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Influenza A virus is an important human pathogen causing significant morbidity and mortality. Numerous host factors and cellular responses are dysregulated during influenza A virus infection. This includes arrest of autophagic flux dependent on the influenza M2 ion channel, but little is known which host factors participate in this autophagic dysfunction. Sarco/endoplasmic reticulum calcium ATPase (SERCA) is known to regulate transport of calcium ions between the cytoplasm and the sarco/endoplasmic reticulum, and has been positively correlated with autophagic flux. Herein, we found that SERCA activity was suppressed in influenza A virus infected human lung cells (H1395) and that CDN1163, an activator of SERCA, restored autophagic flux and thus reduced autophagosome accumulation caused by the influenza A virus. Activating SERCA activity with CDN1163 also decreased expression of inflammatory cytokines and chemokines and attenuated mitochondrial dysfunction in IAV-infected H1395 cells. Conversely, SERCA inhibition or genetic ablation aggravated the autophagy dysfunction, mitochondria, and inflammatory responses in the cells infected with influenza A virus. Further study showed that SERCA might regulate the inflammatory response by modulating phosphorylation of MAPK-JNK pathway. These findings showed that the influenza A virus induced autophagic flux blocking, inflammatory response and mitochondrial dysfunction by inhibiting SERCA activity. This study provides further understanding of the host-viral interactions between the influenza virus, SERCA activity, autophagy, inflammatory response, and mitochondrial function. SERCA may be a potential host target for decreasing inflammatory and superoxide injury during influenza A virus infection.IMPORTANCE:IAV is a major cause of infectious respiratory diseases, characterized by a marked respiratory tract inflammatory response that causes morbidity and mortality in seasonal epidemics, or pandemic outbreaks. SERCA is a critical component in maintaining cellular calcium levels, and is positively correlated with autophagic flux. Here, we discovered that SERCA is suppressed in IAV-infected human lung cells and influenza A virus induces blocking of autophagic flux, inflammatory response and mitochondrial dysfunction by inhibiting SERCA. We posit that the pharmacological activation of SERCA can be a powerful intervention strategy to prevent autophagy arrest, inflammatory response, and mitochondrial dysfunction in IAV-infected cells. Therefore, SERCA activity modulation could be a potential therapeutic strategy for managing clinical symptoms of severe influenza disease.
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Since the first outbreak in 2013, the influenza A (H7N9) virus has continued emerging and has caused over five epidemic waves. Suspected antigenic changes of the H7N9 virus based on hemagglutination inhibition (HI) assay during the fifth outbreak have prompted the update of H7N9 candidate vaccine viruses (CVVs). In this study, we comprehensively compared the serological cross-reactivities induced by the hemagglutinins (HAs) of the earlier CVV A/Anhui/1/2013 (H7/AH13) and the updated A/Guangdong/17SF003/2016 (H7/GD16). We found that although H7/GD16 showed poor HI cross-reactivity to immune sera from mice and rhesus macaques vaccinated with either H7/AH13 or H7/GD16, the cross-reactive neutralizing antibodies between H7/AH13 and H7/GD16 were comparably high. Passive transfer of H7/AH13 immune sera also provided complete protection against the lethal challenge of H7N9/GD16 virus in mice. Analysis of amino acid mutations in the HAs between H7/AH13 and H7/GD16 revealed that L226Q substitution increases the HA binding avidity to sialic acid receptors on red blood cells, leading to decreased HI titers against viruses containing HA Q226 and thus resulting in a biased antigenic evaluation based on HI assay. These results suggest that amino acids located in the receptor-binding site could mislead the evaluation of antigenic variation by solely impacting the receptor-binding avidity to red blood cells without genuine contribution to antigenic drift. Our study highlighted that viral receptor-binding avidity and combination of multiple serological assays should be taken into consideration in evaluating and selecting a candidate vaccine virus of H7N9 and other subtypes of influenza viruses.IMPORTANCE The HI assay is a standard method for profiling the antigenic characterization of influenza viruses. Suspected antigenic changes based on HI divergency in H7N9 viruses during the 2016-2017 wave prompted the recommendation of new H7N9 candidate vaccine viruses (CVVs). In this study, we found that the L226Q substitution in HA of A/Guangdong/17SF003/2016 (H7/GD16) increased the viral receptor-binding avidity to red blood cells with no impact on the antigenicity of H7N9 virus. Although immune sera raised by an earlier vaccine strain (H7/AH13) showed poor HI titers against H7/GD16, the H7/AH13 immune sera had potent cross-neutralizing antibody titers against H7/GD16 and could provide complete passive protection against H7N9/GD16 virus challenge in mice. Our study highlights that receptor-binding avidity might lead to biased antigenic evaluation by using the HI assay. Other serological assays, such as the microneutralization (MN) assay, should be considered a complementary indicator for analysis of antigenic variation and selection of influenza CVVs.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Subtipo H7N9 do Vírus da Influenza A , Mutação de Sentido Incorreto , Infecções por Orthomyxoviridae , Substituição de Aminoácidos , Animais , Cães , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/imunologia , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Macaca mulatta , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologiaRESUMO
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread worldwide through person-to-person contact, causing a public health emergency of international concern. At present, there is no specific antiviral treatment recommended for SARS-CoV-2 infection. Liu Shen capsule (LS), a traditional Chinese medicine, has been proven to have a wide spectrum of pharmacological properties, such as anti-inflammatory, antiviral and immunomodulatory activities. However, little is known about the antiviral effect of LS against SARS-CoV-2. Herein, the study was designed to investigate the antiviral activity of SARS-CoV-2 and its potential effect in regulating the host's immune response. The inhibitory effect of LS against SARS-CoV-2 replication in Vero E6 cells was evaluated by using the cytopathic effect (CPE) and plaque reduction assay. The number of virions of SARS-CoV-2 was observed under transmission electron microscope after treatment with LS. Proinflammatory cytokine expression levels upon SARS-CoV-2 infection in Huh-7 cells were measured by real-time quantitative PCR assays. The results showed that LS could significantly inhibit SARS-CoV-2 replication in Vero E6 cells, and reduce the number of virus particles and it could markedly reduce pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, IL-8, CCL-2/MCP-1 and CXCL-10/IP-10) production at the mRNA levels. Moreover, the expression of the key proteins in the NF-κB/MAPK signaling pathway was detected by western blot and it was found that LS could inhibit the expression of p-NF-κB p65, p-IκBα and p-p38 MAPK, while increasing the expression of IκBα. These findings indicate that LS could inhibit SARS-CoV-2 virus infection via downregulating the expression of inflammatory cytokines induced virus and regulating the activity of NF-κB/MAPK signaling pathway in vitro, making its promising candidate treatment for controlling COVID-19 disease.
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Betacoronavirus/efeitos dos fármacos , Misturas Complexas/farmacologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Antivirais/farmacologia , COVID-19 , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Humanos , Mediadores da Inflamação/metabolismo , Pandemias , Pneumonia Viral/virologia , SARS-CoV-2 , Vírion/efeitos dos fármacosRESUMO
Influenza A virus infection can arrest autophagy, as evidenced by autophagosome accumulation in infected cells. Here, we report that this autophagosome accumulation can be inhibited by amantadine, an antiviral proton channel inhibitor, in amantadine-sensitive virus infected cells or cells expressing influenza A virus matrix protein 2 (M2). Thus, M2 proton channel activity plays a role in blocking the fusion of autophagosomes with lysosomes, which might be a key mechanism for arresting autophagy.
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Autofagia , Interações Hospedeiro-Patógeno , Vírus da Influenza A/fisiologia , Proteínas da Matriz Viral/metabolismo , Humanos , PrótonsRESUMO
Adenovirus has been extensively exploited as a vector platform for delivering vaccines. However, preexisting antiadenovirus immunity is the major stumbling block for application of adenovirus-vectored vaccines. In this study, we found that freshly isolated peripheral blood mononuclear cells (PBMCs), mostly CD14(+) cells, from adenovirus serotype 5 (Ad5)-seropositive primates (humans and rhesus macaques) can be efficiently infected with Ad5 in vitro. On the basis of this observation, a novel strategy based on adenoviral vector-infected PBMC (AVIP) immunization was explored to circumvent antivector immunity. Autologous infusion of Ad5-SIVgag-infected PBMCs elicited a strong Gag-specific cellular immune response but induced weaker Ad5-neutralizing antibody (NAb) in Ad5-seronegative macaques than in macaques intramuscularly injected with Ad5-SIVgag. Moreover, Ad5-seropositive macaques receiving multiple AVIP immunizations with Ad5-SIVenv, Ad5-SIVgag, and Ad5-SIVpol vaccines elicited escalated Env-, Gag-, and Pol-specific immune responses after each immunization that were significantly greater than those in macaques intramuscularly injected with these Ad5-SIV vaccines. After challenged intravenously with a highly pathogenic SIVmac239 virus, macaques receiving AVIP immunization demonstrated a significant reduction in viral load at both the peak time and set-point period compared with macaques without Ad5-SIV vaccines. Our study warranted further research and development of the AVIP immunization as a platform for repeated applications of adenovirus-vectored vaccines.
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Adenoviridae/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra a SAIDS/genética , Vacinas contra a SAIDS/imunologia , Vacinas contra Adenovirus , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Produtos do Gene pol/imunologia , Vetores Genéticos , Humanos , Leucócitos Mononucleares/virologia , Receptores de Lipopolissacarídeos/biossíntese , Macaca mulatta , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Vacinas Virais/imunologiaRESUMO
Background: Influenza virus infection complicated by secondary bacterial pneumonia contributes significantly to death during seasonal or pandemic influenza. Secondary infection of Pseudomonas aeruginosa (P. aeruginosa) in influenza virus-infected patients contributes to morbidity and mortality. Methods: Mice were first infected with PR8 influenza virus, followed by a secondary infection of P. aeruginosa. Body weights and survival rate of mice was monitored daily over 20 days. Bronchoalveolar lavage fluids (BALFs) and lung homogenates were harvested for measuring bacterial titers. Lung tissue section slides were stained with hematoxylin and eosin for microscopic observation. After vaccination with inactivated P. aeruginosa cells or recombinant PcrV protein, the mice were subjected to PR8 influenza virus infection followed by a secondary infection of a P. aeruginosa. The inhibition against P. aeruginosa of serum was evaluated by detecting the growth of P. aeruginosa in broth containing diluted sera. Results: The prior influenza infection greatly enhanced the susceptibility to secondary infection of P. aeruginosa and increased morbidity and mortality in mice. Active immunization with inactivated P. aeruginosa cells could protect mice from secondary P. aeruginosa challenge in influenza virus infected mice. Conclusions: To develop an effective P. aeruginosa vaccine might be a promising strategy to decrease the threat of secondary P. aeruginosa infection in influenza patients.
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Live attenuated influenza vaccines offer broader and longer-lasting protection in comparison to inactivated influenza vaccines. The neuraminidase (NA) surface glycoprotein of influenza A virus is essential for the release and spread of progeny viral particles from infected cells. In this study, we de novo synthesized the NA gene, in which 62% of codons were synonymously changed based on mammalian codon bias usage. The codon-reprogrammed NA (repNA) gene failed to be packaged into the viral genome, which was achievable with partial restoration of wild-type NA sequence nucleotides at the 3' and 5' termini. Among a series of rescued recombinant viruses, we selected 20/13repNA, which contained 20 and 13 nucleotides of wild-type NA at the 3' and 5' termini of repNA, respectively, and evaluated its potential as a live attenuated influenza vaccine. The 20/13repNA is highly attenuated in mice, and the calculated LD50 was about 10,000-fold higher than that of the wild-type (WT) virus. Intranasal inoculation of the 20/13repNA virus in mice induced viral-specific humoral, cell-mediated, and mucosal immune responses. Mice vaccinated with the 20/13repNA virus were protected from the lethal challenge of both homologous and heterologous viruses. This strategy may provide a new method for the development of live, attenuated influenza vaccines for a better and more rapid response to influenza threats.
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Replication-competent influenza viruses carrying reporter genes are of great use for basic research, screening of antiviral drugs, and neutralizing of antibodies. In this study, two recombinant influenza A viruses with a neuraminidase (NA) segment harboring enhanced green fluorescent protein (EGFP) in the background of A/PR/8/34 (PR8) were generated. The viral RNA (vRNA)-specific packaging signals for NA were largely retained for efficient packaging. An "autocleave" 2A peptide sequence, which was inserted at the N terminus or the COOH terminus of NA to link with EGFP, enabled NA and EGFP to be expressed monocistronically. Further analysis demonstrated that both viruses, named rPR8-EGFP+NA and rPR8-NA+EGPF, although with some characteristic differences in growth and EGFP expression, could replicate in noncomplementary cells and propagate to large quantities while maintaining genome stability after multiple passages in embryonated eggs. These replication-competent influenza viruses carrying reporter genes are a great addition to the tool set for developing antiviral therapeutics and vaccines and for in vivo studies of viral dissemination and pathogenicity.
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Genes Reporter , Engenharia Genética , Vírus da Influenza A/fisiologia , Neuraminidase/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Animais , Linhagem Celular , Embrião de Galinha , Cães , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Vírus da Influenza A/enzimologia , Vírus da Influenza A/genética , Neuraminidase/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Virais/genética , Montagem de VírusRESUMO
Penicillium polonicum MCCC3A00951 is a fungus with influenza neuraminidase (NA) inhibition activity derived from a sediment of the mangrove forest of Zhangjiangkou in Fujian province, China. Chemical investigation on an ethyl acetate extract of its fermentation led to the isolation of a new compound, 7-hydroxy-3,10-dehydrocyclopeptine (1), and 13 known compounds (2-14). The new compound was comprehensively characterised by high-resolution electrospray ionisation-mass spectrometry, and 1D, 2D nuclear magnetic resonance (NMR) spectra. The anti-influenza NA assay was performed to evaluate the potential biological activity. Surprisingly, Cyclopenin (2) showed potent influenza NA inhibition with an IC50 value of 5.02 µM. Besides, molecular docking simulation was performed to investigate the binding model of cyclopenin (2) with influenza NA. Consequently, cyclopenin (2) could be further optimised to be a potential anti-influenza NA candidate.
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Antivirais/farmacologia , Produtos Biológicos/farmacologia , Fungos/química , Neuraminidase/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Organismos Aquáticos , China , Simulação de Acoplamento Molecular , Estrutura Molecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Neuraminidase inhibitors (NAIs) are the mainstay antiviral drugs against influenza infection. In this study, a ligand fishing protocol was developed to screen NAIs using neuraminidase immobilized magnetic beads (NA-MB). After verifying the feasibility of NA-MB with an artificial mixture including NA inhibitors and non-inhibitors, the developed ligand fishing protocol was applied to screen NAIs from the crude extracts of Duchesnea indica Andr. Twenty-four NA binding compounds were identified from the normal butanol (n-BuOH) extract of D. indica as potential NAIs by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS) assisted with Compound Structure Identification (CSI):FingerID, including 12 ellagitannins, 4 brevifolin derivatives, 3 ellagic acid derivatives, and 4 flavonoids. Among them, 9 compounds were isolated and tested for in vitro NA inhibitory activities against NA from Clostridium perfringens, and from oseltamivir sensitive and resistant influenza A virus strains. The results indicate that compound B23 has the NA inhibitory activities in both the oseltamivir sensitive and resistant viral NA, with half maximal inhibitory concentration (IC50) values of 197.9 and 125.4 µmol/L, respectively. Moreover, B23 can obviously reduce the replication of oseltamivir sensitive and resistant viruses in Madin-Darby canine kidney (MDCK) cells at the concentrations of 40 and 200 µmol/L.
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Highly pathogenic influenza H7N9 viruses that emerged in the fifth wave of H7N9 outbreak pose a risk to human health. The World Health Organization has updated the candidate vaccine viruses for H7N9 viruses recently. In this study, we evaluated the immune response to an updated H7N9 candidate vaccine virus, which derived from the highly pathogenic A/Guangdong/17SF003/2016 (GD/16) in mice and rhesus macaques. GD/16 vaccination elicited robust neutralizing, virus-specific immunoglobulin G antibodies and effective protection, but poor hemagglutination inhibition antibody titers. Furthermore, mouse and rhesus macaque serum raised against the previous H7N9 CVV A/Anhui/1/2013 (AH/13) were tested for its cross-reactivity to GD/16 virus. We found that although AH/13-immune serum has poor hemagglutination inhibition reactivity against GD/16 virus, AH/13 elicit efficient cross-neutralizing antibodies and in vivo protection against GD/16. Further studies showed that the hemagglutinin of GD/16 has strong receptor binding avidity, which might be associated with the decreased hemagglutination inhibition assay sensitivity. This study underscores the point that receptor binding avidity should be taken into account when performing quantitative interpretation of hemagglutination inhibition data. A combination of multiple serological assays is required for accurate vaccine evaluation and antigenic analysis of influenza viruses.
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Anticorpos Antivirais/sangue , Imunidade , Imunização Passiva , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Neutralizantes/sangue , Reações Cruzadas/imunologia , Feminino , Subtipo H7N9 do Vírus da Influenza A , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , VacinaçãoRESUMO
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has extensively and rapidly spread in the world, causing an outbreak of acute infectious pneumonia. However, no specific antiviral drugs or vaccines can be used. Phillyrin (KD-1), a representative ingredient of Forsythia suspensa, possesses anti-inflammatory, anti-oxidant, and antiviral activities. However, little is known about the antiviral abilities and mechanism of KD-1 against SARS-CoV-2 and human coronavirus 229E (HCoV-229E). PURPOSE: The study was designed to investigate the antiviral and anti-inflammatory activities of KD-1 against the novel SARS-CoV-2 and HCoV-229E and its potential effect in regulating host immune response in vitro. METHODS: The antiviral activities of KD-1 against SARS-CoV-2 and HCoV-229E were assessed in Vero E6 cells using cytopathic effect and plaque-reduction assay. Proinflammatory cytokine expression levels upon infection with SARS-CoV-2 and HCoV-229E infection in Huh-7 cells were measured by real-time quantitative PCR assays. Western blot assay was used to determine the protein expression of nuclear factor kappa B (NF-κB) p65, p-NF-κB p65, IκBα, and p-IκBα in Huh-7 cells, which are the key targets of the NF-κB pathway. RESULTS: KD-1 could significantly inhibit SARS-CoV-2 and HCoV-229E replication in vitro. KD-1 could also markedly reduce the production of proinflammatory cytokines (TNF-α, IL-6, IL-1ß, MCP-1, and IP-10) at the mRNA levels. Moreover, KD-1 could significantly reduce the protein expression of p-NF-κB p65, NF-κB p65, and p-IκBα, while increasing the expression of IκBα in Huh-7 cells. CONCLUSIONS: KD-1 could significantly inhibit virus proliferation in vitro, the up-regulated expression of proinflammatory cytokines induced by SARS-CoV-2 and HCoV-229E by regulating the activity of the NF-кB signaling pathway. Our findings indicated that KD-1 protected against virus attack and can thus be used as a novel strategy for controlling the coronavirus disease 2019.
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Anti-Inflamatórios/farmacologia , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Coronavirus Humano 229E/efeitos dos fármacos , Infecções por Coronavirus , Glucosídeos/farmacologia , NF-kappa B/metabolismo , Pandemias , Pneumonia Viral , Animais , COVID-19 , Chlorocebus aethiops , Coronavirus/efeitos dos fármacos , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Citocinas/metabolismo , Forsythia/química , Humanos , Fitoterapia , Extratos Vegetais/farmacologia , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , SARS-CoV-2 , Síndrome Respiratória Aguda Grave/virologia , Transdução de Sinais/efeitos dos fármacos , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
The rapid spread of coronavirus SARS-CoV-2 greatly threatens global public health but no prophylactic vaccine is available. Here, we report the generation of a replication-incompetent recombinant serotype 5 adenovirus, Ad5-S-nb2, carrying a codon-optimized gene encoding Spike protein (S). In mice and rhesus macaques, intramuscular injection with Ad5-S-nb2 elicits systemic S-specific antibody and cell-mediated immune (CMI) responses. Intranasal inoculation elicits both systemic and pulmonary antibody responses but weaker CMI response. At 30 days after a single vaccination with Ad5-S-nb2 either intramuscularly or intranasally, macaques are protected against SARS-CoV-2 challenge. A subsequent challenge reveals that macaques vaccinated with a 10-fold lower vaccine dosage (1 × 1010 viral particles) are also protected, demonstrating the effectiveness of Ad5-S-nb2 and the possibility of offering more vaccine dosages within a shorter timeframe. Thus, Ad5-S-nb2 is a promising candidate vaccine and warrants further clinical evaluation.
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Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Vacinas Virais/administração & dosagem , Adenoviridae/genética , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19 , Vacinas contra COVID-19 , Infecções por Coronavirus/imunologia , Relação Dose-Resposta Imunológica , Feminino , Células HEK293 , Humanos , Imunidade Celular , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia Viral/imunologia , Sistema Respiratório/patologia , Sistema Respiratório/virologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Sintéticas/administração & dosagemRESUMO
Zika virus (ZIKV) infection can cause neonatal microcephaly and neurological disorders. Currently, there is no designated drug for treating ZIKV infection and preventing neonatal microcephaly. In this study, we evaluated the effect of chloroquine, an anti-malaria drug, in ZIKV infected cells and mouse models. Chloroquine significantly inhibited ZIKV infection in multiple mammalian cell lines. Chloroquine treatment significantly improved the survival of ZIKV-infected 1-day old suckling SCID Beige mice and reduced viremia in adult SCID Beige mice. Importantly, chloroquine protected the fetus from maternal infection by reducing placenta to fetus viral transmission. We found that chloroquine exerts at least two mechanisms in protecting against ZIKV infection: 1) inhibiting endosomal disassembly of the internalized virus and thus reducing the release of viral RNA to the cytoplasm for replication; 2) inhibiting ZIKV RNA replication through blocking ZIKV induced autophagy. Our study suggests that chloroquine treatment warrants to be considered as a mitigation strategy for treating ZIKV infection and preventing ZIKV-associated microcephaly in pregnant women.
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Autofagia/efeitos dos fármacos , Cloroquina/farmacologia , Endossomos/efeitos dos fármacos , RNA Viral/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Infecção por Zika virus/prevenção & controle , Zika virus/efeitos dos fármacos , Animais , Antimaláricos/farmacologia , Linhagem Celular , Chlorocebus aethiops , Modelos Animais de Doenças , Transmissão de Doença Infecciosa/prevenção & controle , Feminino , Feto , Humanos , Camundongos , Camundongos SCID , Placenta , Gravidez , Células Vero , Viremia/tratamento farmacológico , Internalização do Vírus/efeitos dos fármacos , Infecção por Zika virus/transmissãoRESUMO
Rhesus macaques (Macaca mulatta) are used as a human-relevant animal species for the evaluation of vaccines and as a source for cloning monoclonal antibodies (mAbs) that are highly similar to human-derived antibodies. Although antibody-secreting plasmablasts in humans are well-defined and can be easily isolated for mAb cloning, it remains unclear whether the same phenotypic markers could be applied for isolating antibody-secreting plasmablasts from Chinese rhesus macaques. In this study, we evaluated a series of cell surface and intracellular markers and identified the phenotypic markers of plasmablasts in Chinese rhesus macaques as CD3-CD14-CD56-CD19-CD27-CD20-/lowCD80+HLA-DR+CD95+. After influenza virus vaccination, the plasmablasts in peripheral blood mononuclear cells (PBMCs) increased transiently, peaked at day 4-7 after booster vaccination and returned to nearly undetectable levels by day 14. Antigen-specific enzyme-linked immunosorbent spot (ELISPOT) assays confirmed that the majority of the plasmablasts could produce influenza virus-specific antibodies. These plasmablasts showed transcriptional characteristics similar to those of human plasmablasts. Using single-cell PCR for immunoglobulin heavy and light chains, most mAbs cloned from the CD3-CD14-CD56-CD19-CD27-CD20-/lowCD80+HLA-DR+CD95+ plasmablasts after vaccination exhibited specific binding to influenza virus. This study defined the phenotypic markers for isolating antibody-secreting plasmablasts from Chinese rhesus macaques, which has implications for efficient cloning of mAbs and for the evaluation of plasmablast response after vaccination or infection in Chinese rhesus macaques.
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Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Clonagem Molecular , Macaca mulatta , Fenótipo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Animais , Especificidade de Anticorpos/genética , Antígenos CD19/metabolismo , Biologia Computacional , ELISPOT , Imunoglobulina G/imunologia , Imunofenotipagem , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Análise de Célula Única , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , VacinaçãoRESUMO
Human adenoviruses type 4 (HAdV4) and 7 (HAdV7) are two major respiratory pathogens and sporadically cause outbreaks of acute respiratory diseases. The neutralizing antibody (nAb) response to these two adenoviruses in civilian populations, which is important for dissecting previous circulations and predicting potential outbreaks, remains largely unknown. In this study, we generated replication-competent HAdV4 and HAdV7 reporter viruses expressing secreted-alkaline-phosphatase (SEAP), and established neutralization assays to investigate the seroprevalence of pre-existing nAb in healthy volunteers from Hunan Province, southern China. The seropositivity rates are 58.4 and 63.8% for anti-HAdV4 nAb and anti-HAdV7 nAb, respectively. High nAb titers (> 1000) were frequently detected in HAdV4-seropositive individuals, whereas most HAdV7-seropositive volunteers had moderate nAb titers (201-1000). The seropositivity rates of anti-HAdV4 nAb and anti-HAdV7 nAb increase with age, with individuals younger than 20 exhibiting the lowest seropositivity rates. Both seropositivity rates and nAb titers are comparable between different sex groups. Notably, HAdV4-seropositive individuals tend to be HAdV7-seropositive and vice versa. Because HAdV4 antisera showed no neutralizing activity to HAdV7 whereas HAdV7 antisera cannot neutralize HAdV4, a subgroup of individuals might be susceptible to infection by HAdV4 and HAdV7 and thus generate nAb to both of them. These results revealed the continuous circulation of HAdV4 and HAdV7 and the lack of protective immunity in more than 35% of people, which emphasized the surveillance of these two HAdVs and the development of prophylactic vaccines.
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Ebolavirus vaccines based on several adenoviral vectors have been investigated in preclinical studies and clinical trials. The use of adenovirus serotype 2 as a vector for ebolavirus vaccine has not been reported. Herein, we generated rAd2-ZGP, a recombinant replication-incompetent adenovirus serotype 2 expressing codon-optimized Zaire ebolavirus glycoprotein, and evaluated its immunogenicity in mice and rhesus macaques. rAd2-ZGP induced significant antibody and cell-mediated immune responses at 2 weeks after a single immunization. The glycoprotein (GP)-specific immune responses could be further enhanced with a booster immunization. Compared to protein antigens, Zaire ebolavirus GP and Zaire ebolavirus-like particles, rAd2-ZGP could induce stronger cross-reactive antibody and cell-mediated immune responses to heterologous Sudan ebolavirus in mice and rhesus macaques. In rAd2-ZGP-immunized macaques, GP-specific CD8+ T cells could secret IFN-γ and IL-2, indicating a Th1-biased response. In adenovirus serotype 5 seropositive macaques, rAd2-ZGP could induce robust antibody and cell-mediated immune responses, suggesting that the efficacy of rAd2-ZGP is not affected by pre-existing immunity to adenovirus serotype 5. These results demonstrated that rAd2-ZGP can be considered an alternative ebolavirus vaccine for use in adenovirus serotype 5 seropositive subjects or as a sequential booster vaccine after the subjects have been immunized with a recombinant adenovirus serotype 5-based vaccine.
Assuntos
Adenoviridae/genética , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Imunidade Celular , Vacinas Virais/imunologia , Adenoviridae/metabolismo , Animais , Modelos Animais de Doenças , Ebolavirus/genética , Feminino , Glicoproteínas/administração & dosagem , Glicoproteínas/genética , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Doença pelo Vírus Ebola/virologia , Humanos , Interferon gama/imunologia , Interleucina-2/imunologia , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologia , Vacinação , Proteínas Virais/administração & dosagem , Proteínas Virais/genética , Proteínas Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Highly pathogenic avian influenza A (H5N6) virus has been circulating in poultry since 2013 and causes sporadic infections and fatalities in humans. Due to the re-occurrence and continuous evolution of this virus subtype, there is an urgent need to better understand the pathogenicity of the H5N6 virus and to identify effective preventative and therapeutic strategies. We established a mouse model to evaluate the virulence of H5N6 A/Guangzhou/39715/2014 (H5N6/GZ14), which was isolated from an infected patient. BALB/c mice were inoculated intranasally with H5N6/GZ14 and monitored for morbidity, mortality, cytokine production, lung injury, viral replication, and viral dissemination to other organs. H5N6/GZ14 is highly pathogenic and can kill 50% of mice at a very low infectious dose of 5 plaque-forming units (pfu). Infection with H5N6/GZ14 showed rapid disease progression, viral replication to high titers in the lung, a strongly induced pro-inflammatory cytokine response, and severe lung injury. Moreover, infectious H5N6/GZ14 could be detected in the heart and brain of the infected mice. We also demonstrated that anti-influenza polyclonal antibodies generated by immunizing rhesus macaques could protect mice from lethal infection. Our results provide insights into the pathogenicity of the H5N6 human isolate.
Assuntos
Anticorpos Antivirais/administração & dosagem , Vírus da Influenza A/patogenicidade , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Animais , Citocinas/genética , Citocinas/metabolismo , Feminino , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Influenza Humana/patologia , Pulmão/patologia , Pulmão/virologia , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Virulência , Replicação Viral/efeitos dos fármacosRESUMO
Re-emerging human adenoviruses type 14 (HAdV14) and 55 (HAdV55) represent two highly virulent adenoviruses. The neutralizing antibody (nAb) responses elicited by infection or immunization remain largely unknown. Herein, we generated hexon-chimeric HAdV14 viruses harboring each single or entire hexon hyper-variable-regions (HVR) from HAdV55, and determined the neutralizing epitopes of human and mouse nAbs. In human sera, hexon-targeting nAbs are type-specific and mainly recognize HVR2, 5, and 7. Fiber-targeting nAbs are only detectable in sera cross-neutralizing HAdV14 and HAdV55 and contribute substantially to cross-neutralization. Penton-binding antibodies, however, show no significant neutralizing activities. In mice immunized with HAdV14 or HAdV55, a single immunization mainly elicited hexon-specific nAbs, which recognized HAdV14 HVR1, 2, and 7 and HAdV55 HVR1 and 2, respectively. After a booster immunization, cross-neutralizing fiber-specific nAbs became detectable. These results indicated that hexon elicits type-specific nAbs whereas fiber induces cross-neutralizing nAbs to HAdV14 and HAdV55, which are of significance in vaccine development.