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1.
Cell ; 184(17): 4447-4463.e20, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34363755

RESUMO

TANK binding kinase 1 (TBK1) regulates IFN-I, NF-κB, and TNF-induced RIPK1-dependent cell death (RCD). In mice, biallelic loss of TBK1 is embryonically lethal. We discovered four humans, ages 32, 26, 7, and 8 from three unrelated consanguineous families with homozygous loss-of-function mutations in TBK1. All four patients suffer from chronic and systemic autoinflammation, but not severe viral infections. We demonstrate that TBK1 loss results in hypomorphic but sufficient IFN-I induction via RIG-I/MDA5, while the system retains near intact IL-6 induction through NF-κB. Autoinflammation is driven by TNF-induced RCD as patient-derived fibroblasts experienced higher rates of necroptosis in vitro, and CC3 was elevated in peripheral blood ex vivo. Treatment with anti-TNF dampened the baseline circulating inflammatory profile and ameliorated the clinical condition in vivo. These findings highlight the plasticity of the IFN-I response and underscore a cardinal role for TBK1 in the regulation of RCD.


Assuntos
Inflamação/enzimologia , Proteínas Serina-Treonina Quinases/deficiência , Fator de Necrose Tumoral alfa/farmacologia , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Autoimunidade/efeitos dos fármacos , Encéfalo/diagnóstico por imagem , Morte Celular/efeitos dos fármacos , Citocinas/metabolismo , Enzima Desubiquitinante CYLD/metabolismo , Feminino , Células HEK293 , Homozigoto , Humanos , Quinase I-kappa B/metabolismo , Imunofenotipagem , Inflamação/patologia , Interferon Tipo I/metabolismo , Interferon gama/metabolismo , Mutação com Perda de Função/genética , Masculino , Linhagem , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Receptor 3 Toll-Like/metabolismo , Transcriptoma/genética , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/fisiologia
2.
Cell ; 165(4): 813-26, 2016 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-27114034

RESUMO

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ∼90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B, and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, that encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed, and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans among known broadly neutralizing antibodies that target the glycan-shielded trimer.


Assuntos
HIV-1/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Cristalografia por Raios X , Glicosilação , HIV-1/classificação , HIV-1/imunologia , Evasão da Resposta Imune , Modelos Moleculares , Simulação de Dinâmica Molecular , Polissacarídeos/análise , Polissacarídeos/metabolismo
3.
Nature ; 615(7951): 305-314, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36813963

RESUMO

Down's syndrome (DS) presents with a constellation of cardiac, neurocognitive and growth impairments. Individuals with DS are also prone to severe infections and autoimmunity including thyroiditis, type 1 diabetes, coeliac disease and alopecia areata1,2. Here, to investigate the mechanisms underlying autoimmune susceptibility, we mapped the soluble and cellular immune landscape of individuals with DS. We found a persistent elevation of up to 22 cytokines at steady state (at levels often exceeding those in patients with acute infection) and detected basal cellular activation: chronic IL-6 signalling in CD4 T cells and a high proportion of plasmablasts and CD11c+TbethighCD21low B cells (Tbet is also known as TBX21). This subset is known to be autoimmune-prone and displayed even greater autoreactive features in DS including receptors with fewer non-reference nucleotides and higher IGHV4-34 utilization. In vitro, incubation of naive B cells in the plasma of individuals with DS or with IL-6-activated T cells resulted in increased plasmablast differentiation compared with control plasma or unstimulated T cells, respectively. Finally, we detected 365 auto-antibodies in the plasma of individuals with DS, which targeted the gastrointestinal tract, the pancreas, the thyroid, the central nervous system, and the immune system itself. Together, these data point to an autoimmunity-prone state in DS, in which a steady-state cytokinopathy, hyperactivated CD4 T cells and ongoing B cell activation all contribute to a breach in immune tolerance. Our findings also open therapeutic paths, as we demonstrate that T cell activation is resolved not only with broad immunosuppressants such as Jak inhibitors, but also with the more tailored approach of IL-6 inhibition.


Assuntos
Autoimunidade , Linfócitos T CD4-Positivos , Citocinas , Síndrome de Down , Humanos , Autoanticorpos/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/análise , Citocinas/imunologia , Suscetibilidade a Doenças , Síndrome de Down/imunologia , Síndrome de Down/fisiopatologia , Interleucina-6/imunologia , Receptores de Complemento 3d
4.
Immunity ; 48(3): 500-513.e6, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29548671

RESUMO

Virtually the entire surface of the HIV-1-envelope trimer is recognized by neutralizing antibodies, except for a highly glycosylated region at the center of the "silent face" on the gp120 subunit. From an HIV-1-infected donor, #74, we identified antibody VRC-PG05, which neutralized 27% of HIV-1 strains. The crystal structure of the antigen-binding fragment of VRC-PG05 in complex with gp120 revealed an epitope comprised primarily of N-linked glycans from N262, N295, and N448 at the silent face center. Somatic hypermutation occurred preferentially at antibody residues that interacted with these glycans, suggesting somatic development of glycan recognition. Resistance to VRC-PG05 in donor #74 involved shifting of glycan-N448 to N446 or mutation of glycan-proximal residue E293. HIV-1 neutralization can thus be achieved at the silent face center by glycan-recognizing antibody; along with other known epitopes, the VRC-PG05 epitope completes coverage by neutralizing antibody of all major exposed regions of the prefusion closed trimer.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Polissacarídeos/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Antígenos Virais/química , Antígenos Virais/imunologia , Sítios de Ligação , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Glicopeptídeos/química , Glicopeptídeos/imunologia , Glicosilação , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Polissacarídeos/química , Ligação Proteica/imunologia , Hipermutação Somática de Imunoglobulina/imunologia , Relação Estrutura-Atividade
5.
PLoS Pathog ; 18(3): e1010405, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35333911

RESUMO

Type I interferons (IFN-Is) are a group of potent inflammatory and antiviral cytokines. They induce IFN stimulated genes (ISGs), which act as proinflammatory mediators, antiviral effectors, and negative regulators of the IFN-I signaling cascade itself. One such regulator is interferon stimulated gene 15 (ISG15). Humans with complete ISG15 deficiency express persistently elevated levels of ISGs, and consequently, exhibit broad spectrum resistance to viral infection. Here, we demonstrate that IFN-I primed fibroblasts derived from ISG15-deficient individuals are more resistant to infection with single-cycle HIV-1 compared to healthy control fibroblasts. Complementation with both wild-type (WT) ISG15 and ISG15ΔGG (incapable of ISGylation while retaining negative regulation activity) was sufficient to reverse this phenotype, restoring susceptibility to infection to levels comparable to WT cells. Furthermore, CRISPR-edited ISG15ko primary CD4+ T cells were less susceptible to HIV-1 infection compared to cells treated with non-targeting controls. Transcriptome analysis of these CRISPR-edited ISG15ko primary CD4+ T cells recapitulated the ISG signatures of ISG15 deficient patients. Taken together, we document that the increased broad-spectrum viral resistance in ISG15-deficiency also extends to HIV-1 and is driven by a combination of T-cell-specific ISGs, with both known and unknown functions, predicted to target HIV-1 replication at multiple steps.


Assuntos
Citocinas , Infecções por HIV , HIV-1 , Ubiquitinas , Antivirais/farmacologia , Citocinas/genética , Infecções por HIV/genética , Humanos , Interferon Tipo I , Ubiquitinas/genética
6.
Proc Natl Acad Sci U S A ; 117(30): 17510-17512, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32665439

RESUMO

Type I IFN (IFN-I) is thought to be rapidly internalized and degraded following binding to its receptor and initiation of signaling. However, many studies report the persistent effects mediated by IFN-I for days or even weeks, both ex vivo and in vivo. These long-lasting effects are attributed to downstream signaling molecules or induced effectors having a long half-life, particularly in specific cell types. Here, we describe a mechanism explaining the long-term effects of IFN-I. Following receptor binding, IFN-I is siloed into endosomal compartments. These intracellular "IFN silos" persist for days and can be visualized by fluorescence and electron microscopy. However, they are largely dormant functionally, due to IFN-I-induced negative regulators. By contrast, in individuals lacking these negative regulators, such as ISG15 or USP18, this siloed IFN-I can continue to signal from within the endosome. This mechanism may underlie the long-term effects of IFN-I therapy and may contribute to the pathophysiology of type I interferonopathies.


Assuntos
Endossomos/metabolismo , Interferon Tipo I/metabolismo , Animais , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Endossomos/ultraestrutura , Humanos , Transporte Proteico , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo
7.
J Infect Dis ; 221(6): 890-894, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31637432

RESUMO

ISG15-deficient humans exhibit permanent, low-level expression of antiviral effectors that safely protect them from various viruses. Because the murine ISG15 axis functions differently, we identified animal models that recapitulate the human condition for the development of ISG15-targeting broad-spectrum antivirals. Canine, porcine, and rhesus macaque ISG15, such as human ISG15, stabilize USP18, a potent inhibitor of type I interferon (IFN)-I. Type I Interferon-primed ISG15-knockout porcine and rhesus cells demonstrate enhanced ISG expression and protection against vesicular stomatitis Indiana virus infection compared with wild type. Collectively, we unveil the interspecies diversity of the ability of ISG15/USP18 axis to control IFN-I signaling and reveal the therapeutic potential of ISG15-deficient porcine and rhesus models.


Assuntos
Antivirais/farmacologia , Animais , Células Cultivadas , Citocinas/metabolismo , Deleção de Genes , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Macaca mulatta , Filogenia , Especificidade da Espécie , Suínos , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinas/metabolismo
8.
J Immunol ; 201(12): 3479-3485, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30530500

RESUMO

Type I IFNs (IFN-Is) are powerful cytokines. They provide remarkable protection against viral infections, but their indiscriminate production causes severe self-inflicted damage that can be lethal, particularly in early development. In humans, inappropriately high IFN-I levels caused by defects in the regulatory mechanisms that control IFN-I production and response result in clinical conditions known as type I interferonopathies. In essence, type I interferonopathies define the upper limit of safe, IFN-related inflammation in vivo. Conversely, the loss of IFN-I responsiveness increases susceptibility to viral infections, but, surprisingly, most affected individuals survive despite these inborn errors of immunity. These findings suggest that too much IFN-I early in life is toxic, but that insensitivity to IFN-I is perhaps not the death sentence it was initially thought to be. Human genetic analyses have suggested that seemingly insignificant levels of IFN-regulated gene activity may be sufficient for most of the antiviral defenses used by humans in natura.


Assuntos
Antivirais/uso terapêutico , Imunoterapia/métodos , Inflamação/imunologia , Interferon Tipo I/genética , Viroses/imunologia , Animais , Regulação da Expressão Gênica , Humanos , Imunidade , Inflamação/terapia , Interferon Tipo I/metabolismo , Interferon Tipo I/uso terapêutico , Transdução de Sinais , Viroses/terapia
9.
J Virol ; 89(5): 2659-71, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25520506

RESUMO

UNLABELLED: The isolation of broadly neutralizing HIV-1 monoclonal antibodies (MAbs) to distinct epitopes on the viral envelope glycoprotein (Env) provides the potential to use combinations of MAbs for prevention and treatment of HIV-1 infection. Since many of these MAbs have been isolated in the last few years, the potency and breadth of MAb combinations have not been well characterized. In two parallel experiments, we examined the in vitro neutralizing activities of double-, triple-, and quadruple-MAb combinations targeting four distinct epitopes, including the CD4-binding site, the V1V2-glycan region, the V3-glycan supersite, and the gp41 membrane-proximal external region (MPER), using a panel of 125 Env-pseudotyped viruses. All MAb combinations showed substantially improved neutralization breadth compared to the corresponding single MAbs, while the neutralization potency of individual MAbs was maintained. At a 50% inhibitory concentration (IC50) cutoff of 1 µg/ml per antibody, double-MAb combinations neutralized 89 to 98% of viruses, and triple combinations neutralized 98 to 100%. Overall, the improvement of neutralization breadth was closely predicted by an additive-effect model and explained by complementary neutralization profiles of antibodies recognizing distinct epitopes. Subtle but consistent favorable interactions were observed in some MAb combinations, whereas less favorable interactions were observed on a small subset of viruses that are highly sensitive to V3-glycan MAbs. These data demonstrate favorable in vitro combinations of broadly neutralizing HIV-1 MAbs and suggest that such combinations could have utility for HIV-1 prevention and treatment. IMPORTANCE: Over the last 5 years, numerous broadly reactive HIV-1-neutralizing MAbs have been isolated from B cells of HIV-1-infected donors. Each of these MAbs binds to one of the major vulnerable sites (epitopes) on the surface of the viral envelope glycoprotein. Since antibodies to distinct viral epitopes could theoretically act together to provide greater potency and breadth of virus neutralization, we tested physical mixtures of double, triple, and quadruple combinations of neutralizing MAbs targeting four major epitopes on HIV-1 Env. When tested together, antibody combinations showed substantially improved neutralization breadth compared to single MAbs. This improvement could be explained by the complementary neutralization profiles of individual MAbs. We further demonstrated that each antibody maintained its full neutralization potency when used in combination with other MAbs. These data provide a rationale for clinical use of antibody-based combinations for HIV-1 prevention and therapy.


Assuntos
Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Fármacos Anti-HIV/farmacologia , Anticorpos Neutralizantes/farmacologia , Interações Medicamentosas , Anticorpos Anti-HIV/farmacologia , Humanos , Concentração Inibidora 50 , Testes de Neutralização
10.
J Immunol ; 202(8): 2511, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30850478
11.
J Exp Med ; 221(8)2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-38869499

RESUMO

Genetic variation in UNC93B1, a key component in TLR trafficking, can lead to autoinflammation caused by increased TLR activity. Analysis of seven patient variants combined with a comprehensive alanine screen revealed that different regions of UNC93B1 selectively regulate different TLRs (Rael et al. https://doi.org/10.1084/jem.20232005; David et al. https://doi.org/10.1084/jem.20232066).


Assuntos
Receptores Toll-Like , Humanos , Receptores Toll-Like/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Transporte Proteico , Animais
12.
bioRxiv ; 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38979204

RESUMO

Type I interferons (IFN-I) are cytokines with potent antiviral and inflammatory capacities. IFN-I signaling drives the expression of hundreds of IFN-I stimulated genes (ISGs), whose aggregate function results in the control of viral infection. A few of these ISGs are tasked with negatively regulating the IFN-I response to prevent overt inflammation. ISG15 is a negative regulator whose absence leads to persistent, low-grade elevation of ISG expression and concurrent, self-resolving mild autoinflammation. The limited breadth and low-grade persistence of ISGs expressed in ISG15 deficiency are sufficient to confer broad-spectrum antiviral resistance. Inspired by ISG15 deficiency, we have identified a nominal collection of 10 ISGs that recapitulate the broad antiviral potential of the IFN-I system. The expression of the 10 ISG collection in an IFN-I non-responsive cell line increased cellular resistance to Zika, Vesicular Stomatitis, Influenza A (IAV), and SARS-CoV-2 viruses. A deliverable prophylactic formulation of this syndicate of 10 ISGs significantly inhibited IAV PR8 replication in vivo in mice and protected hamsters against a lethal SARS-CoV-2 challenge, suggesting its potential as a broad-spectrum antiviral against many current and future emerging viral pathogens. One-Sentence Summary: Human inborn error of immunity-guided discovery and development of a broad-spectrum RNA antiviral therapy.

13.
Sci Transl Med ; 16(730): eadh9039, 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38232141

RESUMO

The fusion peptide (FP) on the HIV-1 envelope (Env) trimer can be targeted by broadly neutralizing antibodies (bNAbs). Here, we evaluated the ability of a human FP-directed bNAb, VRC34.01, along with two vaccine-elicited anti-FP rhesus macaque mAbs, DFPH-a.15 and DF1W-a.01, to protect against simian-HIV (SHIV)BG505 challenge. VRC34.01 neutralized SHIVBG505 with a 50% inhibitory concentration (IC50) of 0.58 µg/ml, whereas DF1W-a.01 and DFPH-a.15 were 4- or 30-fold less potent, respectively. VRC34.01 was infused into four rhesus macaques at a dose of 10 mg/kg and four rhesus macaques at a dose of 2.5 mg/kg. The animals were intrarectally challenged 5 days later with SHIVBG505. In comparison with all 12 control animals that became infected, all four animals infused with VRC34.01 (10 mg/kg) and three out of four animals infused with VRC34.01 (2.5 mg/kg) remained uninfected. Because of the lower potency of DF1W-a.01 and DFPH-a.15 against SHIVBG505, we infused both Abs at a higher dose of 100 mg/kg into four rhesus macaques each, followed by SHIVBG505 challenge 5 days later. Three of four animals that received DF1W-a.01 were protected against infection, whereas all animals that received DFPH-a.15 were protected. Overall, the protective serum neutralization titers observed in these animals were similar to what has been observed for other bNAbs in similar SHIV infection models and in human clinical trials. In conclusion, FP-directed mAbs can thus provide dose-dependent in vivo protection against mucosal SHIV challenges, supporting the development of prophylactic vaccines targeting the HIV-1 Env FP.


Assuntos
Vacinas contra a AIDS , Infecções por HIV , HIV-1 , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Humanos , Macaca mulatta , Anticorpos Amplamente Neutralizantes , Anticorpos Anti-HIV/uso terapêutico , Infecções por HIV/prevenção & controle , Anticorpos Monoclonais , Peptídeos , Anticorpos Neutralizantes
14.
Cell Rep ; 38(6): 110348, 2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35114110

RESUMO

The increasing prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with the ability to escape existing humoral protection conferred by previous infection and/or immunization necessitates the discovery of broadly reactive neutralizing antibodies (nAbs). Utilizing mRNA display, we identify a set of antibodies against SARS-CoV-2 spike (S) proteins and characterize the structures of nAbs that recognize epitopes in the S1 subunit of the S glycoprotein. These structural studies reveal distinct binding modes for several antibodies, including the targeting of rare cryptic epitopes in the receptor-binding domain (RBD) of S that interact with angiotensin-converting enzyme 2 (ACE2) to initiate infection, as well as the S1 subdomain 1. Further, we engineer a potent ACE2-blocking nAb to sustain binding to S RBD with the E484K and L452R substitutions found in multiple SARS-CoV-2 variants. We demonstrate that mRNA display is an approach for the rapid identification of nAbs that can be used in combination to combat emerging SARS-CoV-2 variants.

15.
J Exp Med ; 219(4)2022 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-35258551

RESUMO

Human USP18 is an interferon (IFN)-stimulated gene product and a negative regulator of type I IFN (IFN-I) signaling. It also removes covalently linked ISG15 from proteins, in a process called deISGylation. In turn, ISG15 prevents USP18 from being degraded by the proteasome. Autosomal recessive complete USP18 deficiency is life-threatening in infancy owing to uncontrolled IFN-I-mediated autoinflammation. We report three Moroccan siblings with autoinflammation and mycobacterial disease who are homozygous for a new USP18 variant. We demonstrate that the mutant USP18 (p.I60N) is normally stabilized by ISG15 and efficient for deISGylation but interacts poorly with the receptor-anchoring STAT2 and is impaired in negative regulation of IFN-I signaling. We also show that IFN-γ-dependent induction of IL-12 and IL-23 is reduced owing to IFN-I-mediated impairment of myeloid cells to produce both cytokines. Thus, insufficient negative regulation of IFN-I signaling by USP18-I60N underlies a specific type I interferonopathy, which impairs IL-12 and IL-23 production by myeloid cells, thereby explaining predisposition to mycobacterial disease.


Assuntos
Ubiquitina Tiolesterase , Ubiquitinas , Citocinas/metabolismo , Humanos , Inflamação/genética , Interleucina-12 , Interleucina-23 , Ubiquitina Tiolesterase/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo
16.
Sci Rep ; 11(1): 12740, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140558

RESUMO

The SARS-CoV-2 variants replacing the first wave strain pose an increased threat by their potential ability to escape pre-existing humoral protection. An angiotensin converting enzyme 2 (ACE2) decoy that competes with endogenous ACE2 for binding of the SARS-CoV-2 spike receptor binding domain (S RBD) and inhibits infection may offer a therapeutic option with sustained efficacy against variants. Here, we used Molecular Dynamics (MD) simulation to predict ACE2 sequence substitutions that might increase its affinity for S RBD and screened candidate ACE2 decoys in vitro. The lead ACE2(T27Y/H34A)-IgG1FC fusion protein with enhanced S RBD affinity shows greater live SARS-CoV-2 virus neutralization capability than wild type ACE2. MD simulation was used to predict the effects of S RBD variant mutations on decoy affinity that was then confirmed by testing of an ACE2 Triple Decoy that included an additional enzyme activity-deactivating H374N substitution against mutated S RBD. The ACE2 Triple Decoy maintains high affinity for mutated S RBD, displays enhanced affinity for S RBD N501Y or L452R, and has the highest affinity for S RBD with both E484K and N501Y mutations, making it a viable therapeutic option for the prevention or treatment of SARS-CoV-2 infection with a high likelihood of efficacy against variants.


Assuntos
Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , COVID-19/metabolismo , Descoberta de Drogas/métodos , Simulação de Dinâmica Molecular , SARS-CoV-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , COVID-19/virologia , Humanos , Mutação , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus/efeitos dos fármacos
17.
bioRxiv ; 2021 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-34545362

RESUMO

The increasing prevalence of SARS-CoV-2 variants with the ability to escape existing humoral protection conferred by previous infection and/or immunization necessitates the discovery of broadly-reactive neutralizing antibodies (nAbs). Utilizing mRNA display, we identified a set of antibodies against SARS-CoV-2 spike (S) proteins and characterized the structures of nAbs that recognized epitopes in the S1 subunit of the S glycoprotein. These structural studies revealed distinct binding modes for several antibodies, including targeting of rare cryptic epitopes in the receptor-binding domain (RBD) of S that interacts with angiotensin- converting enzyme 2 (ACE2) to initiate infection, as well as the S1 subdomain 1. A potent ACE2-blocking nAb was further engineered to sustain binding to S RBD with the E484K and L452R substitutions found in multiple SARS-CoV-2 variants. We demonstrate that mRNA display is a promising approach for the rapid identification of nAbs that can be used in combination to combat emerging SARS-CoV-2 variants.

18.
Cell Rep ; 37(5): 109922, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34731616

RESUMO

Recognition of N-linked glycan at residue N276 (glycan276) at the periphery of the CD4-binding site (CD4bs) on the HIV-envelope trimer is a formidable challenge for many CD4bs-directed antibodies. To understand how this glycan can be recognized, here we isolate two lineages of glycan276-dependent CD4bs antibodies. Antibody CH540-VRC40.01 (named for donor-lineage.clone) neutralizes 81% of a panel of 208 diverse strains, while antibody CH314-VRC33.01 neutralizes 45%. Cryo-electron microscopy (cryo-EM) structures of these two antibodies and 179NC75, a previously identified glycan276-dependent CD4bs antibody, in complex with HIV-envelope trimer reveal substantially different modes of glycan276 recognition. Despite these differences, binding of glycan276-dependent antibodies maintains a glycan276 conformation similar to that observed in the absence of glycan276-binding antibodies. By contrast, glycan276-independent CD4bs antibodies, such as VRC01, displace glycan276 upon binding. These results provide a foundation for understanding antibody recognition of glycan276 and suggest its presence may be crucial for priming immunogens seeking to initiate broad CD4bs recognition.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Epitopos , HIV-1/imunologia , Polissacarídeos/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/metabolismo , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Anticorpos Amplamente Neutralizantes/metabolismo , Anticorpos Amplamente Neutralizantes/ultraestrutura , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Microscopia Crioeletrônica , Células HEK293 , HIV-1/metabolismo , Humanos , Modelos Moleculares , Polissacarídeos/metabolismo , Ligação Proteica , Conformação Proteica , Imagem Individual de Molécula , Relação Estrutura-Atividade , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
19.
J Exp Med ; 217(5)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32092142

RESUMO

Type I interferonopathies are monogenic disorders characterized by enhanced type I interferon (IFN-I) cytokine activity. Inherited USP18 and ISG15 deficiencies underlie type I interferonopathies by preventing the regulation of late responses to IFN-I. Specifically, USP18, being stabilized by ISG15, sterically hinders JAK1 from binding to the IFNAR2 subunit of the IFN-I receptor. We report an infant who died of autoinflammation due to a homozygous missense mutation (R148Q) in STAT2. The variant is a gain of function (GOF) for induction of the late, but not early, response to IFN-I. Surprisingly, the mutation does not enhance the intrinsic activity of the STAT2-containing transcriptional complex responsible for IFN-I-stimulated gene induction. Rather, the STAT2 R148Q variant is a GOF because it fails to appropriately traffic USP18 to IFNAR2, thereby preventing USP18 from negatively regulating responses to IFN-I. Homozygosity for STAT2 R148Q represents a novel molecular and clinical phenocopy of inherited USP18 deficiency, which, together with inherited ISG15 deficiency, defines a group of type I interferonopathies characterized by an impaired regulation of late cellular responses to IFN-I.


Assuntos
Mutação com Ganho de Função/genética , Interferon Tipo I/metabolismo , Fator de Transcrição STAT2/genética , Ubiquitina Tiolesterase/deficiência , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Homozigoto , Humanos , Recém-Nascido , Masculino , Linhagem , Fenótipo , Domínios Proteicos , Fator de Transcrição STAT2/química , Ubiquitina Tiolesterase/genética , Sequenciamento do Exoma
20.
Toxicol Sci ; 161(1): 196-206, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29294139

RESUMO

Amphibian metamorphosis is driven by thyroid hormone (TH). We used prometamorphic tadpoles and a cell line of the African clawed frog (Xenopus laevis) to examine immediate effects of dioxin exposure on TH. Gene expression patterns suggest cross-talk between the thyroid hormone receptor (TR) and aryl hydrocarbon receptor (AHR) signaling pathways. In XLK-WG cells, expression of Cytochrome P450 1A6 (cyp1A6), an AHR target, was induced 1000-fold by 100 nM TCDD (2, 3, 7, 8 tetrachlorodibenzo-p-dioxin). Krüppel-Like Factor 9 (klf9), the first gene induced in a cascade of TH responses tied to metamorphosis, was upregulated over 5-fold by 50 nM triiodothyronine (T3) and 2-fold by dioxin. Co-exposure to T3 and TCDD boosted both responses, further inducing cyp1A6 by 75% and klf9 about 60%. Additional canonical targets of each receptor, including trßa and trßb (TR) and udpgt1a (AHR) responded similarly. Induction of TH targets by TCDD in XLK-WG cells predicts that exposure could speed metamorphosis. We tested this hypothesis in two remodeling events: tail resorption and hind limb growth. Resorption of ex vivo cultured tails was accelerated by 10 nM T3, while a modest increase in resorption by 100 nM TCDD lacked statistical significance. Hind limbs doubled in length over four days following 1 nM T3 treatment, but limb length was unaffected by 100 nM TCDD. TCDD co-exposure reduced the T3 effect by nearly 40%, despite TCDD induction of klf9 in whole tadpoles, alone or with T3. These results suggest that tissue-specific TCDD effects limit or reverse the increased metamorphosis rate predicted by klf9 induction.


Assuntos
Disruptores Endócrinos/toxicidade , Larva/efeitos dos fármacos , Metamorfose Biológica/efeitos dos fármacos , Dibenzodioxinas Policloradas/toxicidade , Hormônios Tireóideos/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular , Larva/metabolismo , Metamorfose Biológica/genética , Receptor Cross-Talk/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Hormônios Tireóideos/farmacologia , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologia , Xenopus laevis
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