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1.
Nature ; 584(7820): 274-278, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760003

RESUMO

Colonization by the microbiota causes a marked stimulation of B cells and induction of immunoglobulin, but mammals colonized with many taxa have highly complex and individualized immunoglobulin repertoires1,2. Here we use a simplified model of defined transient exposures to different microbial taxa in germ-free mice3 to deconstruct how the microbiota shapes the B cell pool and its functional responsiveness. We followed the development of the immunoglobulin repertoire in B cell populations, as well as single cells by deep sequencing. Microbial exposures at the intestinal mucosa generated oligoclonal responses that differed from those of germ-free mice, and from the diverse repertoire that was generated after intravenous systemic exposure to microbiota. The IgA repertoire-predominantly to cell-surface antigens-did not expand after dose escalation, whereas increased systemic exposure broadened the IgG repertoire to both microbial cytoplasmic and cell-surface antigens. These microbial exposures induced characteristic immunoglobulin heavy-chain repertoires in B cells, mainly at memory and plasma cell stages. Whereas sequential systemic exposure to different microbial taxa diversified the IgG repertoire and facilitated alternative specific responses, sequential mucosal exposure produced limited overlapping repertoires and the attrition of initial IgA binding specificities. This shows a contrast between a flexible response to systemic exposure with the need to avoid fatal sepsis, and a restricted response to mucosal exposure that reflects the generic nature of host-microbial mutualism in the mucosa.


Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Imunidade nas Mucosas/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Simbiose/imunologia , Administração Intravenosa , Administração Oral , Animais , Clostridiales/imunologia , Clostridiales/isolamento & purificação , Escherichia coli/imunologia , Escherichia coli/isolamento & purificação , Feminino , Vida Livre de Germes , Imunoglobulina A/química , Imunoglobulina A/imunologia , Imunoglobulina G/química , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Memória Imunológica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasmócitos/citologia , Plasmócitos/imunologia , Priming de Repetição
2.
Medicina (B Aires) ; 80 Suppl 3: 1-6, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658841

RESUMO

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab')2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.


Assuntos
Anticorpos Antivirais , Infecções por Coronavirus/terapia , Soros Imunes/imunologia , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/isolamento & purificação , Pandemias , Pneumonia Viral , Glicoproteína da Espícula de Coronavírus , Animais , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Argentina , Betacoronavirus , Cavalos , Humanos , Imunização Passiva , Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/química , Testes de Neutralização
3.
Cell ; 182(4): 828-842.e16, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32645326

RESUMO

Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.


Assuntos
Anticorpos Neutralizantes/química , Betacoronavirus/química , Infecções por Coronavirus/imunologia , Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/química , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/química , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Betacoronavirus/imunologia , Infecções por Coronavirus/sangue , Infecções por Coronavirus/terapia , Reações Cruzadas , Microscopia Crioeletrônica , Mapeamento de Epitopos , Epitopos , Humanos , Imunização Passiva , Fragmentos Fab das Imunoglobulinas/sangue , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Imunoglobulina G/sangue , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/ultraestrutura , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Modelos Moleculares , Pandemias , Pneumonia Viral/sangue , Vírus da SARS/química , Vírus da SARS/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia
4.
J Biol Chem ; 295(36): 12814-12821, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32727845

RESUMO

There is a desperate need for safe and effective vaccines, therapies, and diagnostics for SARS- coronavirus 2 (CoV-2), the development of which will be aided by the discovery of potent and selective antibodies against relevant viral epitopes. Human phage display technology has revolutionized the process of identifying and optimizing antibodies, providing facile entry points for further applications. Herein, we use this technology to search for antibodies targeting the receptor-binding domain (RBD) of CoV-2. Specifically, we screened a naïve human semisynthetic phage library against RBD, leading to the identification of a high-affinity single-chain fragment variable region (scFv). The scFv was further engineered into two other antibody formats (scFv-Fc and IgG1). All three antibody formats showed high binding specificity to CoV-2 RBD and the spike antigens in different assay systems. Flow cytometry analysis demonstrated specific binding of the IgG1 format to cells expressing membrane-bound CoV-2 spike protein. Docking studies revealed that the scFv recognizes an epitope that partially overlaps with angiotensin-converting enzyme 2 (ACE2)-interacting sites on the CoV-2 RBD. Given its high specificity and affinity, we anticipate that these anti-CoV-2 antibodies will be useful as valuable reagents for accessing the antigenicity of vaccine candidates, as well as developing antibody-based therapeutics and diagnostics for CoV-2.


Assuntos
Afinidade de Anticorpos , Anticorpos de Cadeia Única/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sítios de Ligação , Epitopos/química , Epitopos/imunologia , Células HEK293 , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Simulação de Acoplamento Molecular , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Anticorpos de Cadeia Única/química , Glicoproteína da Espícula de Coronavírus/química
5.
Int J Mol Sci ; 21(11)2020 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-32503354

RESUMO

Monoclonal antibodies, engineered antibodies, and antibody fragments have become important biological therapeutic platforms. The IgG format with bivalent binding sites has a modular structure with different biological roles, i.e., effector and binding functions, in different domains. We demonstrated the reconstruction of an IgG-like domain structure in vitro by protein ligation using protein trans-splicing. We produced various binding domains to replace the binding domain of IgG from Escherichia coli and the Fc domain of human IgG from Brevibacillus choshinensis as split-intein fusions. We showed that in vitro protein ligation could produce various Fc-fusions at the N-terminus in vitro from the independently produced domains from different organisms. We thus propose an off-the-shelf approach for the combinatorial production of Fc fusions in vitro with several distinct binding domains, particularly from naturally occurring binding domains. Antiviral lectins from algae are known to inhibit virus entry of HIV and SARS coronavirus. We demonstrated that a lectin could be fused with the Fc-domain in vitro by protein ligation, producing an IgG-like molecule as a "lectibody". Such an Fc-fusion could be produced in vitro by this approach, which could be an attractive method for developing potential therapeutic agents against rapidly emerging infectious diseases like SARS coronavirus without any genetic fusion and expression optimization.


Assuntos
Fragmentos Fc das Imunoglobulinas/metabolismo , Lectinas/metabolismo , Trans-Splicing , Brevibacillus/imunologia , Clorófitas/metabolismo , HIV/fisiologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Lectinas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Vírus da SARS/fisiologia , Internalização do Vírus/efeitos dos fármacos
6.
Cell ; 181(5): 1004-1015.e15, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: covidwho-88549

RESUMO

Coronaviruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics. Here, we describe the isolation of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs neutralize MERS-CoV or SARS-CoV-1 S pseudotyped viruses, respectively. Crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs interfere with receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S and demonstrate that this cross-reactive VHH neutralizes SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Betacoronavirus/imunologia , Anticorpos de Domínio Único/isolamento & purificação , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Camelídeos Americanos/imunologia , Infecções por Coronavirus/terapia , Reações Cruzadas , Imunoglobulina G/química , Imunoglobulina G/imunologia , Modelos Moleculares , Pandemias , Pneumonia Viral/terapia , Domínios Proteicos , Receptores Virais/química , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia
7.
PLoS One ; 15(5): e0232713, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379792

RESUMO

For an antibody to be a successful therapeutic many competing factors require optimization, including binding affinity, biophysical characteristics, and immunogenicity risk. Additional constraints may arise from the need to formulate antibodies at high concentrations (>150 mg/ml) to enable subcutaneous dosing with reasonable volume (ideally <1.0 mL). Unfortunately, antibodies at high concentrations may exhibit high viscosities that place impractical constraints (such as multiple injections or large needle diameters) on delivery and impede efficient manufacturing. Here we describe the optimization of an anti-PDGF-BB antibody to reduce viscosity, enabling an increase in the formulated concentration from 80 mg/ml to greater than 160 mg/ml, while maintaining the binding affinity. We performed two rounds of structure guided rational design to optimize the surface electrostatic properties. Analysis of this set demonstrated that a net-positive charge change, and disruption of negative charge patches were associated with decreased viscosity, but the effect was greatly dependent on the local surface environment. Our work here provides a comprehensive study exploring a wide sampling of charge-changes in the Fv and CDR regions along with targeting multiple negative charge patches. In total, we generated viscosity measurements for 40 unique antibody variants with full sequence information which provides a significantly larger and more complete dataset than has previously been reported.


Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Becaplermina/imunologia , Desenho Assistido por Computador , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Modelos Moleculares , Mutação , Conformação Proteica , Propriedades de Superfície , Viscosidade
8.
Transfusion ; 60(5): 1060-1068, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32369193

RESUMO

BACKGROUND: Anti-red blood cell (RBC) alloantibodies consisting of only the immunoglobulin G (IgG) 4 subtype are typically considered clinically insignificant. A US Food and Drug Administration-approved monoclonal anti-human globulin (16H8) is nonreactive with IgG4, which has been considered a benefit to avoid testing interference from IgG4. However, 16H8 also does not recognize two natural IgG3 variants (IgG3-03 and IgG3-13). Thus, 16H8 may miss clinically significant alloantibodies in some settings. STUDY DESIGN AND METHODS: Novel mouse anti-human IgG hybridomas were generated and screened for reactivity with 32 human variants of anti-KEL1 across different IgG subtypes, as well as mutants to allow epitope mapping. Anti-IgG reactivity was determined using KEL1+ RBCs bound by each IgG variant as targets. Binding of anti-IgG was determined by flow cytometry. RESULTS: 16H8 recognized an epitope involving amino acid 419, which is glutamate in IgG4, IgG3-03, and IgG3-13, explaining the lack of 16H8 reactivity with these subtypes/isoallotypes. A new monoclonal antibody (PUMA8) was isolated that, like 16H8, was nonreactive with IgG4 but without blind spots for known variants of IgG1, IgG2, or IgG3. PUMA8 recognized an epitope containing arginine at position 355, which is glutamine in IgG4. However, a recently described new IgG4 variant with an arginine at position 355 results in PUMA8 reactivity. CONCLUSION: PUMA8 represents an alternative to 16H8 that avoids IgG4 but without blind spots for IgG3 variants. However, PUMA8 reacts with one recently described IgG4 variant. In addition to relevance to immunohematology, these studies highlight the importance of patient variation with regards to assay performance in an era of personalized medicine.


Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Imunoglobulinas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Cricetulus , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Eritrócitos/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Testes Imunológicos , Isoanticorpos/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica , Análise de Sequência de Proteína
9.
Cell ; 181(5): 1004-1015.e15, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32375025

RESUMO

Coronaviruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics. Here, we describe the isolation of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs neutralize MERS-CoV or SARS-CoV-1 S pseudotyped viruses, respectively. Crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs interfere with receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S and demonstrate that this cross-reactive VHH neutralizes SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Betacoronavirus/imunologia , Anticorpos de Domínio Único/isolamento & purificação , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Camelídeos Americanos/imunologia , Infecções por Coronavirus/terapia , Reações Cruzadas , Imunoglobulina G/química , Imunoglobulina G/imunologia , Modelos Moleculares , Pandemias , Pneumonia Viral/terapia , Domínios Proteicos , Receptores Virais/química , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia
10.
Nat Commun ; 11(1): 2070, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: covidwho-116533

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, at the end of 2019, and there are currently no specific antiviral treatments or vaccines available. SARS-CoV-2 has been shown to use the same cell entry receptor as SARS-CoV, angiotensin-converting enzyme 2 (ACE2). In this report, we generate a recombinant protein by connecting the extracellular domain of human ACE2 to the Fc region of the human immunoglobulin IgG1. A fusion protein containing an ACE2 mutant with low catalytic activity is also used in this study. The fusion proteins are then characterized. Both fusion proteins have a high binding affinity for the receptor-binding domains of SARS-CoV and SARS-CoV-2 and exhibit desirable pharmacological properties in mice. Moreover, the fusion proteins neutralize virus pseudotyped with SARS-CoV or SARS-CoV-2 spike proteins in vitro. As these fusion proteins exhibit cross-reactivity against coronaviruses, they have potential applications in the diagnosis, prophylaxis, and treatment of SARS-CoV-2.


Assuntos
Betacoronavirus/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Testes de Neutralização , Peptidil Dipeptidase A/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Animais , Betacoronavirus/metabolismo , Ligação Competitiva/efeitos dos fármacos , Reações Cruzadas , Desenho de Fármacos , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Técnicas In Vitro , Concentração Inibidora 50 , Fusão de Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/farmacocinética , Peptidil Dipeptidase A/farmacologia , Domínios Proteicos/genética , Estabilidade Proteica , Receptores Virais/antagonistas & inibidores , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
11.
Nat Commun ; 11(1): 2070, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332765

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, at the end of 2019, and there are currently no specific antiviral treatments or vaccines available. SARS-CoV-2 has been shown to use the same cell entry receptor as SARS-CoV, angiotensin-converting enzyme 2 (ACE2). In this report, we generate a recombinant protein by connecting the extracellular domain of human ACE2 to the Fc region of the human immunoglobulin IgG1. A fusion protein containing an ACE2 mutant with low catalytic activity is also used in this study. The fusion proteins are then characterized. Both fusion proteins have a high binding affinity for the receptor-binding domains of SARS-CoV and SARS-CoV-2 and exhibit desirable pharmacological properties in mice. Moreover, the fusion proteins neutralize virus pseudotyped with SARS-CoV or SARS-CoV-2 spike proteins in vitro. As these fusion proteins exhibit cross-reactivity against coronaviruses, they have potential applications in the diagnosis, prophylaxis, and treatment of SARS-CoV-2.


Assuntos
Betacoronavirus/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Testes de Neutralização , Peptidil Dipeptidase A/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Animais , Betacoronavirus/metabolismo , Ligação Competitiva/efeitos dos fármacos , Reações Cruzadas , Desenho de Fármacos , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Técnicas In Vitro , Concentração Inibidora 50 , Fusão de Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/farmacocinética , Peptidil Dipeptidase A/farmacologia , Domínios Proteicos/genética , Estabilidade Proteica , Receptores Virais/antagonistas & inibidores , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo
12.
PLoS One ; 15(4): e0230962, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32282813

RESUMO

A designed disulfide-rich ß-hairpin peptide that dimerizes spontaneously served as a hinge-type connection between proteins. Here, we analyze the range of dynamics of this hinge dimer with the aim of proposing new applications for the DNA-encodable peptide and establishing guidelines for the computational analysis of other disulfide hinges. A recent structural analysis based on nuclear magnetic resonance spectroscopy and ion mobility spectrometry revealed an averaged conformation in the hinge region which motivated us to investigate the dynamic behavior using a combination of molecular dynamics simulation, metadynamics and free energy surface analysis to characterize the conformational space available to the hinge. Principal component analysis uncovered two slow modes of the peptide, namely, the opening and closing motion and twisting of the two ß-hairpins assembling the hinge. Applying a collective variable (CV) that mimics the first dominating mode, led to a major expansion of the conformational space. The description of the dynamics could be achieved by analysis of the opening angle and the twisting of the ß-hairpins and, thus, offers a methodology that can also be transferred to other derivatives. It has been demonstrated that the hinge peptide's lowest energy conformation consists of a large opening angle and strong twist but is separated by small energy barriers and can, thus, adopt a closed and untwisted structure. With the aim of proposing further applications for the hinge peptide, we simulated its behavior in the sterically congested environment of a four-helix bundle. Preliminary investigations show that one helix is pushed out and a three-helix bundle forms. The insights gained into the dynamics of the tetra-disulfide peptide and analytical guidelines developed in this study may contribute to the understanding of the structure and function of more complex hinge-type proteins, such as the IgG antibody family.


Assuntos
Peptídeos/química , DNA/química , Dissulfetos/química , Imunoglobulina G/química , Simulação de Dinâmica Molecular , Conformação Proteica
13.
Nat Commun ; 11(1): 1816, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286330

RESUMO

Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequences with desired properties is labour-intensive and time-consuming. Here we show that an assay in the periplasm of E. coli linking aggregation directly to antibiotic resistance acts as a sensor for the innate (un-accelerated) aggregation of antibody fragments. Using this assay as a directed evolution screen, we demonstrate the generation of aggregation resistant scFv sequences when reformatted as IgGs. This powerful tool can thus screen and evolve 'manufacturable' biopharmaceuticals early in industrial development. By comparing the mutational profiles of three different immunoglobulin scaffolds, we show the applicability of this method to investigate protein aggregation mechanisms important to both industrial manufacture and amyloid disease.


Assuntos
Agregados Proteicos , Sequência de Aminoácidos , Substituição de Aminoácidos , Regiões Determinantes de Complementaridade/química , Escherichia coli/metabolismo , Humanos , Imunoglobulina G/química , Viabilidade Microbiana , Mutação/genética , Anticorpos de Cadeia Única/química , beta-Lactamases/química
14.
J Dairy Sci ; 103(6): 5662-5667, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32307161

RESUMO

We studied changes in chemical composition, somatic cell count, and immunoglobulin G (IgG) and M (IgM) content in red deer (Cervus elaphus) colostrum during the transition to milk at different times after parturition (<5 h, 24 h, 48 h, 2 wk, and 4 wk). The production level was higher at 2 and 4 wk of lactation than during the first day after parturition, with intermediate values at 48 h postpartum. Fat content did not vary during the study period. However, total protein and casein contents were particularly high in the initial 5 h after parturition, decreasing to approximately 50% after 24 h postpartum. Conversely, lactose concentration was low in the beginning (<5 h), increasing gradually throughout the study. Similarly, dry matter dropped during the first 24 h and then remained constant throughout the study. Urea content decreased during the study, showing a slight recovery at 4 wk. Somatic cell count was higher during the first hours after parturition and gradually decreased throughout the study period. The IgG content was higher before 5 h postpartum than at 24 h postpartum. After 5 h, the level of IgG decreased progressively until it reached 0.18 mg/mL at 4 wk of lactation. We observed a similar pattern for IgM content, but it decreased more quickly than IgG and was not detected after 2 wk. In the case of deer, milk should be considered transitional from 24 to 48 h after parturition, and samples collected after 2 wk can be considered mature milk.


Assuntos
Colostro/química , Cervos/fisiologia , Lactação/fisiologia , Leite/química , Animais , Caseínas/análise , Contagem de Células , Feminino , Imunoglobulina G/química , Lactose/análise , Gravidez
15.
Biochim Biophys Acta Proteins Proteom ; 1868(6): 140410, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32169581

RESUMO

Isomerization of aspartate (Asp) is a common non-enzymatic posttranslational modification. Isomerized residues accumulate in proteins associated with age-related human disorders such as cataract and are well known to affect protein structure and function. We previously detected d-Asp-containing peptides in human serum. In this study, we investigated whether isomerized Asp residues are present in human immunoglobulin G (IgG) kappa chain by a qualitative d-amino acid analysis based on diastereomer formation and liquid chromatography tandem mass spectrometry (LC-MS/MS). We also investigated the d/l ratio of Asp residues in the IgG kappa chain in serum from donors aged 25, 37, 41, 54 and 67 years. As a result, two isomerized Asp residues, Asp151 and Asp170, were detected in the IgG kappa chain, and the d/l ratio of these residues was found to increase with aging. To assess the effects of this isomerization, we synthesized four isomeric peptides of IgG kappa chain containing lα-, lß-, dα-, or dß-Asp at position 170, and compared their secondary structures by CD spectroscopy. Peptide containing normal lα-Asp170 showed type II ß-turn structure, while the other isomeric peptides showed random structure, clearly indicating that substitution of a single Asp isomer alters the secondary structure of the peptide. Because IgG is a main component of humoral immunity, Asp isomerization in IgG may reflect changes of structure and decrease in immune function. Proteome research on serum from the standpoint of racemization might enable us to develop new kinds of biomarker and new directions to study the aging process.


Assuntos
Envelhecimento/metabolismo , Ácido Aspártico/química , Imunoglobulina G/química , Fatores Imunológicos/química , Adulto , Idoso , Biomarcadores , Catarata/metabolismo , Cromatografia Líquida/métodos , Humanos , Imunoglobulina G/metabolismo , Cadeias kappa de Imunoglobulina , Isomerismo , Pessoa de Meia-Idade , Modelos Moleculares , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteoma , Soro , Estereoisomerismo , Espectrometria de Massas em Tandem/métodos
16.
PLoS One ; 15(3): e0229027, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32182240

RESUMO

Human immunoglobulin G isotype 4 (IgG4) antibodies are suitable for use in either the antagonist or agonist format because their low effector functions prevent target cytotoxicity or unwanted cytokine secretion. However, while manufacturing therapeutic antibodies, they are exposed to low pH during purification, and IgG4 is more susceptible to low-pH-induced aggregation than IgG1. Therefore, we investigated the underlying mechanisms of IgG4 aggregation at low pH and engineered an IgG4 with enhanced stability. By swapping the constant regions of IgG1 and IgG4, we determined that the constant heavy chain (CH3) domain is critical for aggregate formation, but a core-hinge-stabilizing S228P mutation in IgG4 is insufficient for preventing aggregation. To identify the aggregation-prone amino acid, we substituted the CH3 domain of IgG4 with that of IgG1, changing IgG4 Arg409 to a Lys, thereby preventing the aggregation of the IgG4 variant as effectively as in IgG1. A stabilizing effect was also recorded with other variable-region variants. Analysis of thermal stability using differential scanning calorimetry revealed that the R409K substitution increased the Tm value of CH3, suggesting that the R409K mutation contributed to the structural strengthening of the CH3-CH3 interaction. The R409K mutation did not influence the binding to antigens/human Fcγ receptors; whereas, the concurrent S228P and R409K mutations in IgG4 suppressed Fab-arm exchange drastically and as effectively as in IgG1, in both in vitro and in vivo in mice models. Our findings suggest that the IgG4 R409K variant represents a potential therapeutic IgG for use in low-effector-activity format that exhibits increased stability.


Assuntos
Substituição de Aminoácidos , Imunoglobulina G/química , Anticorpos Monoclonais/química , Varredura Diferencial de Calorimetria , Linhagem Celular , Desenho de Fármacos , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/genética , Agregados Proteicos/efeitos dos fármacos , Domínios Proteicos , Estabilidade Proteica
17.
PLoS One ; 15(3): e0230033, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32150580

RESUMO

Therapy regimens for Chronic lymphocytic leukemia (CLL) commonly include chemotherapy and immunotherapy, which act through complement-mediated-cytotoxicity (CDC) and other mechanisms. CDC depends on several factors, including the availability and activity of the complement classical pathway (CP). Recently, a significant decrease in CP activity was shown to be associated with an immunoglobulin-C5a complex (Ig-C5a) and other markers of chronic CP activation in 40% of the patients. The study focused on the involvement of IgG-hexamers, an established CP activator, in the mechanism of chronic CP activation in CLL. Sera from 51 naïve CLL patients and 20 normal controls were collected. CP and alternative pathway (AP) activities were followed by the complement activity marker sC5b-9. Serum high molecular weight (HMW) proteins were collected by gel-filtration chromatography and their complement activation capacity was assessed. The levels of IgM, another established CP activator, were measured. Data were associated with the presence of Ig-C5a. Baseline levels of activation markers negatively correlated with CP and the AP activities, supporting chronic complement activation. In patients with Ig-C5a, HMW proteins that are not IgM, activated the complement. HMW proteins were identified as IgG-aggregates by affinity binding assays and Western blot analysis. The data indicate chronic CP activation, mediated by cell-free IgG-hexamers as a cause of decreased CP activity in part of the CLL population. This mechanism may affect immunotherapy outcomes due to compromised CP activity and CDC.


Assuntos
Via Clássica do Complemento , Imunoglobulina G/sangue , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/imunologia , Idoso , Feminino , Humanos , Imunoglobulina G/química , Masculino , Pessoa de Meia-Idade , Peso Molecular
18.
Biochemistry (Mosc) ; 85(1): 80-89, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32079519

RESUMO

Here, we determined qualitative and quantitative characteristics of the chaperone and immunoglobulin-binding activities of recombinant Skp protein (rSkp) from Yersinia pseudotuberculosis using the methods of dynamic light scattering and surface plasmon resonance. Commercial human polyclonal IgG and Fc and Fab fragments of human IgG were used as substrate proteins. The activity of rSkp strongly depended on the medium pH. The most stable low-molecular-weight complexes with a hydrodynamic radius up to 10 nm were formed by rSkp and protein substrates at acidic pH values. Under these conditions, rSkp exhibited the lowest propensity to self-association and the highest affinity for human IgG and its Fc and Fab fragments, as well as prevented their aggregation most efficiently (i.e., demonstrated the maximal chaperone activity). As the medium pH increased, the affinity of rSkp for IgG and its fragments decreased; rSkp was not able to completely prevent the aggregation of protein substrates, but significantly slowed it down. The obtained information may be of practical interest, since the stability of therapeutic IgG preparations affects their safety and efficacy in medical applications.


Assuntos
Proteínas de Bactérias/química , Chaperonas Moleculares/química , Proteínas Recombinantes/química , Proteínas Quinases Associadas a Fase S/química , Yersinia pseudotuberculosis/metabolismo , Clonagem Molecular , Escherichia coli/genética , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Dobramento de Proteína
19.
Nat Med ; 26(2): 228-235, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32015557

RESUMO

Zika virus (ZIKV) has caused significant disease, with widespread cases of neurological pathology and congenital neurologic defects. Rapid vaccine development has led to a number of candidates capable of eliciting potent ZIKV-neutralizing antibodies (reviewed in refs. 1-3). Despite advances in vaccine development, it remains unclear how ZIKV vaccination affects immune responses in humans with prior flavivirus immunity. Here we show that a single-dose immunization of ZIKV purified inactivated vaccine (ZPIV)4-7 in a dengue virus (DENV)-experienced human elicited potent cross-neutralizing antibodies to both ZIKV and DENV. Using a unique ZIKV virion-based sorting strategy, we isolated and characterized multiple antibodies, including one termed MZ4, which targets a novel site of vulnerability centered on the Envelope (E) domain I/III linker region and protects mice from viremia and viral dissemination following ZIKV or DENV-2 challenge. These data demonstrate that Zika vaccination in a DENV-experienced individual can boost pre-existing flavivirus immunity and elicit protective responses against both ZIKV and DENV. ZPIV vaccination in Puerto Rican individuals with prior flavivirus experience yielded similar cross-neutralizing potency after a single vaccination, highlighting the potential benefit of ZIKV vaccination in flavivirus-endemic areas.


Assuntos
Dengue/imunologia , Doadores de Tecidos , Vacinas Virais/uso terapêutico , Infecção por Zika virus/imunologia , Infecção por Zika virus/prevenção & controle , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Reações Cruzadas , Vírus da Dengue , Mapeamento de Epitopos , Feminino , Flavivirus/metabolismo , Humanos , Imunoglobulina G/química , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ligação Proteica , Domínios Proteicos , Vacinação , Vacinas de Produtos Inativados/uso terapêutico , Células Vero , Viremia , Zika virus
20.
PLoS Comput Biol ; 16(2): e1007636, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32069281

RESUMO

Most current analysis tools for antibody next-generation sequencing data work with primary sequence descriptors, leaving accompanying structural information unharnessed. We have used novel rapid methods to structurally characterize the complementary-determining regions (CDRs) of more than 180 million human and mouse B-cell receptor (BCR) repertoire sequences. These structurally annotated CDRs provide unprecedented insights into both the structural predetermination and dynamics of the adaptive immune response. We show that B-cell types can be distinguished based solely on these structural properties. Antigen-unexperienced BCR repertoires use the highest number and diversity of CDR structures and these patterns of naïve repertoire paratope usage are highly conserved across subjects. In contrast, more differentiated B-cells are more personalized in terms of CDR structure usage. Our results establish the CDR structure differences in BCR repertoires and have applications for many fields including immunodiagnostics, phage display library generation, and "humanness" assessment of BCR repertoires from transgenic animals. The software tool for structural annotation of BCR repertoires, SAAB+, is available at https://github.com/oxpig/saab_plus.


Assuntos
Linfócitos B/imunologia , Diferenciação Celular , Receptores de Antígenos de Linfócitos B/metabolismo , Imunidade Adaptativa , Animais , Animais Geneticamente Modificados , Anticorpos , Linfócitos B/citologia , Análise por Conglomerados , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulina G/química , Camundongos , Camundongos Endogâmicos C57BL , Análise de Componente Principal , Receptores de Antígenos de Linfócitos B/genética , Software
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