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1.
Immunity ; 54(6): 1290-1303.e7, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34022127

RESUMO

Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we used single-cell sequencing to profile SARS-CoV-2-reactive B cells in 38 COVID-19 patients. Using oligo-tagged antigen baits, we isolated B cells specific to the SARS-CoV-2 spike, nucleoprotein (NP), open reading frame 8 (ORF8), and endemic human coronavirus (HCoV) spike proteins. SARS-CoV-2 spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients several months post symptom onset. With severe acute infection, substantial populations of endemic HCoV-reactive antibody-secreting cells were identified and possessed highly mutated variable genes, signifying preexisting immunity. Finally, MBCs exhibited pronounced maturation to NP and ORF8 over time, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal antibody adaptation to non-neutralizing intracellular antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.


Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Linfócitos B/imunologia , COVID-19/imunologia , Interações Hospedeiro-Patógeno/imunologia , Epitopos Imunodominantes/imunologia , SARS-CoV-2/imunologia , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos/genética , Linfócitos B/metabolismo , Biologia Computacional/métodos , Reações Cruzadas/imunologia , Mapeamento de Epitopos , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno/genética , Humanos , Epitopos Imunodominantes/genética , Memória Imunológica , Masculino , Testes de Neutralização , Análise de Célula Única/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Transcriptoma
2.
Immunity ; 43(5): 859-69, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26522985

RESUMO

The precise impact of thymic positive and negative selection on the T cell receptor (TCR) repertoire remains controversial. Here, we used unbiased, high-throughput cloning and retroviral expression of individual pre-selection TCRs to provide a direct assessment of these processes at the clonal level in vivo. We found that 15% of random TCRs induced signaling and directed positive (7.5%) or negative (7.5%) selection, depending on strength of signal, whereas the remaining 85% failed to induce signaling or selection. Most negatively selected TCRs exhibited promiscuous crossreactivity toward multiple other major histocompatibility complex (MHC) haplotypes. In contrast, TCRs that were positively selected or non-selected were minimally crossreactive. Negative selection of crossreactive TCRs led to clonal deletion but also recycling into intestinal CD4(-)CD8ß(-) intraepithelial lymphocytes (iIELs). Thus, broadly crossreactive TCRs arise at low frequency in the pre-selection repertoire but constitute the primary drivers of thymic negative selection and iIEL lineage differentiation.


Assuntos
Reações Cruzadas/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Timócitos/imunologia , Animais , Ativação Linfocitária/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia
3.
Front Immunol ; 15: 1345467, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38504980

RESUMO

The vast diversity of mammalian adaptive antigen receptors allows for robust and efficient immune responses against a wide number of pathogens. The antigen receptor repertoire is built during the recombination of B and T cell receptor (BCR, TCR) loci and hypermutation of BCR loci. V(D)J recombination rearranges these antigen receptor loci, which are organized as an array of separate V, (D), and J gene segments. Transcription activation at the recombining locus leads to changes in the local three-dimensional architecture, which subsequently contributes to which gene segments are utilized for recombination. The endogenous retrovirus (ERV) mouse mammary tumor provirus 8 (Mtv8) resides on mouse chromosome 6 interposed within the large array of light chain kappa V gene segments. As ERVs contribute to changes in genomic architecture by driving high levels of transcription of neighboring genes, it was suggested that Mtv8 could influence the BCR repertoire. We generated Mtv8-deficient mice to determine if the ERV influences V(D)J recombination to test this possibility. We find that Mtv8 does not influence the BCR repertoire.


Assuntos
Receptores de Antígenos de Linfócitos T , Recombinação V(D)J , Animais , Camundongos , Imunoglobulinas/genética , Mamíferos , Receptores de Antígenos de Linfócitos T/genética , Recombinação V(D)J/genética
4.
J Clin Invest ; 133(8)2023 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-36862518

RESUMO

The rapid evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants has emphasized the need to identify antibodies with broad neutralizing capabilities to inform future monoclonal therapies and vaccination strategies. Herein, we identified S728-1157, a broadly neutralizing antibody (bnAb) targeting the receptor-binding site (RBS) that was derived from an individual previously infected with WT SARS-CoV-2 prior to the spread of variants of concern (VOCs). S728-1157 demonstrated broad cross-neutralization of all dominant variants, including D614G, Beta, Delta, Kappa, Mu, and Omicron (BA.1/BA.2/BA.2.75/BA.4/BA.5/BL.1/XBB). Furthermore, S728-1157 protected hamsters against in vivo challenges with WT, Delta, and BA.1 viruses. Structural analysis showed that this antibody targets a class 1/RBS-A epitope in the receptor binding domain via multiple hydrophobic and polar interactions with its heavy chain complementarity determining region 3 (CDR-H3), in addition to common motifs in CDR-H1/CDR-H2 of class 1/RBS-A antibodies. Importantly, this epitope was more readily accessible in the open and prefusion state, or in the hexaproline (6P)-stabilized spike constructs, as compared with diproline (2P) constructs. Overall, S728-1157 demonstrates broad therapeutic potential and may inform target-driven vaccine designs against future SARS-CoV-2 variants.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Cricetinae , Anticorpos , Epitopos , Anticorpos Antivirais , Anticorpos Neutralizantes
5.
J Exp Med ; 219(10)2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-36098746

RESUMO

IgE mediates allergic responses by coating mast cell or basophil surfaces and inducing degranulation upon binding a specific allergen. IgE can also be spontaneously produced in the absence of foreign allergens; yet the origin, regulation, and functions of such "natural" IgE still remain largely unknown. Here, we find that glucocorticoids enhance the production of IgE in B cells both in vivo and ex vivo without antigenic challenge. Such IgE production is promoted by B cell-intrinsic glucocorticoid receptor signaling that reinforces CD40 signaling and synergizes with the IL-4/STAT6 pathway. In addition, we found that rare B cells in the mesenteric lymph nodes are responsible for the production of glucocorticoid-inducible IgE. Furthermore, locally produced glucocorticoids in the gut may induce natural IgE during perturbations of gut homeostasis, such as dysbiosis. Notably, mice preemptively treated with glucocorticoids were protected from subsequent pathogenic anaphylaxis. Together, our results suggest that glucocorticoids, classically considered to be broadly immunosuppressive, have a selective immunostimulatory role in B cells.


Assuntos
Anafilaxia , Glucocorticoides , Alérgenos , Anafilaxia/metabolismo , Animais , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Imunoglobulina E/metabolismo , Mastócitos , Camundongos
6.
mBio ; 12(6): e0297521, 2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34781736

RESUMO

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have arisen that exhibit increased viral transmissibility and partial evasion of immunity induced by natural infection and vaccination. To address the specific antibody targets that were affected by recent viral variants, we generated 43 monoclonal antibodies (mAbs) from 10 convalescent donors that bound three distinct domains of the SARS-CoV-2 spike. Viral variants harboring mutations at K417, E484, and N501 could escape most of the highly potent antibodies against the receptor binding domain (RBD). Despite this, we identified 12 neutralizing mAbs against three distinct regions of the spike protein that neutralize SARS-CoV-2 and variants of concern (VOCs), including B.1.1.7 (alpha), P.1 (gamma), and B.1.617.2 (delta). Notably, antibodies targeting distinct epitopes could neutralize discrete variants, suggesting that different variants may have evolved to disrupt the binding of particular neutralizing antibody classes. These results underscore that humans exposed to the first pandemic wave of prototype SARS-CoV-2 possess neutralizing antibodies against current variants and that it is critical to induce antibodies targeting multiple distinct epitopes of the spike that can neutralize emerging variants of concern. IMPORTANCE We describe the binding and neutralization properties of a new set of human monoclonal antibodies derived from memory B cells of 10 coronavirus disease 2019 (COVID-19) convalescent donors in the first pandemic wave of prototype SARS-CoV-2. There were 12 antibodies targeting distinct epitopes on spike, including two sites on the RBD and one on the N-terminal domain (NTD), that displayed cross-neutralization of VOCs, for which distinct antibody targets could neutralize discrete variants. This work underlines that natural infection by SARS-CoV-2 induces effective cross-neutralization against only some VOCs and supports the need for COVID-19 vaccination for robust induction of neutralizing antibodies targeting multiple epitopes of the spike protein to combat the current SARS-CoV-2 VOCs and any others that might emerge in the future.


Assuntos
Anticorpos Antivirais/sangue , Anticorpos Amplamente Neutralizantes/sangue , COVID-19/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Convalescença , Epitopos/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Pandemias , Plasma/imunologia , Ligação Proteica , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética
7.
Science ; 358(6361)2017 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-28971969

RESUMO

Large quantities of immunoglobulin A (IgA) are constitutively secreted by intestinal plasma cells to coat and contain the commensal microbiota, yet the specificity of these antibodies remains elusive. Here we profiled the reactivities of single murine IgA plasma cells by cloning and characterizing large numbers of monoclonal antibodies. IgAs were not specific to individual bacterial taxa but rather polyreactive, with broad reactivity to a diverse, but defined, subset of microbiota. These antibodies arose at low frequencies among naïve B cells and were selected into the IgA repertoire upon recirculation in Peyer's patches. This selection process occurred independent of microbiota or dietary antigens. Furthermore, although some IgAs acquired somatic mutations, these did not substantially influence their reactivity. These findings reveal an endogenous mechanism driving homeostatic production of polyreactive IgAs with innate specificity to microbiota.


Assuntos
Microbioma Gastrointestinal/imunologia , Imunoglobulina A/imunologia , Plasmócitos/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Bactérias/imunologia , Vida Livre de Germes/imunologia , Imunoglobulina A/genética , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Nódulos Linfáticos Agregados/imunologia , Simbiose
8.
J Immunol Methods ; 438: 67-70, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27600311

RESUMO

Generating monoclonal antibodies from single B cells is a valuable tool for characterizing the specificity and functional properties of humoral responses. We and others developed protocols that have facilitated major advances in our understanding of B cell development, tolerance, and effector responses to HIV and influenza. Here, we demonstrate various refinements and dramatically reduce the time required to produce recombinant antibodies. Further, we present new methods for cloning and isolating antibodies from cells with lower immunoglobulin mRNA levels that may be resistant to traditional techniques. Together, these refinements significantly increase single-cell antibody expression efficiency and are easily integrated into established and novel pipelines.


Assuntos
Anticorpos Monoclonais/biossíntese , Especificidade de Anticorpos/imunologia , Linfócitos B/imunologia , Clonagem Molecular/métodos , Proteínas Recombinantes/biossíntese , Animais , Vetores Genéticos , Humanos , Camundongos
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