exhibited reduced IFN-α expression upon stimulation with whole- and split-virion influenza vaccines. Suppression of IFN-α expression in the Met-DM group was associated with a reduction in the mechanistic target of rapamycin complex-1 pathway and impaired IgG avidity index. Thus, single-dose TIV each year might not be suitable for T2DM. Our data could aid the development of an efficacious influenza vaccine for T2DM.


Assuntos
Formação de Anticorpos/efeitos dos fármacos , Diabetes Mellitus Tipo 2/imunologia , Interferon-alfa/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metformina/farmacologia , Estações do Ano , Transdução de Sinais , Vacinação , Idoso , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos/imunologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Feminino , Glibureto/farmacologia , Glibureto/uso terapêutico , Testes de Inibição da Hemaglutinação , Humanos , Imunoglobulina G/metabolismo , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza B/efeitos dos fármacos , Vírus da Influenza B/imunologia , Vacinas contra Influenza/imunologia , Masculino , Metformina/uso terapêutico , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vírion/efeitos dos fármacos , Vírion/imunologia
18.
Immunohorizons ; 4(2): 93-107, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32086319

RESUMO

TLR7 and TLR8 are pattern recognition receptors that reside in the endosome and are activated by ssRNA molecules. TLR7 and TLR8 are normally part of the antiviral defense response, but they have also been implicated as drivers of autoimmune diseases such as lupus. The receptors have slightly different ligand-binding specificities and cellular expression patterns that suggest they have nonredundant specialized roles. How the roles of TLR7 and TLR8 differ may be determined by which cell types express each TLR and how the cells respond to activation of each receptor. To provide a better understanding of the effects of TLR7/8 activation, we have characterized changes induced by TLR-specific agonists in different human immune cell types and defined which responses are a direct consequence of TLR7 or TLR8 activation and which are secondary responses driven by type I IFN or cytokines produced subsequent to the primary response. Using cell sorting, gene expression analysis, and intracellular cytokine staining, we have found that the IFN regulatory factor (IRF) and NF-κB pathways are differentially activated downstream of the TLRs in various cell types. Studies with an anti-IFNAR Ab in human cells and lupus mice showed that inhibiting IFN activity can block secondary IFN-induced gene expression changes downstream of TLR7/8 activation, but not NF-κB-regulated genes induced directly by TLR7/8 activation at earlier timepoints. In summary, these results elucidate the different roles TLR7 and TLR8 play in immunity and inform strategies for potential treatment of autoimmune diseases driven by TLR7/8 activation.


Assuntos
Fatores Reguladores de Interferon/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , NF-kappa B/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Animais , Autoanticorpos/sangue , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação , Interferon-alfa/farmacologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/genética , Camundongos , Camundongos Endogâmicos DBA , Modelos Biológicos , Células Mieloides/classificação , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Receptor de Interferon alfa e beta/antagonistas & inibidores , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas
19.
Sci Rep ; 10(1): 2747, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066770

RESUMO

Type I interferons, particularly interferon-alpha (IFN-α), play a vital role in the host's anti-viral defenses by interfering with viral replication. However, the virus rapidly evolves to exploit the IFN-α response for its replication, spread, and pathogenic function. In this study, we attempted to determine IFN-α susceptibility and productivity of infectious transmitted/founder (TF) (n = 8) and non-transmitted (NT) viruses (n = 8) derived from HIV-1 infected infants. Independent experiments were carried out to determine IFN-α resistance, replication fitness, and viral productivity in CD4+ T cells over a short period. In vitro studies showed that TF viruses were resistant to IFN-α during the very near moment of transmission, but in the subsequent time points, they became susceptible to IFN-α. We did not observe much difference in replicative fitness of the TF viruses in cultures treated with and without IFN-α, but the difference was significant in the case of NT viruses obtained from the same individual. Despite increased susceptibility to IFN-α, NT viruses produced more viral particles than TF viruses. Similar results were also obtained in cultures treated with maraviroc (MVC). The study identified unique characteristics of TF viruses thus prompting further investigation into virus-host interaction occurring during the early stages of HIV infection.


Assuntos
HIV-1/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interferon-alfa/farmacologia , Receptores CCR5/genética , Vírion/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Antagonistas dos Receptores CCR5/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/virologia , Expressão Gênica , Células HEK293 , Inibidores da Fusão de HIV/farmacologia , HIV-1/genética , HIV-1/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Lactente , Maraviroc/farmacologia , Mariposas , Cultura Primária de Células , Receptores CCR5/imunologia , Carga Viral/efeitos dos fármacos , Vírion/genética , Vírion/metabolismo , Internalização do Vírus/efeitos dos fármacos
20.
PLoS Pathog ; 16(2): e1008338, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32059034

RESUMO

Interferon (IFN) stimulates a whole repertoire of cellular genes, collectively referred to as ISGs (Interferon-stimulated genes). ISG20, a 3´-5´ exonuclease enzyme, has been previously shown to bind and degrade hepatitis B Virus (HBV) transcripts. Here, we show that the N6-methyladenosine (m6A)-modified HBV transcripts are selectively recognized and processed for degradation by ISG20. Moreover, this effect of ISG20 is critically regulated by m6A reader protein, YTHDF2 (YTH-domain family 2). Previously, we identified a unique m6A site within HBV transcripts and confirmed that methylation at nucleotide A1907 regulates HBV lifecycle. In this report, we now show that the methylation at A1907 is a critical regulator of IFN-α mediated decay of HBV RNA. We observed that the HBV RNAs become less sensitive to ISG20 mediated degradation when methyltransferase enzymes or m6A reader protein YTHDF2 are silenced in HBV expressing cells. By using an enzymatically inactive form ISG20D94G, we further demonstrated that ISG20 forms a complex with m6A modified HBV RNA and YTHDF2 protein. Due to terminal redundancy, HBV genomic nucleotide A1907 position is acquired twice by pregenomic RNA (pgRNA) during transcription and therefore the sites of methylation are encoded within 5´ and 3´ epsilon stem loops. We generated HBV mutants that lack m6A site at either one (5´ or 3´) or both the termini (5´& 3´). Using these mutants, we demonstrated that m6A modified HBV RNAs are subjected to ISG20-mediated decay and propose sequence of events, in which ISG20 binds with YTHDF2 and recognizes m6A-modified HBV transcripts to carry out the ribonuclease activity. This is the first study, which identifies a hitherto unknown role of m6A modification of RNA in IFN-α induced viral RNA degradation and proposes a new role of YTHDF2 protein as a cofactor required for IFN-α mediated viral RNA degradation.


Assuntos
Exorribonucleases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Antivirais/farmacologia , Exonucleases/metabolismo , Exorribonucleases/genética , Células Hep G2 , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Humanos , Interferon-alfa/farmacologia , Interferons/metabolismo , Metiltransferases/metabolismo , Estabilidade de RNA/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Replicação Viral/fisiologia
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