RESUMO
The recent emergence and rapid response to severe acute respiratory syndrome coronavirus 2 was enabled by prototype pathogen and vaccine platform approaches, driven by the preemptive application of RNA vaccine technology to the related Middle East respiratory syndrome coronavirus. Recently, the National Institutes of Allergy and Infectious Diseases identified nine virus families of concern, eight enveloped virus families and one nonenveloped virus family, for which vaccine generation is a priority. Although RNA vaccines have been described for a variety of enveloped viruses, a roadmap for their use against nonenveloped viruses is lacking. Enterovirus D68 was recently designated a prototype pathogen within the family Picornaviridae of nonenveloped viruses because of its rapid evolution and respiratory route of transmission, coupled with a lack of diverse anti-enterovirus vaccine approaches in development. Here, we describe a proof-of-concept approach using a clinical stage RNA vaccine platform that induced robust enterovirus D68-neutralizing antibody responses in mice and nonhuman primates and prevented upper and lower respiratory tract infections and neurological disease in mice. In addition, we used our platform to rapidly characterize the antigenic diversity within the six genotypes of enterovirus D68, providing the necessary data to inform multivalent vaccine compositions that can elicit optimal breadth of neutralizing responses. These results demonstrate that RNA vaccines can be used as tools in our pandemic-preparedness toolbox for nonenveloped viruses.
Assuntos
Anticorpos Neutralizantes , Enterovirus Humano D , Infecções por Enterovirus , Animais , Infecções por Enterovirus/prevenção & controle , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Enterovirus Humano D/imunologia , Enterovirus Humano D/genética , Anticorpos Neutralizantes/imunologia , Camundongos , Vacinas Virais/imunologia , Modelos Animais de Doenças , Humanos , Vacinas de mRNA , Anticorpos Antivirais/imunologia , FemininoRESUMO
Enteroviruses are important human pathogens with diverse serotypes, posing a major challenge to develop vaccines for individual serotypes, the success of polio vaccines in controlling and eradicating polio, along with the recent emergence and high prevalence of enterovirus-caused infectious diseases, highlights the importance of enterovirus vaccine development. Given our previous report on enteroviruses weakened by the 2 A S/T125A mutation, we assessed the potential of the EV-A71 2A-125A mutant as a vaccine candidate to address this challenge. We found that the 2A-125A mutant caused transient mild symptoms, low viral loads, and no significant pathological changes mild pathological changes in hSCARB2-KI mice, producing long-lasting cross-neutralizing antibodies against two EV-A71 wild strains. Pre-exposure to the 2A-125A mutant substantially protected against the EV-A71 Isehara wild-type strain, causing minor pathologies, significantly reducing muscle and lung inflammation, and preventing neurological damage, with reduced viral loads in vivo. Pre-exposure also distinctly suppressed the expression of pro-inflammatory cytokines, correlating to the severity of clinical symptoms. Collectively, the EV-A71 2A-125A mutant was attenuated and could generate a robust and protective immune response, suggesting its potential as a vaccine candidate and global solution for specific enterovirus vaccine development.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Enterovirus Humano A , Infecções por Enterovirus , Vacinas Atenuadas , Carga Viral , Vacinas Virais , Animais , Enterovirus Humano A/imunologia , Enterovirus Humano A/genética , Infecções por Enterovirus/prevenção & controle , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Camundongos , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Vacinas Virais/imunologia , Vacinas Virais/genética , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/genética , Humanos , Desenvolvimento de Vacinas , Feminino , Mutação , CitocinasRESUMO
Enterovirus A71 (EV-A71) is a major pathogen causing hand, foot, and mouth disease (HFMD) in children worldwide. It can lead to severe gastrointestinal, pulmonary, and neurological complications. The innate immune system, which rapidly detects pathogens via pathogen-associated molecular patterns or pathogen-encoded effectors, serves as the first defensive line against EV-A71 infection. Concurrently, the virus has developed various sophisticated strategies to evade host antiviral responses and establish productive infection. Thus, the virus-host interactions and conflicts, as well as the ability to govern biological events at this first line of defense, contribute significantly to the pathogenesis and outcomes of EV-A71 infection. In this review, we update recent progress on host innate immune responses to EV-A71 infection. In addition, we discuss the underlying strategies employed by EV-A71 to escape host innate immune responses. A better understanding of the interplay between EV-A71 and host innate immunity may unravel potential antiviral targets, as well as strategies that can improve patient outcomes.
Assuntos
Enterovirus Humano A , Infecções por Enterovirus , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Imunidade Inata , Humanos , Evasão da Resposta Imune/imunologia , Enterovirus Humano A/imunologia , Enterovirus Humano A/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Animais , Doença de Mão, Pé e Boca/imunologia , Doença de Mão, Pé e Boca/virologiaRESUMO
Enteroviruses are the causative agents associated with several human and animal diseases, posing a significant threat to human and animal health. As one of the host immune defense strategies, innate immunity plays a crucial role in defending against invading pathogens, where the host utilizes a variety of mechanisms to inhibit or eliminate the pathogen. Here, we report a new strategy for the host to repress enterovirus replication by the 78 kDa glucose-regulated protein (GRP78), also known as heat shock protein family A member 5 (HSPA5). The GRP78 recognizes the EV-encoded RNA-dependent RNA polymerases (RdRPs) 3D protein and interacts with the nuclear factor kappa B kinase complex (CHUK) and subunit beta gene (IKBKB) to facilitate the phosphorylation and nuclear translocation of NF-κB, which induces the production of inflammatory factors and leads to a broad inhibition of enterovirus replication. These findings demonstrate a new role of GRP78 in regulating host innate immunity in response to viral infection and provide new insights into the mechanism underlying enterovirus replication and NF-κB activation.IMPORTANCEGRP78 is known as a molecular chaperone for protein folding and plays a critical role in maintaining protein folding and participating in cell proliferation, cell survival, apoptosis, and metabolism. However, the functions of GRP78 to participate in enterovirus genome replication and innate immune responses are rarely documented. In this study, we explored the functions of the EV-3D-interacting protein GRP78 and found that GRP78 inhibits enterovirus replication by activating NF-κB through binding to EV-F 3D and interacting with the NF-κB signaling molecules CHUK/IKBKB. This is the first report that GRP78 interacts with CHUK/IKBKB to activate the NF-κB signaling pathway, which leads to the expression of the proinflammatory cytokines and inhibition of enterovirus replication. These results demonstrate a unique mechanism of virus replication regulation by GRP78 and provide insights into the prevention and treatment of viral infections.
Assuntos
Chaperona BiP do Retículo Endoplasmático , Quinase I-kappa B , NF-kappa B , Proteínas Virais , Replicação Viral , Animais , Humanos , Chlorocebus aethiops , Chaperona BiP do Retículo Endoplasmático/metabolismo , Enterovirus/crescimento & desenvolvimento , Enterovirus/imunologia , Enterovirus/metabolismo , Enterovirus/fisiologia , Infecções por Enterovirus/virologia , Infecções por Enterovirus/metabolismo , Infecções por Enterovirus/imunologia , Proteínas de Choque Térmico/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Quinase I-kappa B/metabolismo , Imunidade Inata , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , NF-kappa B/metabolismo , Fosforilação , Ligação Proteica , RNA Polimerase Dependente de RNA/metabolismo , Transdução de Sinais , Células Vero , Proteínas Virais/metabolismoRESUMO
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) is associated with neurological conditions such as acute meningitis and encephalitis. The virus is detected in the bloodstream, and high blood viral loads are associated with central nervous system (CNS) manifestations. We used an in vitro blood-brain barrier (BBB) model made up of human brain-like endothelial cells (hBLECs) and brain pericytes grown in transwell systems to investigate whether three genetically distinct EV-A71 strains (subgenogroups C1, C1-like, and C4) can cross the human BBB. EV-A71 poorly replicated in hBLECs, which released moderate amounts of infectious viruses from their luminal side and trace amounts of infectious viruses from their basolateral side. The barrier properties of hBLECs were not impaired by EV-A71 infection. We investigated the passage through hBLECs of EV-A71-infected white blood cells. EV-A71 strains efficiently replicated in immune cells, including monocytes, neutrophils, and NK/T cells. Attachment to hBLECs of immune cells infected with the C1-like virus was higher than attachment of cells infected with C1-06. EV-A71 infection did not impair the transmigration of immune cells through hBLECs. Overall, EV-A71 targets different white blood cell populations that have the potential to be used as a Trojan horse to cross hBLECs more efficiently than cell-free EV-A71 particles.IMPORTANCEEnterovirus A71 (EV-A71) was first reported in the USA, and numerous outbreaks have since occurred in Asia and Europe. EV-A71 re-emerged as a new multirecombinant strain in 2015 in Europe and is now widespread. The virus causes hand-foot-and-mouth disease in young children and is involved in nervous system infections. How the virus spreads to the nervous system is unclear. We investigated whether white blood cells could be infected by EV-A71 and transmit it across human endothelial cells mimicking the blood-brain barrier protecting the brain from adverse effects. We found that endothelial cells provide a strong roadblock to prevent the passage of free virus particles but allow the migration of infected immune cells, including monocytes, neutrophils, and NK/T cells. Our data are consistent with the potential role of immune cells in the pathogenesis of EV-A71 infections by spreading the virus in the blood and across the human blood-brain barrier.
Assuntos
Barreira Hematoencefálica , Células Endoteliais , Enterovirus Humano A , Infecções por Enterovirus , Barreira Hematoencefálica/virologia , Humanos , Enterovirus Humano A/genética , Enterovirus Humano A/fisiologia , Infecções por Enterovirus/virologia , Infecções por Enterovirus/imunologia , Células Endoteliais/virologia , Replicação Viral , Monócitos/virologia , Monócitos/imunologia , Pericitos/virologia , Leucócitos/virologia , Leucócitos/imunologia , Encéfalo/virologia , Células Matadoras Naturais/imunologia , Neutrófilos/imunologia , Neutrófilos/virologiaRESUMO
Enterovirus 71 (EV71) is a significant causative agent of hand, foot, and mouth disease, with potential serious neurologic complications or fatal outcomes. The lack of effective treatments for EV71 infection is attributed to its elusive pathogenicity. Our study reveals that human plasmacytoid dendritic cells (pDCs), the main type I IFN-producing cells, selectively express scavenger receptor class B, member 2 (SCARB2) and P-selectin glycoprotein ligand 1 (PSGL-1), crucial cellular receptors for EV71. Some strains of EV71 can replicate within pDCs and stimulate IFN-α production. The activation of pDCs by EV71 is hindered by Abs to PSGL-1 and soluble PSGL-1, whereas Abs to SCARB2 and soluble SCARB2 have a less pronounced effect. Our data suggest that only strains binding to PSGL-1, more commonly found in severe cases, can replicate in pDCs and induce IFN-α secretion, highlighting the importance of PSGL-1 in these processes. Furthermore, IFN-α secretion by pDCs can be triggered by EV71 or UV-inactivated EV71 virions, indicating that productive infection is not necessary for pDC activation. These findings provide new insights into the interaction between EV71 and pDCs, suggesting that pDC activation could potentially mitigate the severity of EV71-related diseases.
Assuntos
Células Dendríticas , Enterovirus Humano A , Interferon-alfa , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana , Células Dendríticas/imunologia , Células Dendríticas/virologia , Humanos , Enterovirus Humano A/imunologia , Enterovirus Humano A/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Membrana Lisossomal/imunologia , Interferon-alfa/metabolismo , Interferon-alfa/imunologia , Receptores Depuradores/metabolismo , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Replicação ViralRESUMO
Human enteroviruses are the most common human pathogen with over 300 distinct genotypes. Previous work with poliovirus has suggested that it is possible to generate antibody responses in humans and animals that can recognize members of multiple enterovirus species. However, cross protective immunity across multiple enteroviruses is not observed epidemiologically in humans. Here we investigated whether immunization of mice or baboons with inactivated poliovirus or enterovirus virus-like-particles (VLPs) vaccines generates antibody responses that can recognize enterovirus D68 or A71. We found that mice only generated antibodies specific for the antigen they were immunized with, and repeated immunization failed to generate cross-reactive antibody responses as measured by both ELISA and neutralization assay. Immunization of baboons with IPV failed to generate neutralizing antibody responses against enterovirus D68 or A71. These results suggest that a multivalent approach to enterovirus vaccination is necessary to protect against enterovirus disease in vulnerable populations.
Assuntos
Anticorpos Antivirais , Reações Cruzadas , Infecções por Enterovirus , Vacina Antipólio de Vírus Inativado , Animais , Camundongos , Reações Cruzadas/imunologia , Anticorpos Antivirais/imunologia , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/prevenção & controle , Infecções por Enterovirus/virologia , Vacina Antipólio de Vírus Inativado/imunologia , Vacina Antipólio de Vírus Inativado/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/imunologia , Anticorpos Neutralizantes/imunologia , Papio/imunologia , Humanos , Poliovirus/imunologia , Feminino , Formação de Anticorpos/imunologia , Enterovirus/imunologia , Camundongos Endogâmicos BALB C , Enterovirus Humano D/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
IMPORTANCE: Enterovirus D68 (EV-D68) is an emerging respiratory pathogen associated with acute flaccid myelitis. Currently, no approved vaccines or antiviral drugs are available. Here, we report four functionally independent neutralizing antigenic sites (I to IV) by analyses of neutralizing monoclonal antibody (MAb)-resistant mutants. Site I is located in the VP1 BC loop near the fivefold axis. Site II resides in the VP2 EF loop, and site III is situated in VP1 C-terminus; both sites are located at the south rim of the canyon. Site IV is composed of residue in VP2 ßB strand and residues in the VP3 BC loop and resides around the threefold axis. The developed MAbs targeting the antigenic sites can inhibit viral binding to cells. These findings advance the understanding of the recognition of EV-D68 by neutralizing antibodies and viral evolution and immune escape and also have important implications for the development of novel EV-D68 vaccines.
Assuntos
Anticorpos Neutralizantes , Proteínas do Capsídeo , Enterovirus Humano D , Infecções por Enterovirus , Humanos , Capsídeo , Proteínas do Capsídeo/química , Enterovirus Humano D/genética , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologiaRESUMO
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
Human rhinoviruses (HRVs) are small non-enveloped RNA viruses that belong to the Enterovirus genus within the Picornaviridae family and are known for causing the common cold. Though symptoms are generally mild in healthy individuals, the economic burden associated with HRV infection is significant. A vaccine could prevent disease. The Vero-cell-based viral vaccine platform technology was considered for such vaccine development. Unfortunately, most HRV strains are unable to propagate on Vero cells due to a lack of the major receptor of HRV group A and B, intercellular adhesion molecule (ICAM1, also known as CD54). Therefore, stable human ICAM1 expressing Vero cell clones were generated by transfecting the ICAM1 gene in Vero cells and selecting clones that overexpressed ICAM1 on the cell surface. Cell banks were made and expression of ICAM1 was stable for at least 30 passages. The Vero_ICAM1 cells and parental Vero cells were infected with four HRV prototypes, B14, A16, B37 and A57. Replication of all four viruses was detected in Vero_ICAM1, but not in the parental Vero cells. Altogether, Vero cells expressing ICAM1 could efficiently propagate the tested HRV strains. Therefore, ICAM1-expressing cells could be a useful tool for the development and future production of polyvalent HRV vaccines or other viruses that use ICAM1 as a receptor.
Assuntos
Molécula 1 de Adesão Intercelular , Infecções por Picornaviridae , Rhinovirus , Células Vero , Vacinas Virais , Animais , Humanos , Chlorocebus aethiops , Enterovirus/genética , Enterovirus/imunologia , Infecções por Enterovirus/genética , Infecções por Enterovirus/imunologia , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Infecções por Picornaviridae/genética , Infecções por Picornaviridae/imunologia , Rhinovirus/genética , Rhinovirus/imunologia , Células Vero/imunologia , Vacinas Virais/imunologiaRESUMO
Platelet factor 4 (PF4) or the CXC chemokine CXCL4 is the most abundant protein within the α-granules of platelets. Previous studies found that PF4 regulates infections of several viruses, including HIV-1, H1N1, hepatitis C virus (HCV), and dengue virus. Here, we show that PF4 is an inhibitor of enterovirus A71 (EV71) and coxsackievirus A16 (CA16) infections. The secreted form of PF4 from transfected cells or soluble purified PF4 from Escherichia coli, even lacking signal peptide affected secretion, obviously inhibited the propagation of EV71 and CA16. Mechanistically, we demonstrated that PF4 blocked the entry of the virus into the host cells by interactions with VP3 proteins of EV71/CA16 and the interaction with SCARB2 receptor-mediated EV71 and CA16 endocytosis. As expected, the incubation of anti-PF4 antibody with PF4 blocked PF4 inhibition on EV71 and CA16 infections further supported the above conclusion. Importantly, pretreatment of EV71 viruses with PF4 significantly protected the neonatal mice from EV71 lethal challenge and promoted the survival rate of infected mice. PF4 derived from natural platelets by EV71/CA16 activation also presented strong inhibition on EV71 and CA16. In summary, our study identified a new host factor against EV71 and CA16 infections, providing a novel strategy for EV71 and CA16 treatment. IMPORTANCE The virus's life cycle starts with binding to cell surface receptors, resulting in receptor-mediated endocytosis. Targeting the entry of the virus into target cells is an effective strategy to develop a novel drug. EV71 and CA16 are the major pathogens that cause hand, foot, and mouth disease (HFMD) outbreaks worldwide since 2008. However, the treatment of EV71 and CA16 infections is mainly symptomatic because there is no approved drug. Therefore, the underlying pathogenesis of EV71/CA16 and the interaction between host-EV71/CA16 need to be further investigated to develop an inhibitor. Here, we identified PF4 as a potent entry inhibitor of EV71 and CA16 via binding to VP3 proteins of EV71 and CA16 or binding to receptor SCARB2. In the EV71 infection model, PF4 protected mice from EV71 lethal challenge and promoted the survival rate of EV71-infected mice. Our study suggests that PF4 represents a potential candidate host factor for anti-EV71 and CA16 infections.
Assuntos
Infecções por Coxsackievirus , Infecções por Enterovirus , Fator Plaquetário 4 , Internalização do Vírus , Animais , Infecções por Coxsackievirus/imunologia , Enterovirus , Enterovirus Humano A , Infecções por Enterovirus/imunologia , Fatores Imunológicos/metabolismo , Camundongos , Fator Plaquetário 4/metabolismoRESUMO
Brainstem encephalitis, a manifestation of severe enterovirus 71 (EV71) infection, is an acute excessive inflammatory response. The mechanisms underlying its development remain poorly understood. Usually neurotropic viruses trigger acute host immune response by engaging cell surface or intracellular receptors. Here, we show that EV71 engagement with intracellular receptor TLR9 elicits IL-12p40-iNOS signaling causing encephalitis in mice. We identified IL-12p40 to be the only prominent cytokine-induced at the early infection stage in the brainstem of mice subjected to a lethal dose of EV71. The upregulated IL-12p40 proteins were expressed in glial cells but not neuronal cells. To better understand the role of IL-12p40 in severe EV71 infection, we treated the EV71-infected mice with an antibody against IL-12p40 and found the mortality rate, brainstem inflammation, and gliosis to be markedly reduced, suggesting that the acute IL-12p40 response plays a critical role in the pathogenesis of brainstem encephalitis. Mechanistically, intracellular TLR9 was found essential to the activation of the IL-12p40 response. Blocking TLR9 signaling with CpG-ODN antagonist ameliorated IL-12p40 response, brainstem inflammation, and limb paralysis in mice with EV71-induced encephalitis. We further found the glial IL-12p40 response might damage neurons by inducing excess production of neurotoxic NO by iNOS. Overall, EV71 engagement with intracellular TLR9 was found to elicit a neurotoxic glial response via IL12p40-iNOS signaling contributing to the neurological manifestation of EV71 infection. This pathway could potentially be targeted for the treatment of brainstem encephalitis.
Assuntos
Encefalite , Enterovirus Humano A , Infecções por Enterovirus , Subunidade p40 da Interleucina-12 , Receptor Toll-Like 9 , Animais , Encefalite/imunologia , Encefalite/virologia , Infecções por Enterovirus/imunologia , Inflamação , Subunidade p40 da Interleucina-12/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Receptor Toll-Like 9/metabolismoRESUMO
A substantial amount of epidemiological data has been reported on Enterovirus D68 (EV-D68) infections after the 2014 outbreak. Our goal was to map the case fatality rate (CFR) and prevalence of current and past EV-D68 infections. We conducted a systematic review (PROSPERO, CRD42021229255) with published articles on EV-68 infections in PubMed, Embase, Web of Science and Global Index Medicus up to January 2021. We determined prevalences using a model random effect. Of the 4,329 articles retrieved from the databases, 89 studies that met the inclusion criteria were from 39 different countries with apparently healthy individuals and patients with acute respiratory infections, acute flaccid myelitis and asthma-related diseases. The CFR estimate revealed occasional deaths (7/1353) related to EV-D68 infections in patients with severe acute respiratory infections. Analyses showed that the combined prevalence of current and past EV-D68 infections was 4% (95% CI = 3.1-5.0) and 66.3% (95% CI = 40.0-88.2), respectively. The highest prevalences were in hospital outbreaks, developed countries, children under 5, after 2014, and in patients with acute flaccid myelitis and asthma-related diseases. The present study shows sporadic deaths linked to severe respiratory EV-D68 infections. The study also highlights a low prevalence of current EV-D68 infections as opposed to the existence of EV-D68 antibodies in almost all participants of the included studies. These findings therefore highlight the need to implement and/or strengthen continuous surveillance of EV-D68 infections in hospitals and in the community for the anticipation of the response to future epidemics.
Assuntos
Enterovirus Humano D/isolamento & purificação , Infecções por Enterovirus/epidemiologia , Infecções por Enterovirus/mortalidade , Anticorpos Antivirais , Asma , Viroses do Sistema Nervoso Central , Enterovirus Humano D/imunologia , Infecções por Enterovirus/imunologia , Humanos , Mielite , Doenças Neuromusculares , Prevalência , Infecções RespiratóriasRESUMO
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
Outbreaks of enteroviral infections are associated with morbidity and mortality in susceptible individuals worldwide. There are still no antiviral drugs or vaccines against most circulating enteroviruses. Antibody-mediated immunity is crucial for preventing and limiting enteroviral infections. In this review, we focus on enteroviruses that continue to cause endemics in recent years, such as rhinovirus, enterovirus A71, coxsackievirus, and echovirus, and introduce a structural understanding of the mechanisms of virus neutralization. The mechanisms by which virus-specific antibodies neutralize enteroviruses have been explored not only through study of viral structures, but also through understanding virus-antibody interactions at the amino acid level. Neutralizing epitopes are predominantly mapped on the canyon northern rim, canyon inner surface, canyon southern rim, and twofold and threefold plateaus of the capsid, where surface-exposed loops are located. This review also describes recent progress in deciphering the virus-receptor complex and structural rearrangements involved in the uncoating process, providing insight into plausible virus neutralization mechanisms.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Enterovirus/imunologia , Animais , Antígenos Virais/química , Antígenos Virais/imunologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Doenças Endêmicas , HumanosRESUMO
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.