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1.
Cell Rep ; 35(1): 108962, 2021 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-33826892

RESUMO

Although neutralizing monoclonal antibodies (mAbs) against epitopes within the alphavirus E2 protein can protect against infection, the functional significance of non-neutralizing mAbs is poorly understood. Here, we evaluate the activity of 13 non-neutralizing mAbs against Mayaro virus (MAYV), an emerging arthritogenic alphavirus. These mAbs bind to the MAYV virion and surface of infected cells but fail to neutralize infection in cell culture. Mapping studies identify six mAb binding groups that localize to discrete epitopes within or adjacent to the A domain of the E2 glycoprotein. Remarkably, passive transfer of non-neutralizing mAbs protects against MAYV infection and disease in mice, and their efficacy requires Fc effector functions. Monocytes mediate the protection of non-neutralizing mAbs in vivo, as Fcγ-receptor-expressing myeloid cells facilitate the binding, uptake, and clearance of MAYV without antibody-dependent enhancement of infection. Humoral protection against alphaviruses likely reflects contributions from non-neutralizing antibodies through Fc-dependent mechanisms that accelerate viral clearance.

2.
J Exp Med ; 218(5)2021 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-33831142

RESUMO

Although Powassan virus (POWV) is an emerging tick-transmitted flavivirus that causes severe or fatal neuroinvasive disease in humans, medical countermeasures have not yet been developed. Here, we developed a panel of neutralizing anti-POWV mAbs recognizing six distinct antigenic sites. The most potent of these mAbs bind sites within domain II or III of the envelope (E) protein and inhibit postattachment viral entry steps. A subset of these mAbs cross-react with other flaviviruses. Both POWV type-specific and cross-reactive neutralizing mAbs confer protection in mice against POWV infection when given as prophylaxis or postexposure therapy. Several cross-reactive mAbs mapping to either domain II or III also protect in vivo against heterologous tick-transmitted flaviviruses including Langat and tick-borne encephalitis virus. Our experiments define structural and functional correlates of antibody protection against POWV infection and identify epitopes targeted by broadly neutralizing antibodies with therapeutic potential against multiple tick-borne flaviviruses.

3.
Cell Rep Med ; : 100230, 2021 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-33754147

RESUMO

The deployment of a vaccine that limits transmission and disease likely will be required to end the Coronavirus Disease 2019 (COVID-19) pandemic. We recently described the protective activity of an intranasally-administered chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike (S) protein (ChAd-SARS-CoV-2-S) in the upper and lower respiratory tract of mice expressing the human angiotensin-converting enzyme 2 (ACE2) receptor. Here, we show the immunogenicity and protective efficacy of this vaccine in non-human primates. Rhesus macaques were immunized with ChAd-Control or ChAd-SARS-CoV-2-S and challenged one month later by combined intranasal and intrabronchial routes with SARS-CoV-2. A single intranasal dose of ChAd-SARS-CoV-2-S induces neutralizing antibodies and T cell responses and limits or prevents infection in the upper and lower respiratory tract after SARS-CoV-2 challenge. As this single intranasal dose vaccine confers protection against SARS-CoV-2 in non-human primates, it is a promising candidate for limiting SARS-CoV-2 infection and transmission in humans.

4.
Cell ; 2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33773105

RESUMO

Most human monoclonal antibodies (mAbs) neutralizing SARS-CoV-2 recognize the spike (S) protein receptor-binding domain and block virus interactions with the cellular receptor angiotensin-converting enzyme 2. We describe a panel of human mAbs binding to diverse epitopes on the N-terminal domain (NTD) of S protein from SARS-CoV-2 convalescent donors and found a minority of these possessed neutralizing activity. Two mAbs (COV2-2676 and COV2-2489) inhibited infection of authentic SARS-CoV-2 and recombinant VSV/SARS-CoV-2 viruses. We mapped their binding epitopes by alanine-scanning mutagenesis and selection of functional SARS-CoV-2 S neutralization escape variants. Mechanistic studies showed that these antibodies neutralize in part by inhibiting a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 offered protection either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, natural infection induces a subset of potent NTD-specific mAbs that leverage neutralizing and Fc-mediated activities to protect against SARS-CoV-2 infection using multiple functional attributes.

5.
Nat Med ; 2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33664494

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic. Rapidly spreading SARS-CoV-2 variants may jeopardize newly introduced antibody and vaccine countermeasures. Here, using monoclonal antibodies (mAbs), animal immune sera, human convalescent sera and human sera from recipients of the BNT162b2 mRNA vaccine, we report the impact on antibody neutralization of a panel of authentic SARS-CoV-2 variants including a B.1.1.7 isolate, chimeric strains with South African or Brazilian spike genes and isogenic recombinant viral variants. Many highly neutralizing mAbs engaging the receptor-binding domain or N-terminal domain and most convalescent sera and mRNA vaccine-induced immune sera showed reduced inhibitory activity against viruses containing an E484K spike mutation. As antibodies binding to spike receptor-binding domain and N-terminal domain demonstrate diminished neutralization potency in vitro against some emerging variants, updated mAb cocktails targeting highly conserved regions, enhancement of mAb potency or adjustments to the spike sequences of vaccines may be needed to prevent loss of protection in vivo.

6.
Virology ; 557: 70-85, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33676349

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged into the human population in late 2019 and caused the global COVID-19 pandemic. SARS-CoV-2 has spread to more than 215 countries and infected many millions of people. Despite the introduction of numerous governmental and public health measures to control disease spread, infections continue at an unabated pace, suggesting that effective vaccines and antiviral drugs will be required to curtail disease, end the pandemic, and restore societal norms. Here, we review the current developments in antibody and vaccine countermeasures to limit or prevent disease.


Assuntos
Anticorpos Antivirais/biossíntese , /prevenção & controle , Pandemias , /imunologia , Animais , /imunologia , /virologia , /biossíntese , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Vetores Genéticos/química , Vetores Genéticos/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Imunização Passiva/métodos , Imunogenicidade da Vacina , Segurança do Paciente , /patogenicidade , Vacinas Atenuadas , Vacinas de DNA , Vacinas de Subunidades , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas de Partículas Semelhantes a Vírus/imunologia
8.
Cell ; 2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33756110

RESUMO

Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 µg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.

9.
Artigo em Inglês | MEDLINE | ID: mdl-33681537

RESUMO

There is ongoing debate as to whether cardiac complications of coronavirus disease 2019 (COVID-19) result from myocardial viral infection or are secondary to systemic inflammation and/or thrombosis. We provide evidence that cardiomyocytes are infected in patients with COVID-19 myocarditis and are susceptible to severe acute respiratory syndrome coronavirus 2. We establish an engineered heart tissue model of COVID-19 myocardial pathology, define mechanisms of viral pathogenesis, and demonstrate that cardiomyocyte severe acute respiratory syndrome coronavirus 2 infection results in contractile deficits, cytokine production, sarcomere disassembly, and cell death. These findings implicate direct infection of cardiomyocytes in the pathogenesis of COVID-19 myocardial pathology and provides a model system to study this emerging disease.

10.
Cell ; 2021 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-33691138

RESUMO

The biosafety level 3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research. Here, we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing ORF3 and envelope gene deletions, as well as mutated transcriptional regulator sequences, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. Thus, the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.

11.
Mucosal Immunol ; 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674763

RESUMO

Although they globally cause viral gastroenteritis in children, astroviruses are understudied due to the lack of well-defined animal models. While murine astroviruses (muAstVs) chronically infect immunodeficient mice, a culture system and understanding of their pathogenesis is lacking. Here, we describe a platform to cultivate muAstV using air-liquid interface (ALI) cultures derived from mouse enteroids, which support apical infection and release. Chronic muAstV infection occurs predominantly in the small intestine and correlates with higher interferon-lambda (IFN-λ) expression. MuAstV stimulates IFN-λ production in ALI, recapitulating our in vivo findings. We demonstrate that goblet cells and enterocytes are targets for chronic muAstV infection in vivo, and that infection is enhanced by parasite co-infection or type 2 cytokine signaling. Depletion of goblet cells from ALI limits muAstV infection in vitro. During chronic infection, muAstV stimulates IFN-λ production in infected cells and induces ISGs throughout the intestinal epithelium in an IFN-λ-receptor-dependent manner. Collectively, our study provides insights into the cellular tropism and innate immune responses to muAstV and establishes an enteroid-based culture system to propagate muAstV in vitro.

12.
Cell Rep ; 34(10): 108756, 2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33691097

RESUMO

Itaconate is a unique regulatory metabolite that is induced upon Toll-like receptor (TLR) stimulation in myeloid cells. Here, we demonstrate major inflammatory tolerance and cell death phenotypes associated with itaconate production in activated macrophages. We show that endogenous itaconate is a key regulator of the signal 2 of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation after long lipopolysaccharide (LPS) priming, which establishes tolerance to late NLRP3 inflammasome activation. We show that itaconate acts synergistically with inducible nitric oxide synthase (iNOS) and that the ability of various TLR ligands to establish NLRP3 inflammasome tolerance depends on the pattern of co-expression of IRG1 and iNOS. Mechanistically, itaconate accumulation upon prolonged inflammatory stimulation prevents full caspase-1 activation and processing of gasdermin D, which we demonstrate to be post-translationally modified by endogenous itaconate. Altogether, our data demonstrate that metabolic rewiring in inflammatory macrophages establishes tolerance to NLRP3 inflammasome activation that, if uncontrolled, can result in pyroptotic cell death and tissue damage.

13.
Cell ; 184(7): 1804-1820.e16, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33691139

RESUMO

SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes and CD8+ T cells for optimal clinical and virological benefit. Thus, potently neutralizing mAbs utilize Fc effector functions during therapy to mitigate lung infection and disease.

14.
Cell Host Microbe ; 29(3): 477-488.e4, 2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33535027

RESUMO

Neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of COVID-19 vaccines and have received emergency use authorization as therapeutics. However, viral escape mutants could compromise efficacy. To define immune-selected mutations in the S protein, we exposed a VSV-eGFP-SARS-CoV-2-S chimeric virus, in which the VSV glycoprotein is replaced with the S protein, to 19 neutralizing monoclonal antibodies (mAbs) against the receptor-binding domain (RBD) and generated 50 different escape mutants. Each mAb had a unique resistance profile, although many shared residues within an epitope of the RBD. Some variants (e.g., S477N) were resistant to neutralization by multiple mAbs, whereas others (e.g., E484K) escaped neutralization by convalescent sera. Additionally, sequential selection identified mutants that escape neutralization by antibody cocktails. Comparing these antibody-mediated mutations with sequence variation in circulating SARS-CoV-2 revealed substitutions that may attenuate neutralizing immune responses in some humans and thus warrant further investigation.


Assuntos
Anticorpos Monoclonais/sangue , Anticorpos Antivirais/sangue , Mutação , Testes de Neutralização/métodos , Glicoproteína da Espícula de Coronavírus/genética , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , /imunologia , Chlorocebus aethiops , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Ligação Proteica , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
15.
Cell ; 184(5): 1214-1231.e16, 2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33636133

RESUMO

Although enteric helminth infections modulate immunity to mucosal pathogens, their effects on systemic microbes remain less established. Here, we observe increased mortality in mice coinfected with the enteric helminth Heligmosomoides polygyrus bakeri (Hpb) and West Nile virus (WNV). This enhanced susceptibility is associated with altered gut morphology and transit, translocation of commensal bacteria, impaired WNV-specific T cell responses, and increased virus infection in the gastrointestinal tract and central nervous system. These outcomes were due to type 2 immune skewing, because coinfection in Stat6-/- mice rescues mortality, treatment of helminth-free WNV-infected mice with interleukin (IL)-4 mirrors coinfection, and IL-4 receptor signaling in intestinal epithelial cells mediates the susceptibility phenotypes. Moreover, tuft cell-deficient mice show improved outcomes with coinfection, whereas treatment of helminth-free mice with tuft cell-derived cytokine IL-25 or ligand succinate worsens WNV disease. Thus, helminth activation of tuft cell-IL-4-receptor circuits in the gut exacerbates infection and disease of a neurotropic flavivirus.

16.
Nature ; 591(7849): 293-299, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33494095

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.


Assuntos
/virologia , Furina/metabolismo , Mutação , /patogenicidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , /fisiopatologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Feminino , Humanos , Pneumopatias/patologia , Pneumopatias/fisiopatologia , Pneumopatias/virologia , Masculino , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteólise , /metabolismo , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Replicação Viral/genética
17.
J Clin Invest ; 131(1)2021 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-33393505

RESUMO

Human herpes simplex virus 1 (HSV-1) encephalitis can be caused by inborn errors of the TLR3 pathway, resulting in impairment of CNS cell-intrinsic antiviral immunity. Deficiencies of the TLR3 pathway impair cell-intrinsic immunity to vesicular stomatitis virus (VSV) and HSV-1 in fibroblasts, and to HSV-1 in cortical but not trigeminal neurons. The underlying molecular mechanism is thought to involve impaired IFN-α/ß induction by the TLR3 recognition of dsRNA viral intermediates or by-products. However, we show here that human TLR3 controls constitutive levels of IFNB mRNA and secreted bioactive IFN-ß protein, and thereby also controls constitutive mRNA levels for IFN-stimulated genes (ISGs) in fibroblasts. Tlr3-/- mouse embryonic fibroblasts also have lower basal ISG levels. Moreover, human TLR3 controls basal levels of IFN-ß secretion and ISG mRNA in induced pluripotent stem cell-derived cortical neurons. Consistently, TLR3-deficient human fibroblasts and cortical neurons are vulnerable not only to both VSV and HSV-1, but also to several other families of viruses. The mechanism by which TLR3 restricts viral growth in human fibroblasts and cortical neurons in vitro and, by inference, by which the human CNS prevents infection by HSV-1 in vivo, is therefore based on the control of early viral infection by basal IFN-ß immunity.

18.
Cell ; 184(1): 15-17, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33338422

RESUMO

Complementary genome-wide CRISPR-Cas9 screens performed by multiple groups reveal new insights into SARS-CoV-2 biology including aspects of viral entry, translation, replication, egress, and the genes regulating these processes. Comparisons with other coronaviruses enhances our understanding of the cellular life cycle of this medically important family of emerging viruses.

19.
bioRxiv ; 2020 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-33299991

RESUMO

The development of an effective vaccine against SARS-CoV-2, the etiologic agent of COVID-19, is a global priority. Here, we compared the protective capacity of intranasal and intramuscular delivery of a chimpanzee adenovirus-vectored vaccine encoding a pre-fusion stabilized spike protein (ChAd-SARS-CoV-2-S) in Golden Syrian hamsters. While immunization with ChAd-SARS-CoV-2-S induced robust spike protein specific antibodies capable or neutralizing the virus, antibody levels in serum were higher in hamsters immunized by an intranasal compared to intramuscular route. Accordingly, ChAd-SARS-CoV-2-S immunized hamsters were protected against a challenge with a high dose of SARS-CoV-2. After challenge, ChAd-SARS-CoV-2-S-immunized hamsters had less weight loss and showed reductions in viral RNA and infectious virus titer in both nasal swabs and lungs, and reduced pathology and inflammatory gene expression in the lungs, compared to ChAd-Control immunized hamsters. Intranasal immunization with ChAd-SARS-CoV-2-S provided superior protection against SARS-CoV-2 infection and inflammation in the upper respiratory tract. These findings support intranasal administration of the ChAd-SARS-CoV-2-S candidate vaccine to prevent SARS-CoV-2 infection, disease, and possibly transmission.

20.
Cell ; 2020 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-33301709

RESUMO

Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics.

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