RESUMO
BACKGROUND: Ebola virus disease (EVD) survivors experience ocular sequelae including retinal lesions, cataracts, and vision loss. While monoclonal antibodies targeting the Ebola virus glycoprotein (EBOV-GP) have shown promise in improving prognosis, their effectiveness in mitigating ocular sequelae remains uncertain. METHODS: We developed and characterized a BSL-2-compatible immunocompetent mouse model to evaluate therapeutics targeting EBOV-GP by inoculating neonatal mice with vesicular stomatitis virus expressing EBOV-GP (VSV-EBOV). To examine the impact of anti-EBOV-GP antibody treatment on acute retinitis and ocular sequelae, VSV-EBOV-infected mice were treated with polyclonal antibodies or monoclonal antibody preparations with antibody-dependent cellular cytotoxicity (ADCC-mAb) or neutralizing activity (NEUT-mAb). FINDINGS: Treatment with all anti-EBOV-GP antibodies tested dramatically reduced viremia and improved survival. Further, all treatments reduced the incidence of cataracts. However, NEUT-mAb alone or in combination with ADCC-mAb reduced viral load in the eyes, downregulated the ocular immune and inflammatory responses, and minimized retinal damage more effectively. INTERPRETATION: Anti-EBOV-GP antibodies can improve survival among EVD patients, but improved therapeutics are needed to reduce life altering sequelae. This animal model offers a new platform to examine the acute and long-term effect of the virus in the eye and the relative impact of therapeutic candidates targeting EBOV-GP. Results indicate that even antibodies that improve systemic viral clearance and survival can differ in their capacity to reduce acute ocular inflammation, and long-term retinal pathology and corneal degeneration. FUNDING: This study was partly supported by Postgraduate Research Fellowship Awards from ORISE through an interagency agreement between the US DOE and the US FDA.
Assuntos
Anticorpos Antivirais , Modelos Animais de Doenças , Ebolavirus , Doença pelo Vírus Ebola , Animais , Camundongos , Ebolavirus/imunologia , Ebolavirus/patogenicidade , Doença pelo Vírus Ebola/virologia , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/farmacologia , Humanos , Carga Viral , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Proteínas do Envelope Viral/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Citotoxicidade Celular Dependente de AnticorposRESUMO
Perineuronal nets (PNNs) form a specialized extracellular matrix that predominantly surrounds parvalbumin (PV)-expressing GABAergic inhibitory interneurons and help regulate neuronal activity. Their formation early in the postnatal period is regulated by neuronal signaling and glial activation raising concerns that part of the long-term effects ascribed to perinatal viral infections could be mediated by altered PNN formation. Previously, we developed a model of neonatal Zika virus (ZIKV) infection where mice have lifelong neurological sequelae that includes motor disfunction and reduced anxiety coupled with a persistent low-grade expression in proinflammatory markers despite resolving the acute infection. Here, we demonstrate that ZIKV infection to P1 neonatal mice results in a reduction of PNN formation during the acute disease with significant reduction in Wisteria floribunda agglutinin (WFA) staining at the peak of infection [15 days post infection (dpi)] that persisted after the symptoms resolved (30 dpi). At 60 dpi, when there is residual inflammation in the CNS, the number of WFA+ cells and the level of WFA staining as well as levels of aggrecan and brevican in the brains of convalescent mice were not different from those in uninfected controls, however, there was increased frequency of PNNs with an immature phenotype. Over time the impact of the perinatal infection became less evident and there were no clear differences in PNN morphology between the groups at 1 year post infection. Of note, the reduction in PNNs during acute ZIKV infection was not associated with decreased mRNA levels of aggrecan or brevican, but increased levels of degraded aggrecan and brevican indicating increased PNN degradation. These changes were associated with increased expression of matrix metalloproteinase 12 (MMP12) and MMP19, but not MMP9, a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) or ADAMTS5. Together our findings indicate that infection at the time of PNN development interferes with PNN formation, but the nets can reform once the infection and inflammation subside.
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Unintended immunogenicity can affect the safety and efficacy of therapeutic proteins and peptides, so accurate assessments of immunogenicity risk can aid in the selection, development, and regulation of biologics. Product- and process- related impurities can act as adjuvants that activate the local or systemic innate immune response increasing the likelihood of product immunogenicity. Thus, assessing whether products have innate immune response modulating impurities (IIRMI) is a key component of immunogenicity risk assessments. Identifying trace levels of individual IIRMI can be difficult and testing individually for all potential impurities is not feasible. Therefore, to mitigate the risk, cell-based assays that use human blood cells or monocyte-macrophage reporter cell lines are being developed to detect minute quantities of impurities capable of eliciting innate immune activation. As these are cell-based assays, there is concern that excipients could blunt the cell responses, masking the presence of immunogenic IIRMI. Here, we explore the impact of frequently used excipients (non-ionic detergents, sugars, amino acids, bulking agents) on the sensitivity of reporter cell lines (THP-1- and RAW-Blue cells) and fresh human blood cells to detect purified TLR agonists as model IIRMI. We show that while excipients do not modulate the innate immune response elicited by TLR agonists in vivo, they can impact on the sensitivity of cell-based IIRMI assays. Reduced sensitivity to detect LPS, FSL-1, and other model IIRMI was also evident when testing 3 different recombinant drug products, product A (a representative mAb), B (a representative growth factor), C (a representative peptide), and their corresponding formulations. These results indicate that product formulations need to be considered when developing and validating cell-based assays for assessing clinically relevant levels of IIRMI in therapeutic proteins. Optimization of reporter cells, culture conditions and drug product concentration appear to be critical to minimize the impact of excipients and attain sensitive and reproducible assays.
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Produtos Biológicos , Excipientes , Adjuvantes Imunológicos , Amino Açúcares , Detergentes , Excipientes/química , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intercelular , Lipopolissacarídeos , PeptídeosRESUMO
Since first reported in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is rapidly acquiring mutations, particularly in the spike protein, that can modulate pathogenicity, transmission and antibody evasion leading to successive waves of COVID19 infections despite an unprecedented mass vaccination necessitating continuous adaptation of therapeutics. Small animal models can facilitate understanding host-pathogen interactions, target selection for therapeutic drugs, and vaccine development, but availability and cost of studies in BSL3 facilities hinder progress. To generate a BSL2-compatible in vivo system that specifically recapitulates spike protein mediated disease we used replication competent, GFP tagged, recombinant Vesicular Stomatitis Virus where the VSV glycoprotein was replaced by the SARS-CoV-2 spike protein (rVSV-SARS2-S). We show that infection requires hACE2 and challenge of neonatal but not adult, K18-hACE2 transgenic mice (hACE2tg) leads to productive infection of the lungs and brains. Although disease progression was faster in SARS-CoV-2 infected mice, infection with both viruses resulted in neuronal infection and encephalitis with increased expression of Interferon-stimulated Irf7, Bst2, Ifi294, as well as CxCL10, CCL5, CLC2, and LILRB4, and both models were uniformly lethal. Further, prophylactic treatment targeting the Spike protein (Receptor Binding Domain) with antibodies resulted in similar levels of protection from lethal infection against rVSV-SARS2-S and SARS-CoV-2 viruses. Strikingly, challenge of neonatal hACE2tg mice with SARS-CoV-2 Variants of Concern (SARS-CoV-2-α, -ß, Ï, or Δ) or the corresponding rVSV-SARS2-S viruses (rVSV-SARS2-Spike-α, rVSV-SARS2-Spike-ß, rVSV-SARS2-Spike-Ï or rVSV-SARS2-Spike-Δ) resulted in increased lethality, suggesting that the Spike protein plays a key role in determining the virulence of each variant. Thus, we propose that rVSV-SARS2-S virus can be used to understand the effect of changes to SARS-CoV-2 spike protein on infection and to evaluate existing or experimental therapeutics targeting spike protein of current or future VOC of SARS-CoV-2 under BSL-2 conditions.
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COVID-19 , Glicoproteína da Espícula de Coronavírus , Animais , Modelos Animais de Doenças , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Receptores Imunológicos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
An early inflammatory insult is the most recognized risk factor associated with neurodevelopmental psychiatric disorders, even more so than genetic variants. Notably, complement component 4 (C4), a molecule involved in inflammatory responses, has been strongly associated with schizophrenia (SZ) and its role in other neurodevelopmental disorders, such as autism (ASD), is an area of active investigation. However, while C4 in SZ has been implicated in the context of synaptic pruning, little is known about its neuroinflammatory role. The subventricular zone (SVZ) is a region heavily involved in neurodevelopment and neuroimmune interactions through the lifespan; thus, it is a region wherein C4 may play a vital role in disease pathology. Using in situ hybridization with radioactive riboprobes and RNAscope, we identified robust astrocytic expression of C4 in the SVZ and in the septum pellucidum. C4 was also expressed in ependyma, neurons, and Ki67+ progenitor cells. Examination of mRNA levels showed elevated C4 in both ASD and SZ, with higher expression in SZ compared to controls. Targeted transcriptomic analysis of inflammatory pathways revealed a strong association of complement system genes with SZ, and to a lesser extent, ASD, as well as generalized immune dysregulation without a strong association with known infectious pathways. Analysis of differentially expressed genes (DEGs) showed that ASD DEGs were enriched in adaptive immune system functions such as Th cell differentiation, while SZ DEGs were enriched in innate immune system functions, including NF-κB and toll like receptor signaling. Moreover, the number of Ki67+ cells was significantly higher in ASD compared to SZ and controls. Taken together, these results support a role for C4 into inflammatory-neuroimmune dysregulation observed in SZ and ASD pathology.
Assuntos
Transtorno do Espectro Autista , Complemento C4 , Esquizofrenia , Transtorno do Espectro Autista/genética , Complemento C4/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Ventrículos Laterais/patologia , NF-kappa B/metabolismo , RNA MensageiroRESUMO
Immune cells express an array of inhibitory checkpoint receptors that are upregulated upon activation and limit tissue damage associated with excessive response to pathogens or allergens. Mouse leukocyte immunoglobulin like receptor B4 (LILRB4), also known as glycoprotein 49B (gp49B), is an inhibitory checkpoint receptor constitutively expressed in myeloid cells and upregulated in B cells, T cells, and NK cells upon activation. Here, we report that expression of LILRB4, which binds Zika virus (ZIKV), was increased in microglia and myeloid cells infiltrating the brains of neonatal mice with ZIKV-associated meningoencephalitis. Importantly, while C57BL/6 mice developed transient neurological symptoms but survived infection, mice lacking LILRB4/gp49B (LILRB4 KO) exhibited more severe signs of neurological disease and succumbed to disease. Their brains showed increased cellular infiltration but reduced control of viral burden. The reduced viral clearance was associated with altered NK cell function in the absence of LILRB4/gp49B. In naive animals, this manifested as reduced granzyme B responses to stimulation, but in ZIKV-infected animals, NK cells showed phenotypic changes that suggested altered maturation, diminished glucose consumption, reduced IFN-γ and granzyme B production, and impaired cytotoxicity. Together, our data reveal LILRB4/gp49B as an important regulator of NK cell function during viral infections.
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Linfócitos B/imunologia , Regulação da Expressão Gênica , Células Matadoras Naturais/imunologia , Glicoproteínas de Membrana/genética , Receptores Imunológicos/genética , Infecção por Zika virus/imunologia , Zika virus , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Modelos Animais de Doenças , Feminino , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Masculino , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA/genética , Receptores Imunológicos/biossíntese , Receptores Imunológicos/metabolismo , Linfócitos T/metabolismo , Infecção por Zika virus/genética , Infecção por Zika virus/metabolismoRESUMO
Ebola virus (EBOV) infections cause haemorrhagic fever, multi-organ failure and death, and survivors can experience neurological sequelae. Licensing of monoclonal antibodies targeting EBOV glycoprotein (EBOV-GP) improved its prognosis, however, this treatment is primarily effective during early stages of disease and its effectiveness in reducing neurological sequela remains unknown. Currently, the need for BSL4 containment hinders research and therapeutic development; development of an accessible BSL-2 in vivo mouse model would facilitate preclinical studies to screen and select therapeutics. Previously, we have shown that a subcutaneous inoculation with replicating EBOV-GP pseudotyped vesicular stomatitis virus (rVSVΔG-EBOV-GP or VSV-EBOV) in neonatal mice causes transient viremia and infection of the mid and posterior brain resulting in overt neurological symptoms and death. Here, we demonstrate that the model can be used to test therapeutics that target the EBOV-GP, by using an anti-EBOV-GP therapeutic (SAB-139) previously shown to block EBOV infection in mice and primates. We show that SAB-139 treatment decreases the severity of neurological symptoms and improves survival when administered before (1 day prior to infection) or up to 3â dpi, by which time animals have high virus titres in their brains. Improved survival was associated with reduced viral titres, microglia loss, cellular infiltration/activation, and inflammatory responses in the brain. Interestingly, SAB-139 treatment significantly reduced the severe VSV-EBOV-induced long-term neurological sequalae although convalescent mice showed modest evidence of abnormal fear responses. Together, these data suggest that the neonatal VSV-EBOV infection system can be used to facilitate assessment of therapeutics targeting EBOV-GP in the preclinical setting.
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Anticorpos Monoclonais/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Ebolavirus/imunologia , Doença pelo Vírus Ebola/tratamento farmacológico , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Neutralizantes/administração & dosagem , Modelos Animais de Doenças , Ebolavirus/genética , Ebolavirus/fisiologia , Doença pelo Vírus Ebola/virologia , Humanos , Camundongos Endogâmicos C57BL , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/fisiologia , Proteínas do Envelope Viral/genéticaRESUMO
The neurodevelopmental defects associated with ZIKV infections early in pregnancy are well documented, however the potential defects and long-term consequences associated with milder infections in late pregnancy and perinatal period are less well understood. To model these, we challenged 1 day old (P1) immunocompetent C57BL/6 mice with ZIKV. The animals developed a transient neurological syndrome including unsteady gait, kinetic tremors, severe ataxia and seizures 10-15 days post-infection (dpi) but symptoms subsided after a week, and most animals survived. Despite apparent recovery, MRI of convalescent mice show reduced cerebellar volume that correlates with altered coordination and motor function as well as hyperactivity and impulsivity. Persistent mRNA levels of pro-inflammatory genes including Cd80, Il-1α, and Ifn-γ together with Cd3, Cd8 and perforin (PrfA), suggested persistence of low-grade inflammation. Surprisingly, the brain parenchyma of convalescent mice harbor multiple small discrete foci with viral antigen, active apoptotic processes in neurons, and cellular infiltrates, surrounded by activated astrocytes and microglia as late as 1-year post-infection. Detection of negative-sense strand viral RNA and isolation of infectious virus derived from these convalescent mice by blinded passage in Vero cells confirmed long-term persistence of replicating ZIKV in CNS of convalescent mice. Although the infection appears to persist in defined reservoirs within CNS, the resulting inflammation could increase the risk of neurodegenerative disorders. This raises concern regarding possible long-term effects in asymptomatic children exposed to the virus and suggests that long-term neurological and behavioral monitoring as well as anti-viral treatment to clear virus from the CNS may be useful in patients exposed to ZIKV at an early age.
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Inflamação/fisiopatologia , Infecção por Zika virus/complicações , Infecção por Zika virus/fisiopatologia , Animais , Encéfalo/virologia , Chlorocebus aethiops , Modelos Animais de Doenças , Feminino , Inflamação/complicações , Camundongos , Camundongos Endogâmicos C57BL , Microcefalia/complicações , Microcefalia/virologia , Neurônios/virologia , Gravidez , Complicações Infecciosas na Gravidez/virologia , Células Vero , Zika virus/imunologia , Zika virus/metabolismo , Zika virus/patogenicidade , Infecção por Zika virus/virologiaRESUMO
Arboviruses including alphavirus are responsible for most emerging infectious diseases worldwide. Recent outbreaks of chikungunya virus serve as a stark reminder to their pathogenic potential. There are no vaccines or therapeutics currently available to contain alphavirus outbreaks. In this study we evaluated the effect of immunomodulatory CpG ODN on the clinical progression of neurotropic Sindbis virus infection. Neonatal C57Bl-6 mice challenged with Sindbis virus AR339 (25 PFU Subcutaneous) infect neurons in the CNS leading to the development of ataxia, seizures, paralysis, and death. We show that systemic administration of CpG ODN modulates the cytokine and chemokine gene expression levels in the CNS and ultimately protects neonatal mice from lethal neurotropic infection. The protection conferred by CpG ODN is controlled by innate immune response and T and B cells were dispensable. Further, protection required Type I, Type II interferons, and TNF as well as functional NK cells, but did not involve iNOS. This study confirms that administration of innate immune modulators can be used as a strategy to boost host innate immune responses and protect against neurotropic viruses reducing their pathogenic footprint.
Assuntos
Infecções por Alphavirus/prevenção & controle , Encefalite Viral/prevenção & controle , Interferons/fisiologia , Células Matadoras Naturais/fisiologia , Oligodesoxirribonucleotídeos/uso terapêutico , Sindbis virus , Fator de Necrose Tumoral alfa/fisiologia , Animais , Chlorocebus aethiops , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/fisiologia , Células VeroRESUMO
INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death globally. In outpatient care, the self-management of COPD is essential, but patient adherence to this remains suboptimal. The objective of this study is to examine whether an innovative mobile health (mHealth)-enabled care programme (MH-COPD) will improve the patient self-management and relevant health outcomes. METHODS AND ANALYSIS: A prospective open randomised controlled trial has been designed. In the trial, patients with COPD will be recruited from The Prince Charles Hospital, Brisbane, Australia. They will then be randomised to participate in either the MH-COPD intervention group (n=50 patients), or usual care control group (UC-COPD) (n=50 patients) for 6 months. The MH-COPD programme has been designed to integrate an mHealth system within a clinical COPD care service. In the programme, participants will use a mHealth application at home to review educational videos, monitor COPD symptoms, use an electronic action plan, modify the risk factors of cigarette smoking and regular physical activity, and learn to use inhalers optimally. All participants will be assessed at baseline, 3 months and 6 months. The primary outcomes will be COPD symptoms and quality of life. The secondary outcomes will be patient adherence, physical activity, smoking cessation, use of COPD medicines, frequency of COPD exacerbations and hospital readmissions, and user experience of the mobile app. ETHICS AND DISSEMINATION: The clinical trial has been approved by The Prince Charles Hospital Human Research Ethics Committee (HREC/16/QPCH/252). The recruitment and follow-up of the trial will be from January 2019 to December 2020. The study outcomes will be disseminated according to the Consolidated Standards of Reporting Trials statement through a journal publication, approximately 6 months after finishing data collection. TRIAL REGISTRATION NUMBER: ACTRN12618001091291.
Assuntos
Doença Pulmonar Obstrutiva Crônica/terapia , Autogestão/educação , Smartphone , Telemedicina/métodos , Promoção da Saúde/métodos , Humanos , Educação de Pacientes como Assunto/métodos , Estudos Prospectivos , Qualidade de Vida , Projetos de Pesquisa , Autocuidado/métodos , Abandono do Hábito de Fumar/métodosRESUMO
Zaire Ebola virus (ZEBOV) survivors experience visual and CNS sequelae that suggests the ZEBOV glycoprotein can mediate neurotropism. Replication-competent rVSVΔG-ZEBOV-GP vaccine candidate is generally well tolerated; however, its potential neurotropism requires careful study. Here, we show that a single inoculation of rVSVΔG-ZEBOV-GP virus in neonatal C57BL/6 mice results in transient viremia, neurological symptoms, high viral titers in eyes and brains, and death. rVSVΔG-ZEBOV-GP infects the inner layers of the retina, causing severe retinitis. In the cerebellum, rVSVΔG-ZEBOV-GP infects neurons in the granular and Purkinje layers, resulting in progressive foci of apoptosis and neurodegeneration. The susceptibility to infection is not due to impaired type I IFN responses, although MDA5-/-, IFNß-/-, and IFNAR1-/- mice have accelerated mortality. However, boosting interferon levels by co-administering poly(I:C) reduces viral titers in CNS and improves survival. Although these data should not be directly extrapolated to humans, they challenge the hypothesis that VSV-based vaccines are non-neurotropic.
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Sistema Nervoso Central/patologia , Doenças Neurodegenerativas/genética , Retina/patologia , Animais , Animais Recém-Nascidos , Apoptose , Humanos , Camundongos , NeurôniosRESUMO
Therapeutic proteins can induce immune responses that affect their safety and efficacy. Product aggregates and innate immune response modulating impurities (IIRMI) are risk factors of product immunogenicity. In this study, we use Intravenous Immunoglobulin (IVIG), Avastin, and Human Serum Albumin (HSA) to explore whether increased aggregates activate innate immune cells or modify the response to IIRMI. We show that increased aggregates (shaken or stirred) in IVIG and Avastin, but not HSA, induced activation of MAPKs (pp38, pERK and pJNK) and transcription of immune-related genes including IL8, IL6, IL1ß, CSF1, CCL2, CCL7, CCL3, CCL24, CXCL2, IRAK1, EGR2, CEBPß, PPARg and TNFSF15 in human PBMC. The immunomodulatory effect was primarily mediated by FcγR, but not by TLR. Interestingly, increased aggregates in IVIG or Avastin magnified innate immune responses to TLR2/4 agonists, but diminished responses to TLR3/9 agonists. This study shows that IIRMI and aggregates can modify the activity of immune cells potentially modifying the milieu where the products are delivered highlighting the complex interplay of different impurities on product immunogenicity risk. Further, we show that aggregates could modify the sensitivity of PBMC-based assays designed to detect IIRMI. Understanding and managing immunogenicity risk is a critical component of product development and regulation.
Assuntos
Imunidade Inata/imunologia , Imunoglobulinas Intravenosas/imunologia , Albumina Sérica Humana/imunologia , Animais , Formação de Anticorpos/imunologia , Bevacizumab/imunologia , Células Cultivadas , Células HEK293 , Humanos , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/imunologia , Transcrição Gênica/imunologiaRESUMO
Zika-infected patients can have eye involvement ranging from mild conjunctivitis to severe chorioretinal lesions, however the possible long-term sequelae of infection and timeline to recovery remain unknown. Here we describe the partial recovery of chorioretinal lesions in an immunocompetent patient diagnosed with bilateral posterior uveitis associated with Zika infection and show that some lesions resolved with focal atrophy evident as pigmentary changes on funduscopy. To better understand the progression of the lesions and correlate the changes in fundus imaging with local viral load, immune responses, and retinal damage, we developed a symptomatic mouse model of ocular Zika virus infection. Imaging of the fundus revealed multiple hypopigmentary patches indicative of chorioretinal degeneration as well as thinning of the retina that mirror the lesions in patients. Microscopically, the virus primarily infected the optic nerve, retinal ganglion cells, and inner nuclear layer cells, showing thinning of the outer plexiform layer. During acute infection, the eyes showed retinal layer disorganization, retinitis, vitritis, and focal choroiditis, with mild cellular infiltration and increased expression of tumor necrosis factor, interferon-γ, granzyme B, and perforin. Focal areas of gliosis and retinal degeneration persisted 60 dpi. The model recapitulates features of ZIKA infections in patients and should help elucidate the mechanisms underlying the damage to the eyes and aid in the development of effective therapeutics.
Assuntos
Coriorretinite/virologia , Retina/virologia , Uveíte Posterior/virologia , Infecção por Zika virus/patologia , Zika virus/isolamento & purificação , Adulto , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Conjuntivite Viral/virologia , Humanos , Células Matadoras Naturais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nervo Óptico/virologia , Células Ganglionares da Retina/virologiaRESUMO
We applied an in vitro selection approach using two different plant lectins that bind to α2,3- or α2,6-linked sialic acids to determine which genetic changes of the A/California/04/09 (H1N1) virus alter hemagglutinin (HA) receptor binding toward α2,3- or α2,6-linked glycans. Consecutive passages of the A/California/04/09 virus with or without lectins in human lung epithelial Calu-3 cells led to development of three HA1 amino acid substitutions, N129D, G155E, and S183P, and one mutation in the neuraminidase (NA), G201E. The S183P mutation significantly increased binding to several α2,6 SA-linked glycans, including YDS, 6'SL(N), and 6-Su-6'SLN, compared to the wild-type virus (↑3.6-fold, P < 0.05). Two other HA1 mutations, N129D and G155E, were sufficient to significantly increase binding to α2,6-linked glycans, 6'SLN and 6-Su-6'SLN, compared to S183P (↑4.1-fold, P < 0.05). These HA1 mutations also increased binding affinity for 3'SLN glycan compared to the wild-type virus as measured by Biacore surface plasmon resonance method. In addition, the HA1 N129D and HA1 G155E substitutions were identified as antigenic mutations. Furthermore, the G201E mutation in NA reduced the NA enzyme activity (↓2.3-fold). These findings demonstrate that the A/California/04/09 (H1N1) virus can acquire enhanced receptor affinity for both α2,3- and α2,6-linked sialic receptors under lectin-induced selective pressure. Such changes in binding affinity are conferred by selection of beneficial HA1 mutations that affect receptor specificity, antigenicity, and/or functional compatibility with the NA protein.
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Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Neuraminidase/genética , Neuraminidase/metabolismo , Lectinas de Plantas/metabolismo , Receptores Virais/fisiologia , Substituição de Aminoácidos , Animais , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/metabolismo , Linhagem Celular , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Células Madin Darby de Rim Canino , Neuraminidase/química , Polissacarídeos/química , Polissacarídeos/genética , Polissacarídeos/metabolismo , Ligação Proteica , Seleção Genética , Ressonância de Plasmônio de SuperfícieRESUMO
The inability of infants to mount proper follicular helper T (TFH) cell response renders this age group susceptible to infectious diseases. Initial instruction of T cells by antigen presenting cells and subsequent differentiation into TFH cells are controlled by T cell receptor signal strength, co-stimulatory molecules and cytokines such as IL-6 and IL-21. In immunized adults, IL-6 promotes TFH development by increasing the expression of CXCR5 and the TFH master transcription factor, B cell lymphoma 6. Underscoring the importance of IL-6 in TFH generation, we found improved antibody responses accompanied by increased TFH cells and decreased follicular regulatory helper T (TFR) cells, a Foxp3 expressing inhibitory CD4+ T cell occupying the germinal center (GC), when a tetanus toxoid conjugated pneumococcal polysaccharide type 14 vaccine was injected in adult mice together with IL-6. Paradoxically, in neonates IL-6 containing PPS14-TT vaccine suppressed the already impaired TFH development and antibody responses in addition to increasing TFR cell population. Supporting the diminished TFH development, we detected lower frequency of phospho-STAT-3+ TFH in immunized neonatal T cells after IL-6 stimulation than adult cells. Moreover, IL-6 induced more phospho-STAT-3+ TFR in neonatal cells than adult cells. We also measured lower expression of IL-6R on TFH cells and higher expression on TFR cells in neonatal cells than adult cells, a possible explanation for the difference in IL-6 induced signaling in different age groups. Supporting the flow cytometry findings, microscopic examination revealed the localization of Treg cells in the splenic interfollicular niches of immunized adult mice compared to splenic follicles in neonatal mice. In addition to the limitations in the formation of IL-21 producing TFH cells, neonatal mice GC B cells also expressed lower levels of IL-21R in comparison to the adult mice cells. These findings point to diminished IL-6 activity on neonatal TFH cells as an underlying mechanism of the increased TFR: TFH ratio in immunized neonatal mice.
Assuntos
Centro Germinativo/imunologia , Imunogenicidade da Vacina , Interleucina-6/imunologia , Vacinas Meningocócicas/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Fatores Etários , Animais , Animais Recém-Nascidos , Diferenciação Celular/imunologia , Feminino , Centro Germinativo/citologia , Centro Germinativo/metabolismo , Subunidade alfa de Receptor de Interleucina-21/imunologia , Subunidade alfa de Receptor de Interleucina-21/metabolismo , Interleucina-6/administração & dosagem , Interleucinas/imunologia , Interleucinas/metabolismo , Vacinas Meningocócicas/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Receptores de Interleucina-6/imunologia , Receptores de Interleucina-6/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Toxoide Tetânico/administração & dosagem , Toxoide Tetânico/imunologia , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/imunologiaRESUMO
Healthcare is currently being transformed by the introduction of genomic sequencing - a major advancement in personalised medicine. This advent provides new opportunities for clinicians to use genomic data in decision making about patient diagnosis and treatment, but this can only be achieved through access to data and support in its use. Engaging with clinicians in the development of decision support tools will optimise relevance and adoption of genomic sequencing in healthcare. In this study, existing data from clinician workshops and interviews together with horizon scanning of relevant technologies were used to define clinician portal specifications. We describe a preliminary structure of a decision support tool for use by clinicians and the manner in which the technology may be evaluated.
Assuntos
Mapeamento Cromossômico , Técnicas de Apoio para a Decisão , Genômica , Tomada de Decisões , HumanosRESUMO
Neonates are at increased risk of viral encephalopathies that can result in neurological dysfunction, seizures, permanent disability and even death. The neurological damage results from the combined effect of the virus and the immune response it elicits, thus finding tools to facilitate viral clearance from central nervous system (CNS) while minimizing neuron damage remains a critical challenge. Neonatal mice inoculated intraperitoneally with Tacaribe virus (TCRV) develop seizures, hindlimb paralysis and death within 15 days of inoculation. TCRV localizes to the CNS within days of challenge, primarily infecting astrocytes in the cerebellum and brain stem. We show that infection leads to inflammation, T cell and monocyte infiltration into the cerebellar parenchyma, apoptosis of astrocytes, neuronal degeneration and loss of Purkinje cells. Infiltrating antigen-specific T cells fail to clear the virus but drive the disease, as T-cell-deficient CD3É KO mice survive TCRV infection with minimal inflammation or clinical manifestations despite no difference in CNS viral loads in comparison with T-cell sufficient mice. CD8+ T cells drive the pathology, which even in the absence of CD4+ T-cell help, infiltrate the parenchyma and mediate the apoptotic loss of cerebellar astrocytes, neurodegeneration and loss of Purkinje cells resulting in loss of balance, paralysis and death. CD4+ T cells are also pathogenic inducing gliosis and inflammation in the cerebellum and cerebrum that are associated with wasting and death several weeks after CD4+ T-cell transfer. These data demonstrate distinct pathogenic effects of CD4+ and CD8+ T cells and identify them as possible therapeutic targets.
Assuntos
Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/virologia , Arenavirus/fisiologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Meningoencefalite/imunologia , Meningoencefalite/virologia , Animais , Animais Recém-Nascidos , Apoptose , Infecções por Arenaviridae/patologia , Astrócitos/patologia , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Gliose/patologia , Meningoencefalite/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/patologia , Degeneração Neural/patologia , Neurônios/patologia , Células de Purkinje/patologia , Linfócitos T/imunologiaRESUMO
The recent spread of Zika virus (ZIKV) and its association with increased rates of Guillain Barre and other neurological disorders as well as congenital defects that include microcephaly has created an urgent need to develop animal models to examine the pathogenesis of the disease and explore the efficacy of potential therapeutics and vaccines. Recently developed infection models for ZIKV utilize mice defective in interferon responses. In this study we establish and characterize a new model of peripheral ZIKV infection using immunocompetent neonatal C57BL/6 mice and compare its clinical progression, virus distribution, immune response, and neuropathology with that of C57BL/6-IFNAR KO mice. We show that while ZIKV infected IFNAR KO mice develop bilateral hind limb paralysis and die 5-6 days post-infection (dpi), immunocompetent B6 WT mice develop signs of neurological disease including unsteady gait, kinetic tremors, severe ataxia and seizures by 13 dpi that subside gradually over 2 weeks. Immunohistochemistry show viral antigen predominantly in cerebellum at the peak of the disease in both models. However, whereas IFNAR KO mice showed infiltration by neutrophils and macrophages and higher expression of IL-1, IL-6 and Cox2, B6 WT mice show a cellular infiltration in the CNS composed predominantly of T cells, particularly CD8+ T cells, and increased mRNA expression levels of IFNg, GzmB and Prf1 at peak of disease. Lastly, the CNS of B6 WT mice shows evidence of neurodegeneration predominantly in the cerebellum that are less prominent in mice lacking the IFN response possibly due to the difference in cellular infiltrates and rapid progression of the disease in that model. The development of the B6 WT model of ZIKV infection will provide insight into the immunopathology of the virus and facilitate assessments of possible therapeutics and vaccines.
Assuntos
Encéfalo/patologia , Modelos Animais de Doenças , Degeneração Neural/virologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/patologia , Animais , Animais Recém-Nascidos , Citometria de Fluxo , Imunofluorescência , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/imunologia , Degeneração Neural/patologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Dimerization of Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) heterodimers is critical for both MyD88- and TIR-domain-containing adapter-inducing IFN-ß (TRIF)-mediated signaling pathways. Recently, Zanoni et al. [(2011) Cell 147(4):868-880] reported that cluster of differentiation 14 (CD14) is required for LPS-/Escherichia coli- induced TLR4 internalization into endosomes and activation of TRIF-mediated signaling in macrophages. We confirmed their findings with LPS but report here that CD14 is not required for receptor endocytosis and downstream signaling mediated by TLR4/MD2 agonistic antibody (UT12) and synthetic small-molecule TLR4 ligands (1Z105) in murine macrophages. CD14 deficiency completely ablated the LPS-induced TBK1/IRF3 signaling axis that mediates production of IFN-ß in murine macrophages without affecting MyD88-mediated signaling, including NF-κB, MAPK activation, and TNF-α and IL-6 production. However, neither the MyD88- nor TRIF-signaling pathways and their associated cytokine profiles were altered in the absence of CD14 in UT12- or 1Z105-treated murine macrophages. Eritoran (E5564), a lipid A antagonist that binds the MD2 "pocket," completely blocked LPS- and 1Z105-driven, but not UT12-induced, TLR4 dimerization and endocytosis. Furthermore, TLR4 endocytosis is induced in macrophages tolerized by exposure to either LPS or UT12 and is independent of CD14. These data indicate that TLR4 receptor endocytosis and the TRIF-signaling pathway are dissociable and that TLR4 internalization in macrophages can be induced by UT12, 1Z105, and during endotoxin tolerance in the absence of CD14.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Western Blotting , Células Cultivadas , Dissacarídeos/farmacologia , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Citometria de Fluxo , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Ligantes , Receptores de Lipopolissacarídeos/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fosfatos Açúcares/farmacologia , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/antagonistas & inibidoresRESUMO
Genetic defects in MOGS, the gene encoding mannosyl-oligosaccharide glucosidase (the first enzyme in the processing pathway of N-linked oligosaccharide), cause the rare congenital disorder of glycosylation type IIb (CDG-IIb), also known as MOGS-CDG. MOGS is expressed in the endoplasmic reticulum and is involved in the trimming of N-glycans. We evaluated two siblings with CDG-IIb who presented with multiple neurologic complications and a paradoxical immunologic phenotype characterized by severe hypogammaglobulinemia but limited clinical evidence of an infectious diathesis. A shortened immunoglobulin half-life was determined to be the mechanism underlying the hypogammaglobulinemia. Impaired viral replication and cellular entry may explain a decreased susceptibility to infections.