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1.
Nat Commun ; 12(1): 4817, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376662

RESUMO

Engineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. We applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. We demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, our analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/ultraestrutura , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/ultraestrutura , Microscopia Crioeletrônica/métodos , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Glicosilação , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/ultraestrutura , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , HIV-1/genética , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Macaca mulatta , Modelos Moleculares , Conformação Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/ultraestrutura
2.
Mol Cell ; 81(17): 3650-3658.e5, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34390675

RESUMO

CRISPR-inspired systems have been extensively developed for applications in genome editing and nucleic acid detection. Here, we introduce a CRISPR-based peptide display technology to facilitate customized, high-throughput in vitro protein interaction studies. We show that bespoke peptide libraries fused to catalytically inactive Cas9 (dCas9) and barcoded with unique single guide RNA (sgRNA) molecules self-assemble from a single mixed pool to programmable positions on a DNA microarray surface for rapid, multiplexed binding assays. We develop dCas9-displayed saturation mutagenesis libraries to characterize antibody-epitope binding for a commercial anti-FLAG monoclonal antibody and human serum antibodies. We also show that our platform can be used for viral epitope mapping and exhibits promise as a multiplexed diagnostics tool. Our CRISPR-based peptide display platform and the principles of complex library self-assembly using dCas9 could be adapted for rapid interrogation of varied customized protein libraries or biological materials assembly using DNA scaffolding.


Assuntos
Epitopos/genética , Edição de Genes/métodos , Biblioteca de Peptídeos , RNA Guia/genética , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/imunologia , Epitopos/imunologia , Humanos , Mutagênese/genética , Ligação Proteica/genética , Ligação Proteica/imunologia , RNA Guia/imunologia
3.
J Immunol ; 207(4): 1138-1149, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34341168

RESUMO

Group A streptococcal infections are a significant cause of global morbidity and mortality. A leading vaccine candidate is the surface M protein, a major virulence determinant and protective Ag. One obstacle to the development of M protein-based vaccines is the >200 different M types defined by the N-terminal sequences that contain protective epitopes. Despite sequence variability, M proteins share coiled-coil structural motifs that bind host proteins required for virulence. In this study, we exploit this potential Achilles heel of conserved structure to predict cross-reactive M peptides that could serve as broadly protective vaccine Ags. Combining sequences with structural predictions, six heterologous M peptides in a sequence-related cluster were predicted to elicit cross-reactive Abs with the remaining five nonvaccine M types in the cluster. The six-valent vaccine elicited Abs in rabbits that reacted with all 11 M peptides in the cluster and functional opsonic Abs against vaccine and nonvaccine M types in the cluster. We next immunized mice with four sequence-unrelated M peptides predicted to contain different coiled-coil propensities and tested the antisera for cross-reactivity against 41 heterologous M peptides. Based on these results, we developed an improved algorithm to select cross-reactive peptide pairs using additional parameters of coiled-coil length and propensity. The revised algorithm accurately predicted cross-reactive Ab binding, improving the Matthews correlation coefficient from 0.42 to 0.74. These results form the basis for selecting the minimum number of N-terminal M peptides to include in potentially broadly efficacious multivalent vaccines that could impact the overall global burden of group A streptococcal diseases.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Reações Cruzadas/imunologia , Vacinas Estreptocócicas/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Epitopos/imunologia , Feminino , Humanos , Masculino , Camundongos , Peptídeos/imunologia , Vacinas Sintéticas/imunologia
4.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445142

RESUMO

It is difficult to treat allergic diseases including asthma completely because its pathogenesis remains unclear. House dust mite (HDM) is a critical allergen and Toll-like receptor (TLR) 4 is a member of the toll-like receptor family, which plays an important role in allergic diseases. The purpose of this study was to characterize a novel allergen, Der f 38 binding to TLR4, and unveil its role as an inducer of allergy. Der f 38 expression was detected in the body and feces of Dermatophagoides farinae (DF). Electron microscopy revealed that it was located in the granule layer, the epithelium layer, and microvilli of the posterior midgut. The skin prick test showed that 60% of allergic subjects were Der f 38-positive. Der f 38 enhanced surface 203c expression in basophils of Der f 38-positive allergic subjects. By analysis of the model structure of Der p 38, the expected epitope sites are exposed on the exterior side. In animal experiments, Der f 38 triggered an infiltration of inflammatory cells. Intranasal (IN) administration of Der f 38 increased neutrophils in the lung. Intraperitoneal (IP) and IN injections of Der f 38 induced both eosinophils and neutrophils. Increased total IgE level and histopathological features were found in BALB/c mice treated with Der f 38 by IP and IN injections. TLR4 knockout (KO) BALB/c mice exhibited less inflammation and IgE level in the sera compared to wild type (WT) mice. Der f 38 directly binds to TLR4 using biolayer interferometry. Der f 38 suppressed the apoptosis of neutrophils and eosinophils by downregulating proteins in the proapoptotic pathway including caspase 9, caspase 3, and BAX and upregulating proteins in the anti-apoptotic pathway including BCL-2 and MCL-1. These findings might shed light on the pathogenic mechanisms of allergy to HDM.


Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Dermatophagoides farinae/imunologia , Hipersensibilidade/imunologia , Ligação Proteica/imunologia , Receptor 4 Toll-Like/imunologia , Sequência de Aminoácidos , Animais , Epitopos/imunologia , Feminino , Humanos , Imunoglobulina E/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Pyroglyphidae/metabolismo , Testes Cutâneos/métodos
5.
Cell Rep ; 36(10): 109679, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34464610

RESUMO

A wide range of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing monoclonal antibodies (mAbs) have been reported, most of which target the spike glycoprotein. Therapeutic implementation of these antibodies has been challenged by emerging SARS-CoV-2 variants harboring mutated spike versions. Consequently, re-assessment of previously identified mAbs is of high priority. Four previously selected mAbs targeting non-overlapping epitopes are now evaluated for binding potency to mutated RBD versions, reported to mediate escape from antibody neutralization. In vitro neutralization potencies of these mAbs, and two NTD-specific mAbs, are evaluated against two frequent SARS-CoV-2 variants of concern, the B.1.1.7 Alpha and the B.1.351 Beta. Furthermore, we demonstrate therapeutic potential of three selected mAbs by treatment of K18-human angiotensin-converting enzyme 2 (hACE2) transgenic mice 2 days post-infection with each virus variant. Thus, despite the accumulation of spike mutations, the highly potent MD65 and BL6 mAbs retain their ability to bind the prevalent viral mutants, effectively protecting against B.1.1.7 and B.1.351 variants.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/química , Afinidade de Anticorpos , COVID-19/terapia , COVID-19/virologia , Epitopos/genética , Epitopos/imunologia , Humanos , Imunização Passiva , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Testes de Neutralização , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Resultado do Tratamento
6.
Nat Commun ; 12(1): 4887, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373446

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that is spreading rapidly, which seriously impacts global public health and economy. Thus, developing effective drugs remains urgent. We identify two potent antibodies, nCoVmab1 and nCoVmab2, targeting the SARS-CoV-2 spike protein receptor-binding domain (RBD) with high affinities from a naïve human phage-displayed Fab library. nCoVmab1 and nCoVmab2 neutralize authentic SARS-CoV-2 with picomolar and nanomolar IC50 values, respectively. No detectable defects of nCoVmab1 and nCoVmab2 are found during the preliminary druggability evaluation. nCoVmab1 could reduce viral titer and lung injury when administered prophylactically and therapeutically in human angiotensin-converting enzyme II (hACE2)-transgenic mice. Therefore, phage display platform could be efficiently used for rapid development of neutralizing monoclonal antibodies (nmabs) with clinical potential against emerging infectious diseases. In addition, we determinate epitopes in RBD of these antibodies to elucidate the neutralizing mechanism. We also convert nCoVmab1 and nCoVmab2 to their germline formats for further analysis, which reveals the contribution of somatic hypermutation (SHM) during nCoVmab1 and nCoVmab2 maturation. Our findings not only provide two highly potent nmabs against SARS-CoV-2 as prophylactic and therapeutic candidates, but also give some clues for development of anti-SARS-CoV-2 agents (e.g., drugs and vaccines) targeting the RBD.


Assuntos
Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , SARS-CoV-2/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/genética , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Sítios de Ligação , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Chlorocebus aethiops , Epitopos/imunologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Ligação Proteica , Receptores Virais/efeitos dos fármacos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus , Células Vero
7.
Nat Commun ; 12(1): 4676, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344900

RESUMO

Interventions against variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Stable and potent nanobodies (Nbs) that target the receptor binding domain (RBD) of SARS-CoV-2 spike are promising therapeutics. However, it is unknown if Nbs broadly neutralize circulating variants. We found that RBD Nbs are highly resistant to variants of concern (VOCs). High-resolution cryoelectron microscopy determination of eight Nb-bound structures reveals multiple potent neutralizing epitopes clustered into three classes: Class I targets ACE2-binding sites and disrupts host receptor binding. Class II binds highly conserved epitopes and retains activity against VOCs and RBDSARS-CoV. Cass III recognizes unique epitopes that are likely inaccessible to antibodies. Systematic comparisons of neutralizing antibodies and Nbs provided insights into how Nbs target the spike to achieve high-affinity and broadly neutralizing activity. Structure-function analysis of Nbs indicates a variety of antiviral mechanisms. Our study may guide the rational design of pan-coronavirus vaccines and therapeutics.


Assuntos
Anticorpos Amplamente Neutralizantes/imunologia , Epitopos/imunologia , SARS-CoV-2/imunologia , Anticorpos de Domínio Único/imunologia , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Anticorpos Amplamente Neutralizantes/química , Anticorpos Amplamente Neutralizantes/classificação , Anticorpos Amplamente Neutralizantes/metabolismo , COVID-19/tratamento farmacológico , COVID-19/prevenção & controle , Epitopos/química , Epitopos/metabolismo , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , SARS-CoV-2/genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/classificação , Anticorpos de Domínio Único/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Relação Estrutura-Atividade
8.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361120

RESUMO

A major limiting factor for systemically delivered gene therapies is the lack of novel tissue specific AAV (Adeno-associated virus) derived vectors. Bispecific antibodies can be used to redirect AAVs to specific target receptors. Here, we demonstrate that the insertion of a short linear epitope "2E3" derived from human proprotein-convertase subtilisin/kexin type 9 (PCSK9) into different surface loops of the VP capsid proteins can be used for AAV de-targeting from its natural receptor(s), combined with a bispecific antibody-mediated retargeting. We chose to target a set of distinct disease relevant membrane proteins-fibroblast activation protein (FAP), which is upregulated on activated fibroblasts within the tumor stroma and in fibrotic tissues, as well as programmed death-ligand 1 (PD-L1), which is strongly upregulated in many cancers. Upon incubation with a bispecific antibody recognizing the 2E3 epitope and FAP or PD-L1, the bispecific antibody/rAAV complex was able to selectively transduce receptor positive cells. In summary, we developed a novel, rationally designed vector retargeting platform that can target AAVs to a new set of cellular receptors in a modular fashion. This versatile platform may serve as a valuable tool to investigate the role of disease relevant cell types and basis for novel gene therapy approaches.


Assuntos
Anticorpos Biespecíficos/imunologia , Proteínas do Capsídeo/imunologia , Capsídeo/imunologia , Dependovirus/genética , Endopeptidases/imunologia , Epitopos/imunologia , Vetores Genéticos/administração & dosagem , Proteínas de Membrana/imunologia , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/imunologia , Pró-Proteína Convertase 9/metabolismo , Transdução Genética
9.
Int J Mol Sci ; 22(15)2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34361054

RESUMO

We addressed the issue of C1q autoantigenicity by studying the structural features of the autoepitopes recognized by the polyclonal anti-C1q antibodies present in Lupus Nephritis (LN) sera. We used six fractions of anti-C1q as antigens and selected anti-idiotypic scFv antibodies from the phage library "Griffin.1". The monoclonal scFv A1 was the most potent inhibitor of the recognition of C1q and its fragments ghA, ghB and ghC, comprising the globular domain gC1q, by the lupus autoantibodies. It was sequenced and in silico folded by molecular dynamics into a 3D structure. The generated 3D model of A1 elucidated CDR similarity to the apical region of gC1q, thus mapping indirectly for the first time a globular autoepitope of C1q. The VH CDR2 of A1 mimicked the ghA sequence GSEAD suggested as a cross-epitope between anti-DNA and anti-C1q antibodies. Other potential inhibitors of the recognition of C1q by the LN autoantibodies among the selected recombinant antibodies were the monoclonal scFv F6, F9 and A12.


Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Autoanticorpos/sangue , Autoantígenos/imunologia , Complemento C1q/imunologia , Epitopos/imunologia , Nefrite Lúpica/imunologia , Anticorpos de Cadeia Única/imunologia , Humanos , Nefrite Lúpica/sangue , Estrutura Terciária de Proteína , Subunidades Proteicas
10.
Signal Transduct Target Ther ; 6(1): 304, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404759

RESUMO

A comprehensive analysis of the humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential in understanding COVID-19 pathogenesis and developing antibody-based diagnostics and therapy. In this work, we performed a longitudinal analysis of antibody responses to SARS-CoV-2 proteins in 104 serum samples from 49 critical COVID-19 patients using a peptide-based SARS-CoV-2 proteome microarray. Our data show that the binding epitopes of IgM and IgG antibodies differ across SARS-CoV-2 proteins and even within the same protein. Moreover, most IgM and IgG epitopes are located within nonstructural proteins (nsps), which are critical in inactivating the host's innate immune response and enabling SARS-CoV-2 replication, transcription, and polyprotein processing. IgM antibodies are associated with a good prognosis and target nsp3 and nsp5 proteases, whereas IgG antibodies are associated with high mortality and target structural proteins (Nucleocapsid, Spike, ORF3a). The epitopes targeted by antibodies in patients with a high mortality rate were further validated using an independent serum cohort (n = 56) and using global correlation mapping analysis with the clinical variables that are associated with COVID-19 severity. Our data provide fundamental insight into humoral immunity during SARS-CoV-2 infection. SARS-CoV-2 immunogenic epitopes identified in this work could also help direct antibody-based COVID-19 treatment and triage patients.


Assuntos
Anticorpos Antivirais/imunologia , COVID-19/imunologia , Imunidade Humoral , SARS-CoV-2/imunologia , Proteínas não Estruturais Virais/imunologia , COVID-19/mortalidade , Estado Terminal , Intervalo Livre de Doença , Epitopos/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Masculino , Análise Serial de Proteínas , Taxa de Sobrevida
11.
Front Immunol ; 12: 632482, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34276644

RESUMO

Recent evidence suggests that inflammation was participated in the pathogenesis of PD, thus, to understand the potential mechanism of gut microbiota in the pathogenesis of Parkinson's disease (PD), we performed a metagenomic analysis of fecal samples from PD patient and controls. Using a two-stage metagenome-wide association strategy, fecal DNA samples from 69 PD patients and 244 controls in three groups (comprising 66 spouses, 97 age-matched, and 81 normal samples, respectively) were analyzed, and differences between candidate gut microbiota and microbiota-associated epitopes (MEs) were compared. In the study, 27 candidate bacterial biomarkers and twenty-eight candidate epitope peptides were significantly different between the PD patients and control groups. Further, enriched 4 and 13 MEs in PD were positively associated with abnormal inflammatory indicators [neutrophil percentage (NEUT.1), monocyte count/percentage (MONO/MONO.1), white blood cell count (WBC)] and five candidate bacterial biomarkers (c_Actinobacteria, f_Bifidobacteriaceae, g_Bifidobacterium, o_Bifidobacteriales, p_Actinobacteria) from Actinobacteria phylum, and they were also positively associated with histidine degradation and proline biosynthesis pathways, respectively. Additionally, enriched 2 MEs and 1 ME in PD were positively associated with above inflammatory indicators and two bacteria (f_Lactobacillaceae, g_Lactobacillus) from Firmicutes phylum, and they were also positively associated with pyruvate fermentation to propanoate I and negatively associated with isopropanol biosynthesis, respectively. Of these MEs, two MEs from GROEL2, RPSC were derived from Mycobacterium tuberculosis, triggered the T cell immune response, as previously reported. Additionally, other candidate epitope peptides derived from Mycobacterium tuberculosis and Mycobacterium leprae may also have potential immune effects in PD. In all, the altered MEs in PD may relate to abnormalities in immunity and glutamate and propionate metabolism, which furthers our understanding of the pathogenesis of PD.


Assuntos
Actinobacteria/imunologia , Epitopos/imunologia , Firmicutes/imunologia , Doença de Parkinson/microbiologia , Actinobacteria/classificação , Actinobacteria/genética , Actinobacteria/metabolismo , Idoso , Biomarcadores , Vias Biossintéticas , Citocinas/sangue , Fezes/microbiologia , Feminino , Firmicutes/classificação , Firmicutes/genética , Firmicutes/metabolismo , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/imunologia , Humanos , Inflamação , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/imunologia
12.
Sci Rep ; 11(1): 14275, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253751

RESUMO

SARS-CoV-2 infection is characterized by a highly variable clinical course with patients experiencing asymptomatic infection all the way to requiring critical care support. This variation in clinical course has led physicians and scientists to study factors that may predispose certain individuals to more severe clinical presentations in hopes of either identifying these individuals early in their illness or improving their medical management. We sought to understand immunogenomic differences that may result in varied clinical outcomes through analysis of T-cell receptor sequencing (TCR-Seq) data in the open access ImmuneCODE database. We identified two cohorts within the database that had clinical outcomes data reflecting severity of illness and utilized DeepTCR, a multiple-instance deep learning repertoire classifier, to predict patients with severe SARS-CoV-2 infection from their repertoire sequencing. We demonstrate that patients with severe infection have repertoires with higher T-cell responses associated with SARS-CoV-2 epitopes and identify the epitopes that result in these responses. Our results provide evidence that the highly variable clinical course seen in SARS-CoV-2 infection is associated to certain antigen-specific responses.


Assuntos
COVID-19/imunologia , Epitopos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , SARS-CoV-2/imunologia , Infecções Assintomáticas/epidemiologia , COVID-19/patologia , COVID-19/virologia , Aprendizado Profundo , Humanos , Receptores de Antígenos de Linfócitos T/genética , SARS-CoV-2/patogenicidade , Linfócitos T/imunologia , Linfócitos T/virologia
13.
Cell Rep ; 36(2): 109353, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34237283

RESUMO

SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier and caused widespread disease in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterize 198 antibodies isolated from four COVID-19+ subjects and identify 14 SARS-CoV-2 neutralizing antibodies. One targets the N-terminal domain (NTD), one recognizes an epitope in S2, and 11 bind the receptor-binding domain (RBD). Three anti-RBD neutralizing antibodies cross-neutralize SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency and antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. All four cross-neutralizing antibodies neutralize the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Sítios de Ligação , Linhagem Celular , Reações Cruzadas , Epitopos/imunologia , Feminino , Células HEK293 , Humanos , Camundongos , Testes de Neutralização , Ligação Proteica/imunologia , Domínios Proteicos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química
14.
Nat Med ; 27(8): 1442-1450, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34282338

RESUMO

Serum antibodies can recognize both pathogens and commensal gut microbiota. However, our current understanding of antibody repertoires is largely based on DNA sequencing of the corresponding B-cell receptor genes, and actual bacterial antigen targets remain incompletely characterized. Here we have profiled the serum antibody responses of 997 healthy individuals against 244,000 rationally selected peptide antigens derived from gut microbiota and pathogenic and probiotic bacteria. Leveraging phage immunoprecipitation sequencing (PhIP-Seq) based on phage-displayed synthetic oligo libraries, we detect a wide breadth of individual-specific as well as shared antibody responses against microbiota that associate with age and gender. We also demonstrate that these antibody epitope repertoires are more longitudinally stable than gut microbiome species abundances. Serum samples of more than 200 individuals collected five years apart could be accurately matched and could serve as an immunologic fingerprint. Overall, our results suggest that systemic antibody responses provide a non-redundant layer of information about microbiota beyond gut microbial species composition.


Assuntos
Epitopos/imunologia , Imunoglobulinas/imunologia , Microbiota , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Aprendizado de Máquina , Masculino , Metagenômica , Pessoa de Meia-Idade , Biblioteca de Peptídeos , Adulto Jovem
15.
Front Immunol ; 12: 618193, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34262556

RESUMO

Corpora amylacea (CA) in the human brain are polyglucosan bodies that accumulate residual substances originated from aging and both neurodegenerative and infectious processes. These structures, which act as waste containers, are released from the brain to the cerebrospinal fluid, reach the cervical lymph nodes via the meningeal lymphatic system and may be phagocytosed by macrophages. Recent studies indicate that CA present certain neoepitopes (NEs) that can be recognized by natural antibodies of the IgM class, and although evidence of different kinds suggests that these NEs may be formed by carbohydrate structures, their precise nature is unknown. Here, we adapted standard techniques to examine this question. We observed that the preadsorption of IgMs with specific carbohydrates has inhibitory effects on the interaction between IgMs and CA, and found that the digestion of CA proteins had no effect on this interaction. These findings point to the carbohydrate nature of the NEs located in CA. Moreover, the present study indicates that, in vitro, the binding between certain natural IgMs and certain epitopes may be disrupted by certain monosaccharides. We wonder, therefore, whether these inhibitions may also occur in vivo. Further studies should now be carried out to assess the possible in vivo effect of glycemia on the reactivity of natural IgMs and, by extension, on natural immunity.


Assuntos
Envelhecimento , Carboidratos/imunologia , Epitopos/imunologia , Hipocampo/imunologia , Corpos de Inclusão/imunologia , Idoso , Idoso de 80 Anos ou mais , Astrócitos/imunologia , Encéfalo/imunologia , Encéfalo/patologia , Epitopos/metabolismo , Feminino , Humanos , Masculino
16.
Nature ; 596(7871): 276-280, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34237773

RESUMO

The SARS-CoV-2 B.1.617 lineage was identified in October 2020 in India1-5. Since then, it has become dominant in some regions of India and in the UK, and has spread to many other countries6. The lineage includes three main subtypes (B1.617.1, B.1.617.2 and B.1.617.3), which contain diverse mutations in the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein that may increase the immune evasion potential of these variants. B.1.617.2-also termed the Delta variant-is believed to spread faster than other variants. Here we isolated an infectious strain of the Delta variant from an individual with COVID-19 who had returned to France from India. We examined the sensitivity of this strain to monoclonal antibodies and to antibodies present in sera from individuals who had recovered from COVID-19 (hereafter referred to as convalescent individuals) or who had received a COVID-19 vaccine, and then compared this strain with other strains of SARS-CoV-2. The Delta variant was resistant to neutralization by some anti-NTD and anti-RBD monoclonal antibodies, including bamlanivimab, and these antibodies showed impaired binding to the spike protein. Sera collected from convalescent individuals up to 12 months after the onset of symptoms were fourfold less potent against the Delta variant relative to the Alpha variant (B.1.1.7). Sera from individuals who had received one dose of the Pfizer or the AstraZeneca vaccine had a barely discernible inhibitory effect on the Delta variant. Administration of two doses of the vaccine generated a neutralizing response in 95% of individuals, with titres three- to fivefold lower against the Delta variant than against the Alpha variant. Thus, the spread of the Delta variant is associated with an escape from antibodies that target non-RBD and RBD epitopes of the spike protein.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , COVID-19/virologia , Convalescença , Evasão da Resposta Imune/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/epidemiologia , Vacinas contra COVID-19/administração & dosagem , Epitopos/química , Epitopos/genética , Epitopos/imunologia , França , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
17.
Sci Rep ; 11(1): 15431, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326355

RESUMO

Currently, no approved vaccine is available against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes severe respiratory disease. The spike glycoprotein is typically considered a suitable target for MERS-CoV vaccine candidates. A computational strategy can be used to design an antigenic vaccine against a pathogen. Therefore, we used immunoinformatics and computational approaches to design a multi-epitope vaccine that targets the spike glycoprotein of MERS-CoV. After using numerous immunoinformatics tools and applying several immune filters, a poly-epitope vaccine was constructed comprising cytotoxic T-cell lymphocyte (CTL)-, helper T-cell lymphocyte (HTL)-, and interferon-gamma (IFN-γ)-inducing epitopes. In addition, various physicochemical, allergenic, and antigenic profiles were evaluated to confirm the immunogenicity and safety of the vaccine. Molecular interactions, binding affinities, and the thermodynamic stability of the vaccine were examined through molecular docking and dynamic simulation approaches, during which we identified a stable and strong interaction with Toll-like receptors (TLRs). In silico immune simulations were performed to assess the immune-response triggering capabilities of the vaccine. This computational analysis suggested that the proposed vaccine candidate would be structurally stable and capable of generating an effective immune response to combat viral infections; however, experimental evaluations remain necessary to verify the exact safety and immunogenicity profile of this vaccine.


Assuntos
Epitopos/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Vacinas/imunologia , Biologia Computacional , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Humanos , Imunogenicidade da Vacina/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Modelos Moleculares , Simulação de Acoplamento Molecular , Filogenia , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas/farmacologia , Vacinas de Subunidades/imunologia , Vacinas Virais/imunologia
18.
J Gen Virol ; 102(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34280085

RESUMO

Pigs are susceptible to foot-and-mouth disease virus (FMDV), and the humoral immune response plays an essential role in protection against FMDV infection. However, little information is available about FMDV-specific mAbs derived from single B cells of pigs. This study aimed to determine the antigenic features of FMDV that are recognized by antibodies from pigs. Therefore, a panel of pig-derived mAbs against FMDV were developed using fluorescence-based single B cell antibody technology. Western blotting revealed that three of the antibodies (1C6, P2-7E and P2-8G) recognized conserved antigen epitopes on capsid protein VP2, and exhibited broad reactivity against both FMDV serotypes A and O. An alanine-substitution scanning assay and sequence conservation analysis elucidated that these porcine mAbs recognized two conserved epitopes on VP2: a linear epitope (2KKTEETTLL10) in the N terminus and a conformational epitope involving residues K63, H65, L66, F67, D68 and L81 on two ß-sheets (B-sheet and C-sheet) that depended on the integrity of VP2. Random parings of heavy and light chains of the IgGs confirmed that the heavy chain is predominantly involved in binding to antigen. The light chain of porcine IgG contributes to the binding affinity toward an antigen and may function as a support platform for antibody stability. In summary, this study is the first to reveal the conserved antigenic profile of FMDV recognized by porcine B cells and provides a novel method for analysing the antibody response against FMDV in its natural hosts (i.e. pigs) at the clonal level.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Vírus da Febre Aftosa/imunologia , Suínos/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , Afinidade de Anticorpos , Antígenos Virais/imunologia , Linfócitos B/imunologia , Proteínas do Capsídeo/química , Mapeamento de Epitopos , Epitopos/imunologia , Vírus da Febre Aftosa/classificação , Genes de Cadeia Pesada de Imunoglobulina , Genes de Cadeia Leve de Imunoglobulina , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Sorogrupo
19.
Nat Commun ; 12(1): 4210, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244522

RESUMO

Neutralizing antibodies (nAbs) to SARS-CoV-2 hold powerful potentials for clinical interventions against COVID-19 disease. However, their common genetic and biologic features remain elusive. Here we interrogate a total of 165 antibodies from eight COVID-19 patients, and find that potent nAbs from different patients have disproportionally high representation of IGHV3-53/3-66 usage, and therefore termed as public antibodies. Crystal structural comparison of these antibodies reveals they share similar angle of approach to RBD, overlap in buried surface and binding residues on RBD, and have substantial spatial clash with receptor angiotensin-converting enzyme-2 (ACE2) in binding to RBD. Site-directed mutagenesis confirms these common binding features although some minor differences are found. One representative antibody, P5A-3C8, demonstrates extraordinarily protective efficacy in a golden Syrian hamster model against SARS-CoV-2 infection. However, virus escape analysis identifies a single natural mutation in RBD, namely K417N found in B.1.351 variant from South Africa, abolished the neutralizing activity of these public antibodies. The discovery of public antibodies and shared escape mutation highlight the intricate relationship between antibody response and SARS-CoV-2, and provide critical reference for the development of antibody and vaccine strategies to overcome the antigenic variation of SARS-CoV-2.


Assuntos
Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Receptores Virais/imunologia , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Sítios de Ligação/imunologia , COVID-19/imunologia , Cricetinae , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Humanos , Masculino , Testes de Neutralização , Receptores de Antígenos de Linfócitos B/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia
20.
MAbs ; 13(1): 1953683, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34313527

RESUMO

The global pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in widespread social and economic disruption. Effective interventions are urgently needed for the prevention and treatment of COVID-19. Neutralizing monoclonal antibodies (mAbs) have demonstrated their prophylactic and therapeutic efficacy against SARS-CoV-2, and several have been granted authorization for emergency use. Here, we discover and characterize a fully human cross-reactive mAb, MW06, which binds to both SARS-CoV-2 and SARS-CoV spike receptor-binding domain (RBD) and disrupts their interaction with angiotensin-converting enzyme 2 (ACE2) receptors. Potential neutralization activity of MW06 was observed against both SARS-CoV-2 and SARS-CoV in different assays. The complex structure determination and epitope alignment of SARS-CoV-2 RBD/MW06 revealed that the epitope recognized by MW06 is highly conserved among SARS-related coronavirus strains, indicating the potential broad neutralization activity of MW06. In in vitro assays, no antibody-dependent enhancement (ADE) of SARS-CoV-2 infection was observed for MW06. In addition, MW06 recognizes a different epitope from MW05, which shows high neutralization activity and has been in a Phase 2 clinical trial, supporting the development of the cocktail of MW05 and MW06 to prevent against future escaping variants. MW06 alone and the cocktail show good effects in preventing escape mutations, including a series of variants of concern, B.1.1.7, P.1, B.1.351, and B.1.617.1. These findings suggest that MW06 recognizes a conserved epitope on SARS-CoV-2, which provides insights for the development of a universal antibody-based therapy against SARS-related coronavirus and emerging variant strains, and may be an effective anti-SARS-CoV-2 agent.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/virologia , Vírus da SARS/imunologia , SARS-CoV-2/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/química , Anticorpos Antivirais/uso terapêutico , Anticorpos Facilitadores , COVID-19/tratamento farmacológico , COVID-19/terapia , Sequência Conservada , Reações Cruzadas , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Humanos , Modelos Moleculares , Testes de Neutralização , Pandemias , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Vírus da SARS/química , Vírus da SARS/genética , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
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