RESUMO
OBJECTIVE: Patients with X linked agammaglobulinemia are susceptible to enterovirus (EV) infections. Similarly, severe EV infections have been described in patients with impaired B-cell response following treatment with anti-CD20 monoclonal antibodies (mAbs), mostly in those treated for haematological malignancies. We aimed to describe severe EV infections in patients receiving anti-CD20 mAbs for immune-mediated inflammatory diseases (IMIDs). METHODS: Patients were included following a screening of data collected through the routine surveillance of EV infections coordinated by the National Reference Center and a review of the literature. Additionally, neutralising antibodies were assessed in a patient with chronic EV-A71 meningoencephalitis. RESULTS: Nine original and 17 previously published cases were retrieved. Meningoencephalitis (n=21/26, 81%) associated with EV-positive cerebrospinal fluid (n=20/22, 91%) was the most common manifestation. The mortality rate was high (27%). EV was the only causal agents in all reported cases. Patients received multiple anti-CD20 mAbs infusions (median 8 (5-10)), resulting in complete B-cell depletion and moderate hypogammaglobulinemia (median 4.9 g/L (4.3-6.7)), and had limited concomitant immunosuppressive treatments. Finally, in a patient with EV-A71 meningoencephalitis, a lack of B-cell response to EV was shown. CONCLUSION: EV infection should be evoked in patients with IMIDs presenting with atypical organ involvement, especially meningoencephalitis. Anti-CD20 mAbs may lead to impaired B-cell response against EV, although an underlying primary immunodeficiency should systematically be discussed.
Assuntos
Anticorpos Monoclonais , Antígenos CD20 , Infecções por Enterovirus , Humanos , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/diagnóstico , Masculino , Feminino , Anticorpos Monoclonais/uso terapêutico , Antígenos CD20/imunologia , Pessoa de Meia-Idade , Adulto , Meningoencefalite/imunologia , Meningoencefalite/virologia , Meningoencefalite/etiologia , Meningoencefalite/diagnóstico , Meningoencefalite/tratamento farmacológico , Idoso , Rituximab/uso terapêutico , Linfócitos B/imunologia , Agamaglobulinemia/imunologia , Agamaglobulinemia/complicações , Inflamação/imunologiaRESUMO
Enterovirus A71 (EV-A71) can induce severe neurological complications and even fatal encephalitis in children, and it has caused several large outbreaks in Taiwan since 1998. We previously generated VP1 codon-deoptimized (VP1-CD) reverse genetics (rg) EV-A71 viruses (rgEV-A71s) that harbor a high-fidelity (HF) 3D polymerase. These VP1-CD-HF rgEV-A71s showed lower replication kinetics in vitro and decreased virulence in an Institute of Cancer Research (ICR) mouse model of EV-A71 infection, while still retaining their antigenicity in comparison to the wild-type virus. In this study, we aimed to further investigate the humoral and cellular immune responses elicited by VP1-CD-HF rgEV-A71s to assess the potential efficacy of these EV-A71 vaccine candidates. Following intraperitoneal (i.p.) injection of VP1-CD-HF rgEV-A71s in mice, we observed a robust induction of EV-A71-specific neutralizing IgG antibodies in the antisera after 21 days. Splenocytes isolated from VP1-CD-HF rgEV-A71s-immunized mice exhibited enhanced proliferative activities and cytokine production (IL-2, IFN-γ, IL-4, IL-6, and TNF-α) upon re-stimulation with VP1-CD-HF rgEV-A71, as compared to control mice treated with adjuvant only. Importantly, administration of antisera from VP1-CD-HF rgEV-A71s-immunized mice protected against lethal EV-A71 challenge in neonatal mice. These findings highlight that our generated VP1-CD-HF rgEV-A71 viruses are capable of inducing both cellular and humoral immune responses, supporting their potential as next-generation EV-A71 vaccines for combating EV-A71 infection.IMPORTANCEEV-A71 can cause severe neurological diseases and cause death in young children. Here, we report the development of synthetic rgEV-A71s with the combination of codon deoptimization and high-fidelity (HF) substitutions that generate genetically stable reverse genetics (rg) viruses as potential attenuated vaccine candidates. Our work provides insight into the development of low-virulence candidate vaccines through a series of viral genetic editing for maintaining antigenicity and genome stability and suggests a strategy for the development of an innovative next-generation vaccine against EV-A71.
Assuntos
Proteínas do Capsídeo , Enterovirus Humano A , Infecções por Enterovirus , RNA Polimerase Dependente de RNA , Animais , Camundongos , Anticorpos Antivirais/imunologia , Códon , Enterovirus Humano A/genética , Infecções por Enterovirus/imunologia , Vacinas Atenuadas , Proteínas do Capsídeo/genética , Imunidade Humoral , Imunidade Celular , Anticorpos Neutralizantes/imunologia , Vacinas Virais , Camundongos Endogâmicos ICR , Camundongos Endogâmicos BALB C , RNA Polimerase Dependente de RNA/genéticaRESUMO
Rhinoviruses and allergens, such as house dust mite are major agents responsible for asthma exacerbations. The influence of pre-existing airway inflammation on the infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely unknown. We analyse mechanisms of response to viral infection in experimental in vivo rhinovirus infection in healthy controls and patients with asthma, and in in vitro experiments with house dust mite, rhinovirus and SARS-CoV-2 in human primary airway epithelium. Here, we show that rhinovirus infection in patients with asthma leads to an excessive RIG-I inflammasome activation, which diminishes its accessibility for type I/III interferon responses, leading to their early functional impairment, delayed resolution, prolonged viral clearance and unresolved inflammation in vitro and in vivo. Pre-exposure to house dust mite augments this phenomenon by inflammasome priming and auxiliary inhibition of early type I/III interferon responses. Prior infection with rhinovirus followed by SARS-CoV-2 infection augments RIG-I inflammasome activation and epithelial inflammation. Timely inhibition of the epithelial RIG-I inflammasome may lead to more efficient viral clearance and lower the burden of rhinovirus and SARS-CoV-2 infections.
Assuntos
Fatores de Restrição Antivirais , Asma , COVID-19 , Proteína DEAD-box 58 , Inflamassomos , Rhinovirus , Humanos , Fatores de Restrição Antivirais/genética , Fatores de Restrição Antivirais/metabolismo , Asma/genética , Asma/imunologia , COVID-19/genética , COVID-19/imunologia , Proteína DEAD-box 58/metabolismo , Infecções por Enterovirus/genética , Infecções por Enterovirus/imunologia , Inflamassomos/genética , Inflamassomos/metabolismo , Inflamação , Interferon Tipo I , Infecções por Picornaviridae/genética , Infecções por Picornaviridae/imunologia , Rhinovirus/metabolismo , Rhinovirus/patogenicidade , SARS-CoV-2RESUMO
Protein kinase B2 (AKT2) is involved in various cardiomyocyte signaling processes, including those important for survival and metabolism. Coxsackievirus B3 (CVB3) is one of the most common pathogens that cause myocarditis in humans. The role of AKT2 in CVB3 infection is not yet well understood. We used a cardiac-specific AKT2 knockout (KO) mouse to determine the role of AKT2 in CVB3-mediated myocarditis. CVB3 was injected intraperitoneally into wild-type (WT) and KO mice. The mice's survival rate was recorded: survival in KO mice was significantly decreased compared with WT mice (WT vs. KO: 73.3 vs. 27.1%). Myocardial damage and inflammation were significantly increased in the hearts of KO mice compared with those of WT mice. Moreover, from surface ECG, AKT2 KO mice showed a prolonged atria and ventricle conduction time (PR interval, WT vs. KO: 47.27 ± 1.17 vs. 64.79 ± 7.17 ms). AKT2 deletion induced severe myocarditis and cardiac dysfunction due to CVB3 infection. According to real-time PCR, the mRNA level of IL-1, IL-6, and TNF-α decreased significantly in KO mice compared with WT mice on Days 5 after infection. In addition, innate immune response antiviral effectors, Type I interferon (interferon-α and ß), and p62, were dramatically suppressed in the heart of KO mice. In particular, the adult cardiac myocytes isolated from the heart showed high induction of TLR4 protein in KO mice in comparison with WT. AKT2 deletion suppressed the activation of Type I interferon and p62 transcription in CVB3 infection. In cardiac myocytes, AKT2 is a key signaling molecule for the heart from damage through the activation of innate immunity during acute myocarditis.
Assuntos
Enterovirus Humano B/imunologia , Infecções por Enterovirus/imunologia , Imunidade Inata , Miocardite/imunologia , Miocárdio/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Doença Aguda , Animais , Enterovirus Humano B/genética , Infecções por Enterovirus/genética , Células HeLa , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/virologia , Camundongos , Camundongos Knockout , Miocardite/genética , Miocardite/virologia , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
Increasing cases related to the pathogenicity of Enterovirus D68 (EV-D68) have made it a growing worldwide public health concern, especially due to increased severe respiratory illness and acute flaccid myelitis (AFM) in children. There are currently no vaccines or medicines to prevent or treat EV-D68 infections. Herein, we performed genome-wide transcriptional profiling of EV-D68-infected human rhabdomyosarcoma (RD) cells to investigate host-pathogen interplay. RNA sequencing and subsequent experiments revealed that EV-D68 infection induced a profound transcriptional dysregulation of host genes, causing significantly elevated inflammatory responses and altered antiviral immune responses. In particular, triggering receptor expressed on myeloid cells 1 (TREM-1) is involved in highly activated TREM-1 signaling processes, acting as an important mediator in EV-D68 infection, and it is related to upregulation of interleukin 8 (IL-8), IL-6, IL-12p70, IL-1ß, and tumor necrosis factor alpha (TNF-α). Further results demonstrated that NF-κB p65 was essential for EV-D68-induced TREM-1 upregulation. Moreover, inhibition of the TREM1 signaling pathway by the specific inhibitor LP17 dampened activation of the p38 mitogen-activated protein kinase (MAPK) signaling cascade, suggesting that TREM-1 mainly transmits activation signals to phosphorylate p38 MAPK. Interestingly, treatment with LP17 to inhibit TREM-1 inhibited viral replication and infection. These findings imply the pathogenic mechanisms of EV-D68 and provide critical insight into therapeutic intervention in enterovirus diseases.
Assuntos
Enterovirus Humano D/patogenicidade , Infecções por Enterovirus/imunologia , Receptor Gatilho 1 Expresso em Células Mieloides/imunologia , Linhagem Celular , Citocinas/biossíntese , Enterovirus Humano D/imunologia , Infecções por Enterovirus/genética , Perfilação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases , Modelos Imunológicos , RNA-Seq , Transdução de Sinais/imunologia , Fator de Transcrição RelA/metabolismo , Receptor Gatilho 1 Expresso em Células Mieloides/antagonistas & inibidores , Receptor Gatilho 1 Expresso em Células Mieloides/genética , Replicação Viral/efeitos dos fármacosRESUMO
Enterovirus (EV) infection rarely results in life-threatening infection of the central nervous system. We report two unrelated children with EV30 and EV71 rhombencephalitis. One patient carries compound heterozygous TLR3 variants (loss-of-function F322fs2* and hypomorphic D280N), and the other is homozygous for an IFIH1 variant (loss-of-function c.1641+1G>C). Their fibroblasts respond poorly to extracellular (TLR3) or intracellular (MDA5) poly(I:C) stimulation. The baseline (TLR3) and EV-responsive (MDA5) levels of IFN-ß in the patients' fibroblasts are low. EV growth is enhanced at early and late time points of infection in TLR3- and MDA5-deficient fibroblasts, respectively. Treatment with exogenous IFN-α2b before infection renders both cell lines resistant to EV30 and EV71, whereas post-infection treatment with IFN-α2b rescues viral susceptibility fully only in MDA5-deficient fibroblasts. Finally, the poly(I:C) and viral phenotypes of fibroblasts are rescued by the expression of WT TLR3 or MDA5. Human TLR3 and MDA5 are critical for cell-intrinsic immunity to EV, via the control of baseline and virus-induced type I IFN production, respectively.
Assuntos
Encefalite Viral/imunologia , Infecções por Enterovirus/imunologia , Helicase IFIH1 Induzida por Interferon/genética , Receptor 3 Toll-Like/genética , Células Cultivadas , Pré-Escolar , Encefalite Viral/genética , Enterovirus/efeitos dos fármacos , Enterovirus/fisiologia , Infecções por Enterovirus/genética , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/virologia , Humanos , Lactente , Interferon alfa-2/farmacologia , Helicase IFIH1 Induzida por Interferon/imunologia , Interferon beta/imunologia , Interferon beta/metabolismo , Mutação com Perda de Função , Masculino , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/imunologia , Poli I-C/farmacologia , Rombencéfalo/virologia , Receptor 3 Toll-Like/imunologia , Replicação Viral/efeitos dos fármacosRESUMO
Previous studies have shown that DEAD (Glu-Asp-Ala-Glu)-box RNA helicases play important roles in viral infection, either as cytosolic sensors of pathogenic molecules or as essential host factors against viral infection. In the current study, we found that DDX6, an RNA helicase belonging to the DEAD-box family of helicase, exhibited anti-Enterovirus 71 activity through augmenting RIG-I-mediated type-I IFN response. Moreover, DDX6 binds viral RNA to form an RNA-protein complex to positively regulate the RIG-I-mediated interferon response; however, EV71 has evolved a strategy to antagonize the antiviral effect of DDX6 by proteolytic degradation of the molecule through its non-structural protein 2A, a virus-encoded protease.
Assuntos
RNA Helicases DEAD-box , Infecções por Enterovirus/imunologia , Interferon Tipo I , Proteínas Proto-Oncogênicas , Proteína DEAD-box 58 , Enterovirus Humano A , Humanos , Interferon Tipo I/imunologia , Helicase IFIH1 Induzida por Interferon , RNA Viral , Receptores ImunológicosRESUMO
The activation of the sympathetic nervous system, release of norepinephrine (NE), and adrenergic receptor signaling participate in and regulate the complicated enterovirus 71 (EV71) brainstem encephalitis (BE). The neurotoxin 6-hydroxydopamine (6-OHDA) selectively ablates sympathetic nerves and markedly depletes NE in innervated organs. Changes in the plasma levels of NE, severity score, cytokine profiles, and percentages of immunophenotype expression in 7-day-old Bltw : CD1 (ICR) mice infected with EV71, with or without 6-OHDA treatment, were compared. The survival rate (76.9%) of EV71-infected and 6-OHDA (30 µg/g)-treated mice was increased significantly. The clinical scores were decreased markedly on days 8-12 in MP4-infected and 6-OHDA-treated mice compared to those without treatment. The results showed that the plasma levels of NE, epinephrine, and dopamine were decreased on days 4-8 after 6-OHDA treatment and at most on day 8. The plasma levels of interleukin (IL)-12p70, tumor necrosis factor, IL-6, and IL-10 did not change significantly after 6-OHDA treatment. Interferon-γ levels decreased evidently on days 4, 6, and 8 after 6-OHDA treatment. The absolute events of CD3+CD4+, CD3+CD8+, and CD3+NK1.1+ cells of peripheral blood mononuclear cells were increased significantly in MP4-infected and 6-OHDA-treated mice compared to those without treatment. In splenocytes, the absolute cells of CD3-NK1.1+, CD3+NK1.1+ and CD11b+Gr-1+ cells of EV71-infected mice were increased significantly after 6-OHDA treatment. These findings suggested that 6-OHDA may be used a probe to explore clinical improvements and immune responses in the complicated EV71 infection. Taken together, peripheral chemical sympathectomy contribute to further understand the immunopathogenesis of EV71 BE with autonomic nervous system dysregulation.
Assuntos
Encefalite Viral/imunologia , Infecções por Enterovirus/imunologia , Simpatectomia Química/métodos , Animais , Tronco Encefálico/imunologia , Tronco Encefálico/patologia , Encefalite Viral/patologia , Enterovirus Humano A , Infecções por Enterovirus/patologia , Camundongos , Camundongos Endogâmicos ICR , OxidopaminaRESUMO
Enterovirus 71 (EV71) is a positive single-stranded RNA virus from the enterovirus genus of the Picornaviridae family. Most young children infected with EV71 develop mild symptoms of hand, foot and mouth disease, but some develop severe symptoms with neurological involvement. Limb paralysis from EV71 infection is presumed to arise mainly from dysfunction of motor neurons in the spinal cord. However, EV71 also targets and damages skeletal muscle, which may also contribute to the debilitating symptoms. In this study, we have delineated the impacts of EV71 infection on skeletal muscle using a mouse model. Mouse pups infected with EV71 developed limb paralysis, starting at day 3 post-infection and peaking at day 5-7 post-infection. At later times, mice recovered gradually but not completely. Notably, severe disease was associated with high levels of myositis accompanied by muscle calcification and persistent motor end plate abnormalities. Interestingly, macrophages exhibited a dynamic change in phenotype, with inflammatory macrophages (CD45+CD11b+Ly6Chi) appearing in the early stage of infection and anti-inflammatory/restorative macrophages (CD45+CD11b+Ly6Clow/-) appearing in the late stage. The presence of inflammatory macrophages was associated with severe inflammation, while the restorative macrophages were associated with recovery. Altogether, we have demonstrated that EV71 infection causes myositis, muscle calcification and structural defects in motor end plates. Subsequent muscle regeneration is associated with a dynamic change in macrophage phenotype.
Assuntos
Enterovirus Humano A , Infecções por Enterovirus/imunologia , Macrófagos/imunologia , Músculo Esquelético/patologia , Miosite/imunologia , Fenótipo , Recuperação de Função Fisiológica/imunologia , Animais , Antígenos Ly/metabolismo , Antígeno CD11b/metabolismo , Calcinose/imunologia , Modelos Animais de Doenças , Infecções por Enterovirus/virologia , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Paralisia/imunologia , Regeneração/imunologiaRESUMO
Enterovirus D68 (EV-D68) is an emerging pathogen associated with respiratory diseases and/or acute flaccid myelitis. Here, two MAbs, 2H12 and 8F12, raised against EV-D68 virus-like particle (VLP), show distinct preference in binding VLP and virion and in neutralizing different EV-D68 strains. A combination of 2H12 and 8F12 exhibits balanced and potent neutralization effects and confers broader protection in mice than single MAbs when given at onset of symptoms. Cryo-EM structures of EV-D68 virion complexed with 2H12 or 8F12 show that both antibodies bind to the canyon region of the virion, creating steric hindrance for sialic acid receptor binding. Additionally, 2H12 binding can impair virion integrity and trigger premature viral uncoating. We also capture an uncoating intermediate induced by 2H12 binding, not previously described for picornaviruses. Our study elucidates the structural basis and neutralizing mechanisms of the 2H12 and 8F12 MAbs and supports further development of the 2H12/8F12 cocktail as a broad-spectrum therapeutic agent against EV-D68 infections in humans.
Assuntos
Anticorpos Monoclonais/imunologia , Enterovirus Humano D/imunologia , Infecções por Enterovirus/imunologia , Vírion/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Linhagem Celular Tumoral , Microscopia Crioeletrônica , Enterovirus Humano D/efeitos dos fármacos , Enterovirus Humano D/fisiologia , Infecções por Enterovirus/tratamento farmacológico , Infecções por Enterovirus/virologia , Feminino , Humanos , Camundongos Endogâmicos BALB C , Ligação Proteica/efeitos dos fármacos , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Tempo para o Tratamento , Resultado do Tratamento , Vírion/efeitos dos fármacos , Vírion/metabolismo , Vírion/ultraestrutura , Desenvelopamento do Vírus/efeitos dos fármacosRESUMO
Enterovirus uncoating receptors bind at the surface depression ("canyon") that encircles each capsid vertex causing the release of a host-derived lipid called "pocket factor" that is buried in a hydrophobic pocket formed by the major viral capsid protein, VP1. Coxsackievirus and adenovirus receptor (CAR) is a universal uncoating receptor of group B coxsackieviruses (CVB). Here, we present five high-resolution cryoEM structures of CVB representing different stages of virus infection. Structural comparisons show that the CAR penetrates deeper into the canyon than other uncoating receptors, leading to a cascade of events: collapse of the VP1 hydrophobic pocket, high-efficiency release of the pocket factor and viral uncoating and genome release under neutral pH, as compared with low pH. Furthermore, we identified a potent therapeutic antibody that can neutralize viral infection by interfering with virion-CAR interactions, destabilizing the capsid and inducing virion disruption. Together, these results define the structural basis of CVB cell entry and antibody neutralization.
Assuntos
Microscopia Crioeletrônica , Enterovirus/metabolismo , Enterovirus/ultraestrutura , Animais , Anticorpos Neutralizantes , Capsídeo/metabolismo , Proteínas do Capsídeo/ultraestrutura , Enterovirus Humano B/metabolismo , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/metabolismo , Infecções por Enterovirus/virologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Receptores Virais , Vírion/metabolismo , Vírion/ultraestrutura , Desenvelopamento do VírusRESUMO
BACKGROUND: Enterovirus 71 (EV71) infection contributes to hand, foot, and mouth disease (HFMD) with severe neurogenic complications, leading to higher morbidity. In addition to their typical roles in coagulation, platelets could serve as essential immune regulatory cells to play a key role in the pathogenesis of this viral infection. METHODS: Platelet parameters were measured using an automatic hematology analyzer. T-helper type 1 (Th1) and Th2 cells were analyzed by flow cytometry. The levels of cytokines and key transcription factors were determined. RESULTS: The levels of platelet count and plateletcrit were positively associated with the severity of HFMD. Th1 and Th2 cells as well as their corresponding cytokines were increased in the severe group compared to the healthy volunteers. Moreover, the levels of platelets were negatively correlated with the level of interferon-γ (IFN-γ), but positively correlated with the frequency of Th1 cells. Coculture of platelets and naive CD4+ T cells showed that platelets from mild patients promote Th1 cell differentiation and IFN-γ secretion. CONCLUSIONS: Our study has shown for the first time that the distinct roles of platelets are responsible for the regulation of pathogenic CD4+ T cell differentiation and function in the pathogenesis of HFMD caused by EV71. IMPACT: Our study has shown for the first time that the distinct roles of platelets are responsible for the regulation of pathogenic CD4+ T cell differentiation and function in the pathogenesis of HFMD caused by EV71. For the first time, we have discovered the role of platelets in children's HFMD caused by EV71 infection, which may provide a better treatment for HFMD in the future. This article describes new discoveries in platelet immunity.
Assuntos
Plaquetas/citologia , Plaquetas/virologia , Enterovirus Humano A/imunologia , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Doença de Mão, Pé e Boca/imunologia , Doença de Mão, Pé e Boca/virologia , Linfócitos T CD4-Positivos/citologia , Ligante de CD40/metabolismo , Diferenciação Celular , Criança , Citocinas/metabolismo , Feminino , Voluntários Saudáveis , Humanos , Masculino , Selectina-P/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Células Th1/citologia , Células Th2/citologiaRESUMO
Human enterovirus D68 (EV-D68) has received considerable attention recently as a global reemergent pathogen because it causes severe respiratory tract infections and acute flaccid myelitis (AFM). The nonstructural protein 2A protease (2Apro) of EVs, which functions in the cleavage of host proteins, comprises a pivotal part of the viral immune evasion process. However, the pathogenic mechanism of EV-D68 is not fully understood. In this study, we found that EV-D68 inhibited antiviral type I interferon responses by cleaving tumor necrosis factor receptor-associated factor 3 (TRAF3), which is the key factor for type I interferon production. EV-D68 inhibited Sendai virus (SEV)-induced interferon regulatory factor 3 (IRF3) activation and beta interferon (IFN-ß) expression in HeLa and HEK293T cells. Furthermore, we demonstrated that EV-D68 and 2Apro were able to cleave the C-terminal region of TRAF3 in HeLa and HEK293T cells, respectively. A cysteine-to-alanine substitution at amino acid 107 (C107A) in the 2Apro protease resulted in the loss of cleavage activity to TRAF3, and mutation of glycine at amino acid 462 to alanine (G462A) in TRAF3 conferred resistance to 2Apro These results suggest that control of TRAF3 by 2Apro may be a mechanism EV-D68 utilizes to subvert host innate immune responses.IMPORTANCE Human enterovirus 68 (EV-D68) has received considerable attention recently as a global reemergent pathogen because it causes severe respiratory tract infections and acute flaccid myelitis. The nonstructural protein 2A protease (2Apro) of EV, which functions in cleavage of host proteins, comprises an essential part of the viral immune evasion process. However, the pathogenic mechanism of EV-D68 is not fully understood. Here, we show for the first time that EV-D68 inhibited antiviral type I interferon responses by cleaving tumor necrosis factor receptor-associated factor 3 (TRAF3). Furthermore, we identified the key cleavage site in TRAF3. Our study may suggest a new mechanism by which the 2Apro of EV facilitates subversion of host innate immune responses. These findings increase our understanding of EV-D68 infection and may help identify new antiviral targets against EV-D68.
Assuntos
Enterovirus Humano D/enzimologia , Infecções por Enterovirus/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/imunologia , Peptídeo Hidrolases/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismo , Proteínas Virais/metabolismo , Infecções por Enterovirus/metabolismo , Infecções por Enterovirus/patologia , Infecções por Enterovirus/virologia , Células HEK293 , Células HeLa , Humanos , Interferon Tipo I/metabolismo , Peptídeo Hidrolases/genética , Proteólise , Fator 3 Associado a Receptor de TNF/genética , Proteínas Virais/genéticaRESUMO
Enteroviruses infect gastrointestinal epithelium cells, cause multiple human diseases, and present public health risks worldwide. However, the mechanisms underlying host immune responses in intestinal mucosa against the early enterovirus infections remain elusive. Here, we showed that human enteroviruses including enterovirus 71 (EV71), coxsackievirus B3 (CVB3), and poliovirus 1 (PV1) predominantly induce type III interferons (IFN-λ1 and IFN-λ2/3), rather than type I interferons (IFN-α and IFN-ß), in cultured human normal and cancerous intestine epithelial cells (IECs), mouse intestine tissues, and human clinical intestine specimens. Mechanistic studies demonstrated that IFN-λ production is induced upon enterovirus infection through the Toll-like receptor 3/interferon regulatory factor 1 (TLR3/IRF1) signaling pathway in IECs. In turn, the supplementation of IFN-λ subsequently induces intrinsically antiviral responses against enterovirus replication. Notably, intraperitoneal injection in neonatal C57BL/6J mice with mouse recombinant IFN-λ2 protein represses EV71 replication and protects mice from viral lethal effects. Altogether, these results revealed a distinct mechanism by which the host elicited immune responses against enterovirus infections in intestine through activating the TLR3/IRF1/type III IFN axis. The new findings would provide an antiviral strategy for the prevention and treatment of enterovirus infections and associated diseases.IMPORTANCE Enterovirus infections are significant sources of human diseases and public health risks worldwide, but little is known about the mechanism of innate immune response in host intestine epithelial surface during the viral replication. We reported the epithelial immune response in cultured human normal and cancerous cells (IECs), mouse tissues, and human clinical intestine specimens following infection with enterovirus 71. The results mechanistically revealed type III interferons (IFN-λ1 and IFN-λ2/3), rather than type I interferons (IFN-α and IFN-ß), as the dominant production through TLR3/IRF1 signaling upon multiple human enterovirus infection, including enterovirus 71 (EV71), coxsackievirus B3 (CVB3), and poliovirus 1 (PV1). IFN-λ subsequently induced antiviral activity against enterovirus replication in vitro and in vivo. These studies uncovered the role of the novel process of type III IFN production involved in the TLR3/IRF1 pathway in host intestine upon enterovirus infection, which highlighted a regulatory manner of antiviral defense in intestine during enterovirus infection.
Assuntos
Infecções por Enterovirus/imunologia , Enterovirus/imunologia , Imunidade Inata , Fator Regulador 1 de Interferon/metabolismo , Interferons/metabolismo , Receptor 3 Toll-Like/metabolismo , Animais , Enterovirus/genética , Enterovirus/fisiologia , Infecções por Enterovirus/virologia , Feminino , Humanos , Fator Regulador 1 de Interferon/genética , Interferons/genética , Intestinos/imunologia , Intestinos/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Receptor 3 Toll-Like/genética , Replicação Viral , Interferon lambdaAssuntos
Anti-Inflamatórios/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Desferroxamina/uso terapêutico , Fatores Imunológicos/uso terapêutico , Quelantes de Ferro/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Animais , Anti-Inflamatórios/farmacologia , Anticorpos Neutralizantes/sangue , Antivirais/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Betacoronavirus , COVID-19 , Desferroxamina/farmacologia , Infecções por Enterovirus/sangue , Infecções por Enterovirus/tratamento farmacológico , Infecções por Enterovirus/imunologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Fatores Imunológicos/farmacologia , Interleucina-6/imunologia , Quelantes de Ferro/farmacologia , Sobrecarga de Ferro/tratamento farmacológico , Camundongos , Pandemias , SARS-CoV-2 , Resultado do Tratamento , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Prophylactic vaccines are critical in preventing hand, foot, and mouth disease (HFMD) primarily caused by human enterovirus 71 (EV71) infection. Children aged less than 5 years are especially susceptible to EV71 infections. In addition to the development of vaccines containing the inactivated virus, those containing virus-like particles (VLPs) with repeated antigens also constitute an effective preventive strategy for EV71 infections, with safety and productivity advantages. We previously developed a fusion protein composed with truncated peptides of the EV71 capsid protein, which assembled into spherical particles. This study aimed to assess the immunoprotective effects of this fusion protein as a vaccine candidate in a mouse model of EV71 infection. METHODS: To evaluate the protective effect of fusion protein vaccine candidate, neonatal mice born by immunized female mice, as well as normal neonatal mice immunized twice were infected with EV71 virus. Whereafter, the survival rates, clinical scores and viral loads were measured. RESULTS: The high dosage and booster immunization helped induce specific serum antibodies with high neutralization titers, which were transferred to neonatal mice, thereby facilitating effective resistance towards EV71 infection. An active immune response was also observed in neonatal mice which generated following immunization. CONCLUSIONS: The present results suggest that this fusion protein is a suitable vaccine candidate in treating EV71 infections.
Assuntos
Enterovirus Humano A/genética , Infecções por Enterovirus/prevenção & controle , Peptídeos/imunologia , Proteínas Virais de Fusão/imunologia , Vacinas Virais/imunologia , Animais , Animais Recém-Nascidos , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linhagem Celular Tumoral , Enterovirus Humano A/imunologia , Infecções por Enterovirus/imunologia , Feminino , Humanos , Imunização Secundária , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/genética , Proteínas Virais de Fusão/administração & dosagem , Carga Viral , Vacinas Virais/genéticaRESUMO
In the past few decades, enterovirus A71 (EVA71) has caused devastating outbreaks in the Asia-Pacific region, resulting in serious sequelae in infected young children. No preventive or therapeutic interventions are currently available for curing EVA71 infection, highlighting a great unmet medical need for this disease. Here, we showed that one novel single-domain antibody (sdAb), F1, isolated from an immunized llama, could alleviate EVA71 infection both in vitro and in vivo We also confirmed that the sdAb clone F1 recognizes EVA71 through a novel conformational epitope comprising the highly conserved region of VP3 capsid protein by using competitive-binding and overlapping-peptide enzyme-linked immunosorbent assays (ELISAs). Because of the virion's icosahedral structure, we reasoned that adjacent epitopes must be clustered within molecular ranges that may be simultaneously bound by an engineered antibody with multiple valency. Therefore, two single-domain binding modules (F1) were fused to generate an sdAb-in-tandem design so that the capture of viral antigens could be further increased by valency effects. We showed that the tetravalent construct F1×F1-hFc, containing two sdAb-in-tandem on a fragment crystallizable (Fc) scaffold, exhibits more potent neutralization activity against EVA71 than does the bivalent sdAb F1-hFc by at least 5.8-fold. We also demonstrated that, using a human scavenger receptor class B member 2 (hSCARB2) transgenic mouse model, a half dose of the F1×F1-hFc provided better protection against EVA71 infection than did the F1-hFc. Thus, our study furnishes important insights into multivalent sdAb engineering against viral infection and provides a novel strategic deployment approach for preparedness of emerging infectious diseases such as EVA71.
Assuntos
Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Enterovirus Humano A/imunologia , Infecções por Enterovirus/terapia , Anticorpos de Domínio Único/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Antígenos Virais/imunologia , Camelídeos Americanos , Linhagem Celular Tumoral , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Camundongos , Camundongos Transgênicos , Anticorpos de Domínio Único/farmacologiaRESUMO
Hand, foot and mouth disease (HFMD), caused by enterovirus A71 (EV-A71), presents mild to severe disease, and sometimes fatal neurological and respiratory manifestations. However, reasons for the severe pathogenesis remain undefined. To investigate this, infection and viral kinetics of EV-A71 isolates from clinical disease (mild, moderate and severe) from Sarawak, Malaysia, were characterised in human rhabdomyosarcoma (RD), neuroblastoma (SH-SY5Y) and peripheral blood mononuclear cells (PBMCs). High resolution transcriptomics was used to decipher EV-A71-host interactions in PBMCs. Ingenuity analyses revealed similar pathways triggered by all EV-A71 isolates, although the extent of activation varied. Importantly, several pathways were found to be specific to the severe isolate, including triggering receptor expressed on myeloid cells 1 (TREM-1) signalling. Depletion of TREM-1 in EV-A71-infected PBMCs with peptide LP17 resulted in decreased levels of pro-inflammatory genes for the moderate and severe isolates. Mechanistically, this is the first report describing the transcriptome profiles during EV-A71 infections in primary human cells, and the potential involvement of TREM-1 in the severe disease pathogenesis, thus providing new insights for future treatment targets.
Assuntos
Enterovirus Humano A/fisiologia , Infecções por Enterovirus/metabolismo , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo , Linfócitos B/virologia , Linhagem Celular Tumoral , Infecções por Enterovirus/imunologia , Regulação da Expressão Gênica , Humanos , Monócitos/virologia , Filogenia , Linfócitos T/virologiaRESUMO
BACKGROUND: Enteroviruses are a group of common non-enveloped RNA viruses that cause symptoms ranging from mild respiratory infections to paralysis. Due to the abundance of enterovirus infections it is hard to distinguish between on-going and previous infections using immunological assays unless the IgM fraction is studied. METHODS: In this study we show using Indirect ELISA and capture IgM ELISA that an IgG antibody response against the nonstructural enteroviral proteins 2A and 3C can be used to distinguish between IgM positive (n = 22) and IgM negative (n = 20) human patients with 83% accuracy and a diagnostic odds ratio of 30. Using a mouse model, we establish that the antibody response to the proteases is short-lived compared to the antibody response to the structural proteins in. As such, the protease antibody response serves as a potential marker for an acute infection. CONCLUSIONS: Antibody responses against enterovirus proteases are shorter-lived than against structural proteins and can differentiate between IgM positive and negative patients, and therefore they are a potential marker for acute infections.
Assuntos
Anticorpos Antivirais/sangue , Enterovirus/enzimologia , Enterovirus/imunologia , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Peptídeo Hidrolases/imunologia , Proteases Virais 3C , Doença Aguda , Adulto , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos , Biomarcadores/sangue , Cisteína Endopeptidases/imunologia , Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Lactente , Camundongos , Camundongos Endogâmicos C57BL , Peptídeo Hidrolases/classificação , Proteínas Virais/imunologiaRESUMO
INTRODUCTION: Chronic enterovirus infections can occur in primary immunodeficiency with hypogammaglobulinemia. They usually associate meningitis and myofasciitis. Such infections have also been described in adults with rituximab-induced hypogammaglobulinemia. CASE REPORT: We report the case of a 33-year-old woman who was given rituximab for immune thrombocytopenia and developed rituximab-induced hypogammaglobulinemia (IgG 4.4g/L). One year after the last rituximab infusion, she developed lower limbs myofasciitis, followed two months later by a chronic lymphocytic meningitis. PCR in the serum and the cerebrospinal fluid at the time of the meningitis and the myofasciitis were positive to the same enterovirus (echovirus 11) while it was negative in the fascia biopsy. Under treatment with intravenous immunoglobulins, all symptoms and laboratory abnormalities improved and enterovirus PCR became negative. CONCLUSION: We report a case of chronic enterovirus infection associating meningitis and myofasciitis in an adult with rituximab-induced hypogammaglobulinemia. Outcome was favorable under treatment with intravenous immunoglobulins.