RESUMEN
Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of the viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Although antibodies that block this interaction are in emergency use as early coronavirus disease 2019 (COVID-19) therapies, the precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that potently block ACE2 binding but exhibit divergent neutralization efficacy against the live virus. Strikingly, these neutralizing antibodies can inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in individuals with COVID-19. As revealed by cryoelectron microscopy, multiple structures of Spike-antibody complexes have distinct binding modes that not only block ACE2 binding but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion with profound implications for COVID-19 pathology and immunity.
Asunto(s)
Anticuerpos Neutralizantes/química , Células Gigantes/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/metabolismo , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/metabolismo , Sitios de Unión , Células CHO , COVID-19/patología , COVID-19/virología , Cricetinae , Cricetulus , Microscopía por Crioelectrón , Células Gigantes/citología , Humanos , Fusión de Membrana , Biblioteca de Péptidos , Unión Proteica , Dominios Proteicos , Estructura Cuaternaria de Proteína , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
The uptake, transport, and presentation of Ags by lung dendritic cells (DCs) are central to the initiation of CD8 T cell responses against respiratory viruses. Although several studies have demonstrated a critical role of CD11b(low/neg)CD103(+) DCs for the initiation of cytotoxic T cell responses against the influenza virus, the underlying mechanisms for its potent ability to prime CD8 T cells remain poorly understood. Using a novel approach of fluorescent lipophilic dye-labeled influenza virus, we demonstrate that CD11b(low/neg)CD103(+) DCs are the dominant lung DC population transporting influenza virus to the posterior mediastinal lymph node as early as 20 h postinfection. By contrast, CD11b(high)CD103(neg) DCs, although more efficient for taking up the virus within the lung, migrate poorly to the lymph node and remain in the lung to produce proinflammatory cytokines instead. CD11b(low/neg)CD103(+) DCs efficiently load viral peptide onto MHC class I complexes and therefore uniquely possess the capacity to potently induce proliferation of naive CD8 T cells. In addition, the peptide transporters TAP1 and TAP2 are constitutively expressed at higher levels in CD11b(low/neg)CD103(+) DCs, providing, to our knowledge, the first evidence of a distinct regulation of the Ag-processing pathway in these cells. Collectively, these results show that CD11b(low/neg)CD103(+) DCs are functionally specialized for the transport of Ag from the lung to the lymph node and also for efficient processing and presentation of viral Ags to CD8 T cells.
Asunto(s)
Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Ganglios Linfáticos/inmunología , Activación de Linfocitos/inmunología , Infecciones por Orthomyxoviridae/inmunología , Animales , Antígenos CD/inmunología , Antígenos Virales/inmunología , Separación Celular , Células Dendríticas/virología , Citometría de Flujo , Antígenos de Histocompatibilidad Clase I/inmunología , Cadenas alfa de Integrinas/inmunología , Pulmón/inmunología , Ganglios Linfáticos/virología , Ratones , Ratones Endogámicos C57BL , Orthomyxoviridae/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Although SARS-CoV-2-neutralizing antibodies are promising therapeutics against COVID-19, little is known about their mechanism(s) of action or effective dosing windows. We report the generation and development of SC31, a potent SARS-CoV-2 neutralizing antibody, isolated from a convalescent patient. Antibody-mediated neutralization occurs via an epitope within the receptor-binding domain of the SARS-CoV-2 Spike protein. SC31 exhibited potent anti-SARS-CoV-2 activities in multiple animal models. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, treatment with SC31 greatly reduced viral loads and attenuated pro-inflammatory responses linked to the severity of COVID-19. Importantly, a comparison of the efficacies of SC31 and its Fc-null LALA variant revealed that the optimal therapeutic efficacy of SC31 requires Fc-mediated effector functions that promote IFNγ-driven anti-viral immune responses, in addition to its neutralization ability. A dose-dependent efficacy of SC31 was observed down to 5mg/kg when administered before viral-induced lung inflammatory responses. In addition, antibody-dependent enhancement was not observed even when infected mice were treated with SC31 at sub-therapeutic doses. In SARS-CoV-2-infected hamsters, SC31 treatment significantly prevented weight loss, reduced viral loads, and attenuated the histopathology of the lungs. In rhesus macaques, the therapeutic potential of SC31 was evidenced through the reduction of viral loads in both upper and lower respiratory tracts to undetectable levels. Together, the results of our preclinical studies demonstrated the therapeutic efficacy of SC31 in three different models and its potential as a COVID-19 therapeutic candidate.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , COVID-19/terapia , SARS-CoV-2/inmunología , Enzima Convertidora de Angiotensina 2/genética , Animales , Anticuerpos Neutralizantes/metabolismo , COVID-19/inmunología , COVID-19/virología , Quimiocinas/sangre , Quimiocinas/genética , Chlorocebus aethiops , Convalecencia , Cricetinae , Citocinas/sangre , Citocinas/genética , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Macaca mulatta , Masculino , Ratones Transgénicos , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero , Carga ViralRESUMEN
Given the ongoing SARS-CoV-2 pandemic, identification of immunogenic targets against the coronavirus spike glycoprotein will provide crucial advances towards the development of sensitive diagnostic tools and potential vaccine candidate targets. In this study, using pools of overlapping linear B-cell peptides, we report two IgG immunodominant regions on SARS-CoV-2 spike glycoprotein that are recognised by sera from COVID-19 convalescent patients. Notably, one is specific to SARS-CoV-2, which is located in close proximity to the receptor binding domain. The other region, which is localised at the fusion peptide, could potentially function as a pan-SARS target. Functionally, antibody depletion assays demonstrate that antibodies targeting these immunodominant regions significantly alter virus neutralisation capacities. Taken together, identification and validation of these neutralising B-cell epitopes will provide insights towards the design of diagnostics and vaccine candidates against this high priority coronavirus.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/inmunología , Neumonía Viral/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , COVID-19 , Infecciones por Coronavirus/sangre , Epítopos de Linfocito B , Humanos , Epítopos Inmunodominantes , Inmunoglobulina G/sangre , Pandemias , Neumonía Viral/sangre , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
In vitro antibody selection against pathogens from naïve combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection1-4. Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction5. Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naïve human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the "up" and the other in the "down" position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.
RESUMEN
Multi-polypeptide proteins such as antibodies are difficult to express in prokaryotic systems such as E. coli due to the complexity of protein folding plus secretion. Thus far, proprietary strains or fermenter cultures have been required for appreciable yields. Previous studies have shown that expression of heterologous proteins in E. coli can be enhanced by the reduction of protein translation rates. In this paper, we demonstrate that useful quantities of full-length IgG can be expressed and purified from the common E. coli laboratory strain HB2151 in standard shaking culture using a simple strategy of reduced inducer concentration combined with delayed induction times to modulate translation rates. Purified IgG had only marginally reduced avidity compared to mammalian derived IgG. This indicates that this technique can be used to derive antibodies of potentially equal utility as those expressed in mammalian cell culture, particularly for applications where effector functions mediated by the glycosylated residues in the Fragment Crystallizable (Fc) portion of the immunoglobulin are not required.