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1.
Arch Virol ; 166(4): 1163-1170, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33554289

RESUMO

The envelope glycoprotein E2 of pestiviruses is a major target for neutralizing antibodies. In this study, we analyzed the E2 DA domain of 43 pestiviruses from Southern Brazil. The isolates were identified as Bovine viral diarrhea virus (BVDV) subtypes 1a and 1b or BVDV-2b. Compared to reference strains, the BVDV-1 and -2 isolates had four and two mutations in the DA domain, respectively. All BVDV-2 isolates had a deletion of residues 724 and 725. All mutated amino acids in the BVDV isolates had the same aa substitution, and all were in previously identified antibody binding sites. It is possible that an immunity-mediated selection is acting on the pestiviruses circulating in Southern Brazil.


Assuntos
Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/isolamento & purificação , Proteínas do Envelope Viral/genética , Animais , Antígenos Virais/genética , Sítios de Ligação de Anticorpos/genética , Doença das Mucosas por Vírus da Diarreia Viral Bovina/epidemiologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Brasil/epidemiologia , Bovinos , Vírus da Diarreia Viral Bovina/classificação , Vírus da Diarreia Viral Bovina/imunologia , Mutação , Filogenia , RNA Viral/genética , Proteínas do Envelope Viral/imunologia
2.
Science ; 371(6531): 823-829, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33495307

RESUMO

The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. We employed a directed evolution approach to engineer three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains and neutralizes representative epidemic sarbecoviruses with high potency. Structural and biochemical studies demonstrate that ADG-2 employs a distinct angle of approach to recognize a highly conserved epitope that overlaps the receptor binding site. In immunocompetent mouse models of SARS and COVID-19, prophylactic administration of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate against clade 1 sarbecoviruses.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , /metabolismo , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Sítios de Ligação , Sítios de Ligação de Anticorpos , Anticorpos Amplamente Neutralizantes/genética , Anticorpos Amplamente Neutralizantes/metabolismo , /terapia , Técnicas de Visualização da Superfície Celular , Evolução Molecular Direcionada , Epitopos/imunologia , Humanos , Imunização Passiva , Fragmentos Fc das Imunoglobulinas/imunologia , Camundongos Endogâmicos BALB C , Domínios Proteicos , Engenharia de Proteínas , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Síndrome Respiratória Aguda Grave/terapia , Glicoproteína da Espícula de Coronavírus/metabolismo
3.
Nat Commun ; 12(1): 708, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514724

RESUMO

We report the development of a platform of dual targeting Fab (DutaFab) molecules, which comprise two spatially separated and independent binding sites within the human antibody CDR loops: the so-called H-side paratope encompassing HCDR1, HCDR3 and LCDR2, and the L-side paratope encompassing LCDR1, LCDR3 and HCDR2. Both paratopes can be independently selected and combined into the desired bispecific DutaFabs in a modular manner. X-ray crystal structures illustrate that DutaFabs are able to bind two target molecules simultaneously at the same Fv region comprising a VH-VL heterodimer. In the present study, this platform is applied to generate DutaFabs specific for VEGFA and PDGF-BB, which show high affinities, physico-chemical stability and solubility, as well as superior efficacy over anti-VEGF monotherapy in vivo. These molecules exemplify the usefulness of DutaFabs as a distinct class of antibody therapeutics, which is currently being evaluated in patients.


Assuntos
Anticorpos Biespecíficos/farmacologia , Neovascularização de Coroide/tratamento farmacológico , Desenvolvimento de Medicamentos/métodos , Fragmentos Fab das Imunoglobulinas/farmacologia , Engenharia de Proteínas , Sequência de Aminoácidos/genética , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Biespecíficos/ultraestrutura , Becaplermina/antagonistas & inibidores , Sítios de Ligação de Anticorpos/genética , Cristalografia por Raios X , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Concentração Inibidora 50 , Injeções Intravítreas , Masculino , Modelos Moleculares , Estudo de Prova de Conceito , Conformação Proteica , Ratos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores
4.
Food Chem Toxicol ; 149: 112009, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33503469

RESUMO

SARS-CoV-2 is the coronavirus that originated in Wuhan in December 2019 and has spread globally. Studies have shown that smokers are less likely to be diagnosed with or be hospitalized for COVID-19 but, once hospitalized, have higher odds for an adverse outcome. We have previously presented the potential interaction between SARS-CoV-2 Spike glycoprotein and nicotinic acetylcholine receptors (nAChRs), due to a "toxin-like" epitope on the Spike glycoprotein, with homology to a sequence of a snake venom toxin. This epitope coincides with the well-described cryptic epitope for the human anti-SARS-CoV antibody CR3022. In this study, we present the molecular complexes of both SARS-CoV and SARS-CoV-2 Spike glycoproteins, at their open or closed conformations, with the model of the human α7 nAChR. We found that all studied protein complexes' interface involves a large part of the "toxin-like" sequences of SARS-CoV and SARS-CoV-2 Spike glycoproteins and toxin binding site of human α7 nAChR. Our findings provide further support to the hypothesis about the protective role of nicotine and other cholinergic agonists. The potential therapeutic role of CR3022 and other similar monoclonal antibodies with increased affinity for SARS-CoV-2 Spike glycoprotein against the clinical effects originating from the dysregulated cholinergic pathway should be further explored.


Assuntos
/virologia , Epitopos , Nicotina/farmacologia , Vírus da SARS/química , Glicoproteína da Espícula de Coronavírus/química , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/química , Anticorpos Antivirais/uso terapêutico , Sítios de Ligação de Anticorpos , /prevenção & controle , Humanos , Modelos Moleculares , Agonistas Nicotínicos/química , Agonistas Nicotínicos/uso terapêutico , Sistema Colinérgico não Neuronal , Pandemias , Fatores de Proteção , Conformação Proteica , Homologia de Sequência , Transdução de Sinais , Fumantes , Fumar , Venenos de Serpentes/química
5.
Science ; 371(6530)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436526

RESUMO

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, we generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. We used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, we engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , /imunologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Substituição de Aminoácidos , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Antígenos Virais/imunologia , Sítios de Ligação de Anticorpos , Linhagem Celular , Microscopia Crioeletrônica , Epitopos , Humanos , Fusão de Membrana , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , /genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Replicação Viral
6.
Methods Mol Biol ; 2253: 175-183, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33315224

RESUMO

We studied the molecular details of the recognition of antigens by the variable domain of their cognate antibodies in as well as those elicited by the constant domains, which do not directly interact with antigens. Such effects are difficult to study experimentally; however, molecular dynamics simulations and subsequent residue interaction network analysis provide insight into the allosteric communication between the antigen-binding CDR region and the constant domain. We performed MD simulations of the complex of Fab and prion-associated peptide in the apo and bound forms and follow the conformational changes in the antibody and cross-talk between its subunits and with antigens. These protocols could be generally applied for studies of other antigens-antibody recognition systems.


Assuntos
Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Príons/química , Príons/imunologia , Regulação Alostérica , Sítio Alostérico , Animais , Sítios de Ligação de Anticorpos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica
7.
Sci Rep ; 10(1): 22370, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33353972

RESUMO

There are currently few approved effective treatments for SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Nanobodies are 12-15 kDa single-domain antibody fragments that can be delivered by inhalation and are amenable to relatively inexpensive large scale production compared to other biologicals. We have isolated nanobodies that bind to the SARS-CoV-2 spike protein receptor binding domain and block spike protein interaction with the angiotensin converting enzyme 2 (ACE2) with 1-5 nM affinity. The lead nanobody candidate, NIH-CoVnb-112, blocks SARS-CoV-2 spike pseudotyped lentivirus infection of HEK293 cells expressing human ACE2 with an EC50 of 0.3 µg/mL. NIH-CoVnb-112 retains structural integrity and potency after nebulization. Furthermore, NIH-CoVnb-112 blocks interaction between ACE2 and several high affinity variant forms of the spike protein. These nanobodies and their derivatives have therapeutic, preventative, and diagnostic potential.


Assuntos
/metabolismo , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos , Descoberta de Drogas/métodos , Domínios e Motivos de Interação entre Proteínas/imunologia , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/química , /genética , Animais , Anticorpos Neutralizantes/imunologia , Sítios de Ligação de Anticorpos/imunologia , /virologia , Camelídeos Americanos , Células HEK293 , Humanos , Imunização/métodos , Masculino , Ligação Proteica , Transdução de Sinais/genética , Glicoproteína da Espícula de Coronavírus/genética , Transdução Genética , Transfecção
8.
PLoS Pathog ; 16(12): e1009103, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33315937

RESUMO

The antibody molecule comprises a variable domain conferring antigen specificity and affinity distinct from the heavy chain constant (CH) domains dictating effector functions. We here interrogate this paradigm by evaluating the unique influence of the CH1α domain on epitope specificity and functions using two mucosal gp41-specific Fab-IgAs (FabA) derived from HIV-1 highly-exposed but persistently seronegative individuals (HESN). These HESN develop selectively affinity-matured HIV-1-specific mucosal IgA that target the gp41 viral envelope and might provide protection although by unclear mechanisms. Isotype-switching FabAs into Fab-IgGs (FabGs) results in a >10-fold loss in affinity for HIV-1 clade A, B, and C gp41, together with reduced neutralization of HIV-1 cross-clade. The FabA conformational epitopes map selectively on gp41 in 6-Helix bundle and pre-fusion conformations cross-clade, unlike FabGs. Finally, we designed in silico, a 12 amino-acid peptide recapitulating one FabA conformational epitope that inhibits the FabA binding to gp41 cross-clade and its neutralizing activity. Altogether, our results reveal that the CH1α domain shapes the antibody paratope through an allosteric effect, thereby strengthening the antibody specificity and functional activities. Further, they clarify the mechanisms by which these HESN IgAs might confer protection against HIV-1-sexual acquisition. The IgA-specific epitope we characterized by reverse vaccinology could help designing a mucosal HIV-1 vaccine.


Assuntos
Especificidade de Anticorpos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Imunoglobulina A/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Proteína gp41 do Envelope de HIV/imunologia , Soronegatividade para HIV/imunologia , Humanos , Imunoglobulina A/química , Imunoglobulina G/imunologia , Domínios Proteicos/imunologia
9.
Int J Mol Sci ; 22(1)2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374673

RESUMO

T cells are a critical part of the adaptive immune system that are able to distinguish between healthy and unhealthy cells. Upon recognition of protein fragments (peptides), activated T cells will contribute to the immune response and help clear infection. The major histocompatibility complex (MHC) molecules, or human leukocyte antigens (HLA) in humans, bind these peptides to present them to T cells that recognise them with their surface T cell receptors (TCR). This recognition event is the first step that leads to T cell activation, and in turn can dictate disease outcomes. The visualisation of TCR interaction with pMHC using structural biology has been crucial in understanding this key event, unravelling the parameters that drive this interaction and their impact on the immune response. The last five years has been the most productive within the field, wherein half of current unique TCR-pMHC-I structures to date were determined within this time. Here, we review the new insights learned from these recent TCR-pMHC-I structures and their impact on T cell activation.


Assuntos
Antígenos de Histocompatibilidade Classe I/química , Peptídeos/química , Receptores de Antígenos de Linfócitos T/química , Animais , Sítios de Ligação de Anticorpos , Reações Cruzadas , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Ativação Linfocitária , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia
10.
Cell Rep ; 33(4): 108322, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33091382

RESUMO

Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, we design constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor. Yields of biotin-labeled probes from transient transfection range from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes are characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe is determined by cryoelectron microscopy. We also characterize antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike ectodomain probes.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Sondas Moleculares/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Especificidade de Anticorpos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Biotinilação , Microscopia Crioeletrônica , Humanos , Pandemias , Peptidil Dipeptidase A/metabolismo , Receptores Virais/metabolismo
11.
Nat Commun ; 11(1): 5337, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087721

RESUMO

The CR3022 antibody, selected from a group of SARS-CoV monoclonal antibodies for its ability to cross-react with SARS-CoV-2, has been examined for its ability to bind to the ectodomain of the SARS-CoV-2 spike glycoprotein. Using cryo-electron microscopy we show that antibody binding requires rearrangements in the S1 domain that result in dissociation of the spike.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Sítios de Ligação de Anticorpos/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Humanos , Testes de Neutralização , Pandemias , Pneumonia Viral/virologia , Domínios Proteicos/imunologia , Células Vero
12.
J Clin Virol ; 131: 104611, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32882666

RESUMO

BACKGROUND: The involvement of SARS-CoV-2 antibodies in mediating immunopathogenetic events in COVID-19 patients has been suggested. By using several experimental approaches, we investigated the potential association between SARS-CoV-2 IgGs recognizing the spike (S) protein receptor-binding domain (RBD), neutralizing antibodies (NtAb) targeting S, and COVID-19 severity. PATIENTS AND METHODS: This unicenter, retrospective, observational study included 51 hospitalized patients (24 at the intensive care unit; ICU). A total of 93 sera from these patients collected at different time points from the onset of symptoms were analyzed. SARS-CoV-2 RBD IgGs were quantitated by ELISA and NtAb50 titers were measured in a GFP reporterbased pseudotyped virus platform. Demographic and clinical data, complete blood counts, as well as serum levels of ferritin, Dimer-D, C reactive protein (CRP), lactose dehydrogenase (LDH), and interleukin-6 (IL-6) were retrieved from clinical charts. RESULTS: The overall correlation between levels of both antibody measurements was good (Rho = 0.82; P = 0 < 0.001). SARS-CoV-2 RBD IgG and NtAb50 levels in sera collected up to day 30 after the onset of symptoms were comparable between ICU and non-ICU patients (P=>0.1). Four ICU patients died; two of these achieved NtAb50 titers ≥1/160 while the other two exhibited a 1/80 titer. Very weak (Rho=>0.0-<0.2) or weak (Rho=>0.2-<0.4) correlations were observed between anti-RBD IgGs, NtAb50, and serum levels pro-inflammatory biomarkers. CONCLUSIONS: The data presented herein do not support an association between SARS-CoV-2 RBD IgG or NtAb50 levels and COVID-19 severity.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Coronavirus/sangue , Hospitalização/estatística & dados numéricos , Inflamação/sangue , Pneumonia Viral/sangue , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Betacoronavirus , Sítios de Ligação de Anticorpos , Biomarcadores/sangue , Infecções por Coronavirus/imunologia , Feminino , Humanos , Inflamação/virologia , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/imunologia , Estudos Retrospectivos , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto Jovem
13.
J Phys Chem Lett ; 11(19): 8084-8093, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32885971

RESUMO

SARS-CoV-2 is a health threat with dire socioeconomical consequences. As the crucial mediator of infection, the viral glycosylated spike protein (S) has attracted the most attention and is at the center of efforts to develop therapeutics and diagnostics. Herein, we use an original decomposition approach to identify energetically uncoupled substructures as antibody binding sites on the fully glycosylated S. Crucially, all that is required are unbiased MD simulations; no prior knowledge of binding properties or ad hoc parameter combinations is needed. Our results are validated by experimentally confirmed structures of S in complex with anti- or nanobodies. We identify poorly coupled subdomains that are poised to host (several) epitopes and potentially involved in large functional conformational transitions. Moreover, we detect two distinct behaviors for glycans: those with stronger energetic coupling are structurally relevant and protect underlying peptidic epitopes, and those with weaker coupling could themselves be prone to antibody recognition.


Assuntos
Epitopos/química , Simulação de Dinâmica Molecular , Glicoproteína da Espícula de Coronavírus/química , Algoritmos , Betacoronavirus/química , Sítios de Ligação de Anticorpos , Glicosilação , Humanos , Modelos Moleculares , Conformação Molecular , Peptídeos/química , Polissacarídeos/química
14.
Biomed Pharmacother ; 130: 110559, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32768882

RESUMO

As the number of people infected with the newly identified 2019 novel coronavirus (SARS-CoV2) is continuously increasing every day, development of potential therapeutic platforms is vital. Based on the comparatively high similarity of receptor-binding domain (RBD) in SARS-CoV2 and SARS-CoV, it seems crucial to assay the cross-reactivity of anti-SARS-CoV monoclonal antibodies (mAbs) with SARS-CoV2 spike (S)-protein. Indeed, developing mAbs targeting SARS-CoV2 S-protein RBD could show novel applications for rapid and sensitive development of potential epitope-specific vaccines (ESV). Herein, we present an overview on the discovery of new CoV followed by some explanation on the SARS-CoV2 S-protein RBD site. Furthermore, we surveyed the novel therapeutic mAbs for targeting S-protein RBD such as S230, 80R, F26G18, F26G19, CR3014, CR3022, M396, and S230.15. Afterwards, the mechanism of interaction of RBD and different mAbs were explained and it was suggested that one of the SARS-CoV-specific human mAbs, namely CR3022, could show the highest binding affinity with SARS-CoV2 S-protein RBD. Finally, some ongoing challenges and future prospects for rapid and sensitive advancement of therapeutic mAbs targeting S-protein RBD were discussed. In conclusion, it may be proposed that this review may pave the way for recognition of RBD and different mAbs to develop potential therapeutic ESV.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pandemias , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Reações Antígeno-Anticorpo , Antígenos Virais/metabolismo , Sítios de Ligação de Anticorpos , Coronavirus/química , Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Epitopos/imunologia , Humanos , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinas Virais/imunologia
15.
Proc Natl Acad Sci U S A ; 117(29): 16949-16960, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32616569

RESUMO

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.


Assuntos
Anticorpos/química , Afinidade de Anticorpos , Arginase/imunologia , Regiões Determinantes de Complementaridade/química , Anticorpos/genética , Anticorpos/imunologia , Sítios de Ligação de Anticorpos , Regiões Determinantes de Complementaridade/imunologia , Humanos
16.
Science ; 369(6506): 1010-1014, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32540901

RESUMO

Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola-one from genetically humanized mice and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, which yielded a large collection of fully human antibodies that were characterized for binding, neutralization, and three-dimensional structure. On the basis of these criteria, we selected pairs of highly potent individual antibodies that simultaneously bind the receptor binding domain of the spike protein, thereby providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single-antibody treatment.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adolescente , Adulto , Animais , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Afinidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Betacoronavirus/química , Sítios de Ligação de Anticorpos , Anticorpos Amplamente Neutralizantes/química , Anticorpos Amplamente Neutralizantes/imunologia , Linhagem Celular , Infecções por Coronavirus/terapia , Citofagocitose , Epitopos , Humanos , Imunização Passiva , Camundongos , Pessoa de Meia-Idade , Modelos Moleculares , Testes de Neutralização , Pandemias , Peptidil Dipeptidase A/metabolismo , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/metabolismo , Vírus da SARS/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Adulto Jovem
17.
PLoS Comput Biol ; 16(5): e1007830, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365091

RESUMO

It is difficult to predict how antibodies will behave when mixed together, even after each has been independently characterized. Here, we present a statistical mechanical model for the activity of antibody mixtures that accounts for whether pairs of antibodies bind to distinct or overlapping epitopes. This model requires measuring n individual antibodies and their [Formula: see text] pairwise interactions to predict the 2n potential combinations. We apply this model to epidermal growth factor receptor (EGFR) antibodies and find that the activity of antibody mixtures can be predicted without positing synergy at the molecular level. In addition, we demonstrate how the model can be used in reverse, where straightforward experiments measuring the activity of antibody mixtures can be used to infer the molecular interactions between antibodies. Lastly, we generalize this model to analyze engineered multidomain antibodies, where components of different antibodies are tethered together to form novel amalgams, and characterize how well it predicts recently designed influenza antibodies.


Assuntos
Anticorpos Monoclonais/imunologia , Modelos Biológicos , Anticorpos Antivirais/imunologia , Sítios de Ligação de Anticorpos , Epitopos/imunologia , Orthomyxoviridae/imunologia
18.
Emerg Microbes Infect ; 9(1): 775-786, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32241249

RESUMO

One of the major goals in HIV-1 vaccine development is to achieve properly folded and stabilized envelope glycoprotein (Env) trimers that mimic the native Env on the mature virion. Here, we design and characterize uncleaved prefusion-optimized (UFO) trimers for 12 Envs currently circulating in China. Biochemical and biophysical characterization of these UFO trimers identified two subtype B/B' Envs, CNE6 and MG13, which exhibited the highest trimer content and stability at a level comparable to the subtype A reference, BG505. Replacing the gp41 ectodomain (gp41ECTO) of CRF01_AE trimers with that of CNE6, MG13, and BG505 resulted in chimeric constructs with significantly improved trimer content and stability. Negative-stain electron microscopy (EM) confirmed the structural integrity of these chimeric UFO trimers with CNE6 gp41ECTO. Antibody binding assays showed that the chimeric trimers shared similar antigenic profiles to those with their original gp41ECTO domains. Our results thus revealed the intrinsic differences among HIV-1 Envs of diverse origins and the critical role of gp41ECTO in stabilizing the trimeric spike. By taking advantage of naturally stable Envs, gp41ECTO swapping may represent a universal approach for the generation of stable trimers with the desired structural and antigenic properties for downstream in vivo evaluation and vaccine development.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/imunologia , Multimerização Proteica/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Anticorpos Neutralizantes/imunologia , Sítios de Ligação de Anticorpos , Desenho de Fármacos , HIV-1 , Humanos , Imunogenicidade da Vacina
19.
Subcell Biochem ; 94: 465-497, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32189312

RESUMO

In vertebrates, immunoglobulins (Igs), commonly known as antibodies, play an integral role in the armamentarium of immune defense against various pathogens. After an antigenic challenge, antibodies are secreted by differentiated B cells called plasma cells. Antibodies have two predominant roles that involve specific binding to antigens to launch an immune response, along with activation of other components of the immune system to fight pathogens. The ability of immunoglobulins to fight against innumerable and diverse pathogens lies in their intrinsic ability to discriminate between different antigens. Due to this specificity and high affinity for their antigens, antibodies have been a valuable and indispensable tool in research, diagnostics and therapy. Although seemingly a simple maneuver, the association between an antibody and its antigen, to make an antigen-antibody complex, is comprised of myriads of non-covalent interactions. Amino acid residues on the antigen binding site, the epitope, and on the antibody binding site, the paratope, intimately contribute to the energetics needed for the antigen-antibody complex stability. Structural biology methods to study antigen-antibody complexes are extremely valuable tools to visualize antigen-antibody interactions in detail; this helps to elucidate the basis of molecular recognition between an antibody and its specific antigen. The main scope of this chapter is to discuss the structure and function of different classes of antibodies and the various aspects of antigen-antibody interactions including antigen-antibody interfaces-with a special focus on paratopes, complementarity determining regions (CDRs) and other non-CDR residues important for antigen binding and recognition. Herein, we also discuss methods used to study antigen-antibody complexes, antigen recognition by antibodies, types of antigens in complexes, and how antigen-antibody complexes play a role in modern day medicine and human health. Understanding the molecular basis of antigen binding and recognition by antibodies helps to facilitate the production of better and more potent antibodies for immunotherapy, vaccines and various other applications.


Assuntos
Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Animais , Sítios de Ligação de Anticorpos , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/imunologia , Epitopos/química , Epitopos/imunologia , Humanos , Modelos Moleculares
20.
PLoS Pathog ; 16(3): e1008373, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32150583

RESUMO

Lasting protection has long been a goal for malaria vaccines. The major surface antigen on Plasmodium falciparum sporozoites, the circumsporozoite protein (PfCSP), has been an attractive target for vaccine development and most protective antibodies studied to date interact with the central NANP repeat region of PfCSP. However, it remains unclear what structural and functional characteristics correlate with better protection by one antibody over another. Binding to the junctional region between the N-terminal domain and central NANP repeats has been proposed to result in superior protection: this region initiates with the only NPDP sequence followed immediately by NANP. Here, we isolated antibodies in Kymab mice immunized with full-length recombinant PfCSP and two protective antibodies were selected for further study with reactivity against the junctional region. X-ray and EM structures of two monoclonal antibodies, mAb667 and mAb668, shed light on their differential affinity and specificity for the junctional region. Importantly, these antibodies also bind to the NANP repeat region with equal or better affinity. A comparison with an NANP-only binding antibody (mAb317) revealed roughly similar but statistically distinct levels of protection against sporozoite challenge in mouse liver burden models, suggesting that junctional antibody protection might relate to the ability to also cross-react with the NANP repeat region. Our findings indicate that additional efforts are necessary to isolate a true junctional antibody with no or much reduced affinity to the NANP region to elucidate the role of the junctional epitope in protection.


Assuntos
Anticorpos Monoclonais Murinos/química , Anticorpos Antiprotozoários/química , Sítios de Ligação de Anticorpos , Epitopos/química , Plasmodium falciparum/química , Proteínas de Protozoários/química , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Antiprotozoários/imunologia , Epitopos/imunologia , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Relação Estrutura-Atividade
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