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1.
Cell ; 184(9): 2316-2331.e15, 2021 04 29.
Artículo en Inglés | MEDLINE | ID: mdl-33773105

RESUMEN

Most human monoclonal antibodies (mAbs) neutralizing SARS-CoV-2 recognize the spike (S) protein receptor-binding domain and block virus interactions with the cellular receptor angiotensin-converting enzyme 2. We describe a panel of human mAbs binding to diverse epitopes on the N-terminal domain (NTD) of S protein from SARS-CoV-2 convalescent donors and found a minority of these possessed neutralizing activity. Two mAbs (COV2-2676 and COV2-2489) inhibited infection of authentic SARS-CoV-2 and recombinant VSV/SARS-CoV-2 viruses. We mapped their binding epitopes by alanine-scanning mutagenesis and selection of functional SARS-CoV-2 S neutralization escape variants. Mechanistic studies showed that these antibodies neutralize in part by inhibiting a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 offered protection either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, natural infection induces a subset of potent NTD-specific mAbs that leverage neutralizing and Fc-mediated activities to protect against SARS-CoV-2 infection using multiple functional attributes.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Sustancias Protectoras/farmacología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Unión Competitiva , COVID-19/inmunología , COVID-19/virología , Quimiocinas/metabolismo , Chlorocebus aethiops , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Pulmón/metabolismo , Ratones Endogámicos C57BL , Modelos Moleculares , Mutagénesis/genética , Pruebas de Neutralización , Dominios Proteicos , Células Vero
2.
Cell ; 184(17): 4430-4446.e22, 2021 08 19.
Artículo en Inglés | MEDLINE | ID: mdl-34416147

RESUMEN

Alphaviruses cause severe arthritogenic or encephalitic disease. The E1 structural glycoprotein is highly conserved in these viruses and mediates viral fusion with host cells. However, the role of antibody responses to the E1 protein in immunity is poorly understood. We isolated E1-specific human monoclonal antibodies (mAbs) with diverse patterns of recognition for alphaviruses (ranging from Eastern equine encephalitis virus [EEEV]-specific to alphavirus cross-reactive) from survivors of natural EEEV infection. Antibody binding patterns and epitope mapping experiments identified differences in E1 reactivity based on exposure of epitopes on the glycoprotein through pH-dependent mechanisms or presentation on the cell surface prior to virus egress. Therapeutic efficacy in vivo of these mAbs corresponded with potency of virus egress inhibition in vitro and did not require Fc-mediated effector functions for treatment against subcutaneous EEEV challenge. These studies reveal the molecular basis for broad and protective antibody responses to alphavirus E1 proteins.


Asunto(s)
Alphavirus/inmunología , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas/inmunología , Proteínas Virales/inmunología , Liberación del Virus/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Neutralizantes/inmunología , Antígenos Virales/inmunología , Línea Celular , Virus Chikungunya/inmunología , Virus de la Encefalitis Equina del Este/inmunología , Encefalomielitis Equina/inmunología , Encefalomielitis Equina/virología , Mapeo Epitopo , Femenino , Caballos , Humanos , Concentración de Iones de Hidrógeno , Articulaciones/patología , Masculino , Ratones Endogámicos C57BL , Modelos Biológicos , Unión Proteica , ARN Viral/metabolismo , Receptores Fc/metabolismo , Temperatura , Virión/metabolismo , Internalización del Virus
3.
Immunity ; 54(4): 781-796.e4, 2021 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-33675683

RESUMEN

Human IGHV1-69-encoded broadly neutralizing antibodies (bnAbs) that target the hepatitis C virus (HCV) envelope glycoprotein (Env) E2 are important for protection against HCV infection. An IGHV1-69 ortholog gene, VH1.36, is preferentially used for bnAbs isolated from HCV Env-immunized rhesus macaques (RMs). Here, we studied the genetic, structural, and functional properties of VH1.36-encoded bnAbs generated by vaccination, in comparison to IGHV1-69-encoded bnAbs from HCV patients. Global B cell repertoire analysis confirmed the expansion of VH1.36-derived B cells in immunized animals. Most E2-specific, VH1.36-encoded antibodies cross-neutralized HCV. Crystal structures of two RM bnAbs with E2 revealed that the RM bnAbs engaged conserved E2 epitopes using similar molecular features as human bnAbs but with a different binding mode. Longitudinal analyses of the RM antibody repertoire responses during immunization indicated rapid lineage development of VH1.36-encoded bnAbs with limited somatic hypermutation. Our findings suggest functional convergence of a germline-encoded bnAb response to HCV Env with implications for vaccination in humans.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Células Germinativas/inmunología , Glicoproteínas/inmunología , Hepacivirus/inmunología , Hepatitis C/inmunología , Macaca mulatta/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Linfocitos B/inmunología , Células CHO , Línea Celular , Cricetulus , Epítopos/inmunología , Células HEK293 , Hepatitis C/virología , Humanos , Estudios Longitudinales , Macaca mulatta/virología , Receptores de Antígenos de Linfocitos B/inmunología , Vacunación/métodos
4.
J Virol ; 96(11): e0007122, 2022 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-35575481

RESUMEN

Zika virus (ZIKV) is a global public health concern due to its ability to cause congenital Zika syndrome and lack of approved vaccine, therapeutic, or other control measures. We discovered eight novel rabbit monoclonal antibodies (MAbs) that bind to distinct ZIKV envelope protein epitopes. The majority of the MAbs were ZIKV specific and targeted the lateral ridge of the envelope (E) protein domain III, while the MAb with the highest neutralizing activity recognized a putative quaternary epitope spanning E protein domains I and III. One of the non-neutralizing MAbs specifically recognized ZIKV precursor membrane protein (prM). Somatic hypermutation of immunoglobulin variable regions increases antibody affinity maturation and triggers antibody class switching. Negative correlations were observed between the somatic hypermutation rate of the immunoglobulin heavy-chain variable region and antibody binding parameters such as equilibrium dissociation constant, dissociation constant, and half-maximal effective concentration value of MAb binding to ZIKV virus-like particles. Complementarity-determining regions recognize the antigen epitopes and are scaffolded by canonical framework regions. Reversion of framework region amino acids to the rabbit germ line sequence decreased anti-ZIKV MAb binding activity of some MAbs. Thus, antibody affinity maturation, including somatic hypermutation and framework region mutations, contributed to the binding and function of these anti-ZIKV MAbs. IMPORTANCE ZIKV is a global health concern against which no vaccine or therapeutics are available. We characterized eight novel rabbit monoclonal antibodies recognizing ZIKV envelope and prM proteins and studied the relationship between somatic hypermutation of complementarity-determining regions, framework regions, mutations, antibody specificity, binding, and neutralizing activity. The results contribute to understanding structural features and somatic mutation pathways by which potent Zika virus-neutralizing antibodies can evolve, including the role of antibody framework regions.


Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antivirales , Hipermutación Somática de Inmunoglobulina , Virus Zika , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/genética , Regiones Determinantes de Complementariedad/genética , Epítopos/genética , Mutación , Conejos , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Virus Zika/inmunología
5.
MAbs ; 14(1): 2002236, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-34967699

RESUMEN

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Técnicas de Visualización de Superficie Celular , Inmunoglobulina G/inmunología , Biblioteca de Péptidos , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/metabolismo , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , COVID-19/metabolismo , COVID-19/prevención & control , COVID-19/virología , Chlorocebus aethiops , Modelos Animales de Enfermedad , Epítopos , Femenino , Interacciones Huésped-Patógeno , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Inmunoglobulina G/farmacología , Mesocricetus , SARS-CoV-2/patogenicidad , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/farmacología , Células Vero
6.
bioRxiv ; 2021 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-33501445

RESUMEN

Most human monoclonal antibodies (mAbs) neutralizing SARS-CoV-2 recognize the spike (S) protein receptor-binding domain and block virus interactions with the cellular receptor angiotensin-converting enzyme 2. We describe a panel of human mAbs binding to diverse epitopes on the N-terminal domain (NTD) of S protein from SARS-CoV-2 convalescent donors and found a minority of these possessed neutralizing activity. Two mAbs (COV2-2676 and COV2-2489) inhibited infection of authentic SARS-CoV-2 and recombinant VSV/SARS-CoV-2 viruses. We mapped their binding epitopes by alanine-scanning mutagenesis and selection of functional SARS-CoV-2 S neutralization escape variants. Mechanistic studies showed that these antibodies neutralize in part by inhibiting a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 offered protection either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, natural infection induces a subset of potent NTD-specific mAbs that leverage neutralizing and Fc-mediated activities to protect against SARS-CoV-2 infection using multiple functional attributes.

7.
Cell Host Microbe ; 27(6): 976-991.e11, 2020 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-32320678

RESUMEN

Marburg virus (MARV) and Ebola virus (EBOV) belong to the family Filoviridae. MARV causes severe disease in humans with high fatality. We previously isolated a large panel of monoclonal antibodies (mAbs) from B cells of a human survivor with previous naturally acquired MARV infection. Here, we characterized functional properties of these mAbs and identified non-neutralizing mAbs targeting the glycoprotein (GP) 2 portion of the mucin-like domain (MLD) of MARV GP, termed the wing region. One mAb targeting the GP2 wing, MR228, showed therapeutic protection in mice and guinea pigs infected with MARV. The protection was mediated by the Fc fragment functions of MR228. Binding of another GP2 wing-specific non-neutralizing mAb, MR235, to MARV GP increased accessibility of epitopes in the receptor-binding site (RBS) for neutralizing mAbs, resulting in enhanced virus neutralization by these mAbs. These findings highlight an important role for non-neutralizing mAbs during natural human MARV infection.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Enfermedad del Virus de Marburg/inmunología , Marburgvirus/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Linfocitos B , Chlorocebus aethiops , Modelos Animales de Enfermedad , Ebolavirus/inmunología , Epítopos/inmunología , Femenino , Glicoproteínas/inmunología , Cobayas , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Sobrevivientes , Células THP-1 , Células Vero , Proteínas del Envoltorio Viral/inmunología
8.
EBioMedicine ; 54: 102733, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32315970

RESUMEN

Antibody-mediated humoral immunity is thought to play a central role in mediating the immunopathogenesis of acute DENV infection, but limited data are available on the diversity, specificity, and functionality of the antibody response at the molecular level elicited by primary or secondary DENV infection. In order to close this functional gap in our understanding of DENV-specific humoral immunity, we utilized high-throughput single cell RNA sequencing to investigate B cells circulating in both primary and secondary natural DENV infections. We captured full-length paired immunoglobulin receptor sequence data from 9,027 B cells from a total of 6 subjects, including 2,717 plasmablasts. In addition to IgG and IgM class-switched cells, we unexpectedly found a high proportion of the DENV-elicited plasmablasts expressing IgA, principally in individuals with primary DENV infections. These IgA class-switched cells were extensively hypermutated even in individuals with a serologically confirmed primary DENV infection. Utilizing a combination of conventional biochemical assays and high-throughput shotgun mutagenesis, we determined that DENV-reactive IgA class-switched antibodies represent a significant fraction of DENV-reactive Igs generated in response to DENV infection, and that they exhibit a comparable epitope specificity to DENV-reactive IgG antibodies. These results provide insight into the molecular-level diversity of DENV-elicited humoral immunity and identify a heretofore unappreciated IgA plasmablast response to DENV infection.


Asunto(s)
Linfocitos B/inmunología , Dengue/inmunología , Inmunoglobulinas/genética , Linfocitos B/citología , Células Cultivadas , Dengue/genética , Humanos , Inmunidad Humoral , Inmunoglobulinas/metabolismo , RNA-Seq , Análisis de la Célula Individual , Transcriptoma
9.
Nat Biomed Eng ; 4(11): 1030-1043, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32747832

RESUMEN

The emergence and re-emergence of highly virulent viral pathogens with the potential to cause a pandemic creates an urgent need for the accelerated discovery of antiviral therapeutics. Antiviral human monoclonal antibodies (mAbs) are promising candidates for the prevention and treatment of severe viral diseases, but their long development timeframes limit their rapid deployment and use. Here, we report the development of an integrated sequence of technologies, including single-cell mRNA-sequence analysis, bioinformatics, synthetic biology and high-throughput functional analysis, that enables the rapid discovery of highly potent antiviral human mAbs, the activity of which we validated in vivo. In a 78-d study modelling the deployment of a rapid response to an outbreak, we isolated more than 100 human mAbs that are specific to Zika virus, assessed their function, identified that 29 of these mAbs have broadly neutralizing activity, and verified the therapeutic potency of the lead candidates in mice and non-human primate models of infection through the delivery of an antibody-encoding mRNA formulation and of the respective IgG antibody. The pipeline provides a roadmap for rapid antibody-discovery programmes against viral pathogens of global concern.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Antivirales/uso terapéutico , Descubrimiento de Drogas/métodos , Virus Zika/inmunología , Animales , Células Cultivadas , Biología Computacional , Humanos , Macaca mulatta , Ratones , ARN Mensajero/inmunología , Análisis de Secuencia de ARN
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