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Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 µg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Sítios de Ligação de Anticorpos , Células CHO , Chlorocebus aethiops , Cricetulus , Epitopos , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , SARS-CoV-2/imunologia , Células VeroRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1009352.].
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Serological and plasmablast responses and plasmablast-derived IgG monoclonal antibodies (MAbs) have been analysed in three COVID-19 patients with different clinical severities. Potent humoral responses were detected within 3 weeks of onset of illness in all patients and the serological titre was elicited soon after or concomitantly with peripheral plasmablast response. An average of 13.7% and 3.5% of plasmablast-derived MAbs were reactive with virus spike glycoprotein or nucleocapsid, respectively. A subset of anti-spike (10 of 32) antibodies cross-reacted with other betacoronaviruses tested and harboured extensive somatic mutations, indicative of an expansion of memory B cells upon SARS-CoV-2 infection. Fourteen of 32 anti-spike MAbs, including five anti-receptor-binding domain (RBD), three anti-non-RBD S1 and six anti-S2, neutralised wild-type SARS-CoV-2 in independent assays. Anti-RBD MAbs were further grouped into four cross-inhibiting clusters, of which six antibodies from three separate clusters blocked the binding of RBD to ACE2 and five were neutralising. All ACE2-blocking anti-RBD antibodies were isolated from two recovered patients with prolonged fever, which is compatible with substantial ACE2-blocking response in their sera. Finally, the identification of non-competing pairs of neutralising antibodies would offer potential templates for the development of prophylactic and therapeutic agents against SARS-CoV-2.
Assuntos
Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Adulto , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/genética , Células Produtoras de Anticorpos/imunologia , Sítios de Ligação , Epitopos , Humanos , Imunoglobulina G/imunologia , Nucleocapsídeo/imunologia , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Cyclic GMP-AMP (cGAMP) is an immunostimulatory molecule produced by cGAS that activates STING. cGAMP is an adjuvant when administered alongside antigens. cGAMP is also incorporated into enveloped virus particles during budding. Here, we investigate whether inclusion of cGAMP within viral vaccine vectors enhances their immunogenicity. We immunise mice with virus-like particles (VLPs) containing HIV-1 Gag and the vesicular stomatitis virus envelope glycoprotein G (VSV-G). cGAMP loading of VLPs augments CD4 and CD8 T-cell responses. It also increases VLP- and VSV-G-specific antibody titres in a STING-dependent manner and enhances virus neutralisation, accompanied by increased numbers of T follicular helper cells. Vaccination with cGAMP-loaded VLPs containing haemagglutinin induces high titres of influenza A virus neutralising antibodies and confers protection upon virus challenge. This requires cGAMP inclusion within VLPs and is achieved at markedly reduced cGAMP doses. Similarly, cGAMP loading of VLPs containing the SARS-CoV-2 Spike protein enhances Spike-specific antibody titres. cGAMP-loaded VLPs are thus an attractive platform for vaccination.
Assuntos
COVID-19 , Vacinas contra Influenza , Vacinas de Partículas Semelhantes a Vírus , Animais , Humanos , Camundongos , Nucleotídeos Cíclicos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Vacinas de Partículas Semelhantes a Vírus/genéticaRESUMO
The Zika virus (ZIKV) became a major worldwide public concern in 2015 due to the congenital syndrome which presents the highest risk during the first trimester of pregnancy and includes microcephaly and eye malformations. Several cellular, genetic and molecular studies have shown alterations in metabolic pathways, endoplasmic reticulum (ER) stress, immunity and dysregulation of RNA and energy metabolism both in vivo and in vitro. Here we summarise the main metabolic complications, with a particular focus on the possibility that brain energy metabolism is altered following ZIKV infection, contributing to developmental abnormalities. Brain energetic failure has been implicated in neurological conditions such as autism disorder and epilepsy, as well as in metabolic diseases with severe neurodevelopmental complications such as Glut-1 deficiency syndrome. Therefore, these energetic alterations are of wide-ranging interest as they might be directly implicated in congenital ZIKV syndrome. Data showing increased glycolysis during ZIKV infection, presumably required for viral replication, might support the idea that the virus can cause energetic stress in the developing brain cells. Consequences may include neuroinflammation, cell cycle dysregulation and cell death. Ketone bodies are non-glycolytic brain fuels that are produced during neonatal life, starvation or fasting, ingestion of high-fat low-carbohydrate diets, and following supplementation with ketone esters. We propose that dietary ketones might alter the course of the disease and could even provide some degree of prevention of ZIKV-associated abnormalities and potentially related neurological conditions characterised by brain glucose impairment.
Assuntos
Encéfalo/metabolismo , Infecção por Zika virus/congênito , Animais , Encéfalo/embriologia , Metabolismo Energético , Glucose/metabolismo , Humanos , Infecção por Zika virus/metabolismoRESUMO
The underlying threat of new Zika virus (ZIKV) outbreaks remains, as no vaccines or therapies have yet been developed. In vitro research has shown that glycolysis is a key factor to enable sustained ZIKV replication in neuroprogenitors. However, neither in vivo nor clinical investigation of glycolytic modulators as potential therapeutics for ZIKV-related fetal abnormalities has been conducted. Accordingly, we tested the therapeutic potential of metabolic modulators in relevant in vitro systems comprising two pools of neuroprogenitors (NPCs), which resemble early and late stages of pregnancy. Effective doses of metabolic modulators [3.0 µM] dimethyl fumarate (DMF), [3.2 mM] dichloroacetate (DCA), and [6.3 µM] VER-246608 were determined for these cells by their effect on lactate release, pyruvate dehydrogenase (PDH) activity and cell survival. The drugs were used in a 24h pre-treatment and kept throughout ZIKV infection of NPCs. Drug effects and ZIKV replication were assessed at 24- and 56-h post-infection. In early NPCs treated with DMF, DCA and VER-246608, there was a significant reduction in the extracellular release of ZIKV potentially by PDH-mediated increased mitochondrial oxidation of glucose. Out of the three drugs, only DCA was observed to reduce viral replication in late NPCs treated with DCA. Altogether, our findings suggest that reduction of anaerobic glycolysis could be of therapeutic potential against ZIKV-related fetal abnormalities and that clinical translation should consider the use of specific glycolytic modulators over different trimesters.
Assuntos
Ácido Dicloroacético , Glucose , Replicação Viral , Infecção por Zika virus , Zika virus , Zika virus/efeitos dos fármacos , Zika virus/fisiologia , Ácido Dicloroacético/farmacologia , Replicação Viral/efeitos dos fármacos , Glucose/metabolismo , Humanos , Infecção por Zika virus/tratamento farmacológico , Infecção por Zika virus/virologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/virologia , Células-Tronco Neurais/metabolismo , Animais , Glicólise/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Antivirais/farmacologiaRESUMO
Defending against future pandemics requires vaccine platforms that protect across a range of related pathogens. Nanoscale patterning can be used to address this issue. Here, we produce quartets of linked receptor-binding domains (RBDs) from a panel of SARS-like betacoronaviruses, coupled to a computationally designed nanocage through SpyTag/SpyCatcher links. These Quartet Nanocages, possessing a branched morphology, induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented in the vaccine. Equivalent antibody responses are raised to RBDs close to the nanocage or at the tips of the nanoparticle's branches. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increase the strength and breadth of an otherwise narrow immune response. A Quartet Nanocage including the Omicron XBB.1.5 'Kraken' RBD induced antibodies with binding to a broad range of sarbecoviruses, as well as neutralizing activity against this variant of concern. Quartet nanocages are a nanomedicine approach with potential to confer heterotypic protection against emergent zoonotic pathogens and facilitate proactive pandemic protection.
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Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Anticorpos Neutralizantes/imunologia , SARS-CoV-2/imunologia , Anticorpos Antivirais/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/química , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/química , COVID-19/prevenção & controle , COVID-19/imunologia , COVID-19/virologia , Humanos , Vacinação/métodos , Camundongos , Nanopartículas/química , FemininoRESUMO
Cortical development consists of an orchestrated process in which progenitor cells exhibit distinct fate restrictions regulated by time-dependent activation of energetic pathways. Thus, the hijacking of cellular metabolism by Zika virus (ZIKV) to support its replication may contribute to damage in the developing fetal brain. Here, we showed that ZIKV replicates differently in two glycolytically distinct pools of cortical progenitors derived from human induced pluripotent stem cells (hiPSCs), which resemble the metabolic patterns of quiescence (early hi-NPCs) and immature brain cells (late hi-NPCs) in the forebrain. This differential replication alters the transcription of metabolic genes in both pools of cortical progenitors but solely upregulates the glycolytic capacity of early hi-NPCs. Analysis using Imagestream® revealed that, during early stages of ZIKV replication, in early hi-NPCs there is an increase in lipid droplet abundance and size. This stage of ZIKV replication significantly reduced the mitochondrial distribution in both early and late hi-NPCs. During later stages of ZIKV replication, late hi-NPCs show reduced mitochondrial size and abundance. The finding that there are alterations of cellular metabolism during ZIKV infection which are specific to pools of cortical progenitors at different stages of maturation may help to explain the differences in brain damage over each trimester.
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Células-Tronco Pluripotentes Induzidas , Células-Tronco Neurais , Infecção por Zika virus , Zika virus , Gravidez , Feminino , Humanos , Zika virus/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Encéfalo/metabolismoRESUMO
Defending against future pandemics may require vaccine platforms that protect across a range of related pathogens. The presentation of multiple receptor-binding domains (RBDs) from evolutionarily-related viruses on a nanoparticle scaffold elicits a strong antibody response to conserved regions. Here we produce quartets of tandemly-linked RBDs from SARS-like betacoronaviruses coupled to the mi3 nanocage through a SpyTag/SpyCatcher spontaneous reaction. These Quartet Nanocages induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented on the vaccine. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increased the strength and breadth of an otherwise narrow immune response. Quartet Nanocages are a strategy with potential to confer heterotypic protection against emergent zoonotic coronavirus pathogens and facilitate proactive pandemic protection.
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The SARS-CoV-2 pandemic continues despite the presence of effective vaccines, and novel vaccine approaches may help to reduce viral spread and associated COVID-19 disease. Current vaccine administration modalities are based on systemic needle-administered immunisation which may be suboptimal for mucosal pathogens. Here we demonstrate in a mouse model that small-volume intranasal administration of purified spike (S) protein in the adjuvant polyethylenemine (PEI) elicits robust antibody responses with modest systemic neutralisation activity. Further, we test a heterologous intranasal immunisation regimen, priming with S and boosting with RBD-Fc. Our data identify small volume PEI adjuvantation as a novel platform with potential for protective mucosal vaccine development.
Assuntos
COVID-19 , Vacinas , Camundongos , Animais , Administração Intranasal , SARS-CoV-2 , Polietilenoimina , COVID-19/prevenção & controleRESUMO
Background: Administration of potent anti-receptor-binding domain (RBD) monoclonal antibodies has been shown to curtail viral shedding and reduce hospitalization in patients with SARS-CoV-2 infection. However, the structure-function analysis of potent human anti-RBD monoclonal antibodies and its links to the formulation of antibody cocktails remains largely elusive. Methods: Previously, we isolated a panel of neutralizing anti-RBD monoclonal antibodies from convalescent patients and showed their neutralization efficacy in vitro. Here, we elucidate the mechanism of action of antibodies and dissect antibodies at the epitope level, which leads to a formation of a potent antibody cocktail. Results: We found that representative antibodies which target non-overlapping epitopes are effective against wild type virus and recently emerging variants of concern, whilst being encoded by antibody genes with few somatic mutations. Neutralization is associated with the inhibition of binding of viral RBD to ACE2 and possibly of the subsequent fusion process. Structural analysis of representative antibodies, by cryo-electron microscopy and crystallography, reveals that they have some unique aspects that are of potential value while sharing some features in common with previously reported neutralizing monoclonal antibodies. For instance, one has a common VH 3-53 public variable region yet is unusually resilient to mutation at residue 501 of the RBD. We evaluate the in vivo efficacy of an antibody cocktail consisting of two potent non-competing anti-RBD antibodies in a Syrian hamster model. We demonstrate that the cocktail prevents weight loss, reduces lung viral load and attenuates pulmonary inflammation in hamsters in both prophylactic and therapeutic settings. Although neutralization of one of these antibodies is abrogated by the mutations of variant B.1.351, it is also possible to produce a bi-valent cocktail of antibodies both of which are resilient to variants B.1.1.7, B.1.351 and B.1.617.2. Conclusions: These findings support the up-to-date and rational design of an anti-RBD antibody cocktail as a therapeutic candidate against COVID-19.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Tratamento Farmacológico da COVID-19 , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Sítios de Ligação , Ligação Competitiva , COVID-19/virologia , Cricetinae , Microscopia Crioeletrônica , Cristalografia por Raios X , Cães , Epitopos , Feminino , Humanos , Células Madin Darby de Rim Canino , Testes de Neutralização , Domínios Proteicos , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Combinatorial antibody libraries not only effectively reduce antibody discovery to a numbers game, but enable documentation of the history of antibody responses in an individual. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has prompted a wider application of this technology to meet the public health challenge of pandemic threats in the modern era. Herein, a combinatorial human antibody library constructed 20 years before the coronavirus disease 2019 (COVID-19) pandemic is used to discover three highly potent antibodies that selectively bind SARS-CoV-2 spike protein and neutralize authentic SARS-CoV-2 virus. Compared to neutralizing antibodies from COVID-19 patients with generally low somatic hypermutation (SHM), these three antibodies contain over 13-22 SHMs, many of which are involved in specific interactions in their crystal structures with SARS-CoV-2 spike receptor binding domain. The identification of these somatically mutated antibodies in a pre-pandemic library raises intriguing questions about the origin and evolution of these antibodies with respect to their reactivity with SARS-CoV-2.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Sítios de Ligação , Ligação Competitiva , Técnicas de Visualização da Superfície Celular , Chlorocebus aethiops , Células HEK293 , Humanos , Biblioteca de Peptídeos , SARS-CoV-2/efeitos dos fármacos , Hipermutação Somática de Imunoglobulina , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células VeroRESUMO
In response to viral infections, the innate immune system rapidly activates expression of several interferon-stimulated genes (ISGs), whose protein and metabolic products are believed to directly interfere with the viral life cycle. Here, we argue that biochemical reactions performed by two specific protein products of ISGs modulate central carbon metabolism to support a broad-spectrum antiviral response. We demonstrate that the metabolites generated by metalloenzymes nitric oxide synthase and the radical S-adenosylmethionine (SAM) enzyme RSAD2 inhibit the activity of the housekeeping and glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). We discuss that this inhibition is likely to stimulate a range of metabolic and signalling processes to support a broad-spectrum immune response. Based on these analyses, we propose that inhibiting GAPDH in individuals with deteriorated cellular innate immune response like elderly might help in treating viral diseases such as COVID-19.
Assuntos
Antivirais/metabolismo , Carbono/metabolismo , Interferons/metabolismo , Proteínas/metabolismo , S-Adenosilmetionina/metabolismo , Antivirais/farmacologia , COVID-19/prevenção & controle , COVID-19/virologia , Células Cultivadas , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Células HEK293 , Humanos , Imunidade Inata/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Macrófagos/metabolismo , Modelos Biológicos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Proteínas/genética , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacosRESUMO
Human induced pluripotent stem cells (iPSCs) and macrophages derived from them are increasingly popular tools for research into both infectious and degenerative diseases. However, as the field strives for greater modeling accuracy, it is becoming ever more challenging to justify the use of undefined and proprietary media for the culture of these cells. Here, we describe a defined, serum-free, open-source medium for the differentiation of iPSC-derived macrophages. This medium is equally capable of maintaining these cells compared with commercial alternatives. The macrophages differentiated in this medium display improved terminally differentiated cell characteristics, reduced basal expression of induced antiviral response genes, and improved polarization capacity. We conclude that cells cultured in this medium are an appropriate and malleable model for tissue-resident macrophages, on which future differentiation techniques can be built.
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Diferenciação Celular , Meios de Cultura Livres de Soro/farmacologia , Células-Tronco Pluripotentes Induzidas/citologia , Macrófagos/citologia , Biomarcadores/metabolismo , Forma Celular/efeitos dos fármacos , Células Cultivadas , Infecções por HIV/patologia , Homeostase , Humanos , Ativação de Macrófagos , Macrófagos/metabolismo , Macrófagos/virologia , Fenótipo , Transcrição Gênica/efeitos dos fármacos , Transcriptoma/genética , Zika virus/fisiologiaRESUMO
The extent to which immune responses to natural infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and immunization with vaccines protect against variants of concern (VOC) is of increasing importance. Accordingly, here we analyse antibodies and T cells of a recently vaccinated, UK cohort, alongside those recovering from natural infection in early 2020. We show that neutralization of the VOC compared to a reference isolate of the original circulating lineage, B, is reduced: more profoundly against B.1.351 than for B.1.1.7, and in responses to infection or a single dose of vaccine than to a second dose of vaccine. Importantly, high magnitude T cell responses are generated after two vaccine doses, with the majority of the T cell response directed against epitopes that are conserved between the prototype isolate B and the VOC. Vaccination is required to generate high potency immune responses to protect against these and other emergent variants.
Assuntos
Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2/imunologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Proteínas de Transporte , Epitopos , Humanos , Imunidade , SARS-CoV-2/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
In the absence of a proven effective vaccine preventing infection by SARS-CoV-2, or a proven drug to treat COVID-19, the positive results of passive immune therapy using convalescent serum provide a strong lead. We have developed a new class of tetravalent, biparatopic therapy, 89C8-ACE2. It combines the specificity of a monoclonal antibody (89C8) that recognizes the relatively conserved N-terminal domain of the viral Spike (S) glycoprotein, and the ectodomain of ACE2, which binds to the receptor-binding domain of S. This molecule shows exceptional performance in vitro, inhibiting the interaction of recombinant S1 to ACE2 and transduction of ACE2-overexpressing cells by S-pseudotyped lentivirus with IC50s substantially below 100 pM, and with potency approximately 100-fold greater than ACE2-Fc itself. Moreover, 89C8-ACE2 was able to neutralize authentic viral infection in a standard 96-h co-incubation assay at low nanomolar concentrations, making this class of molecule a promising lead for therapeutic applications.
Assuntos
Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus , Pandemias , Peptidil Dipeptidase A/efeitos dos fármacos , Pneumonia Viral , Enzima de Conversão de Angiotensina 2 , Anticorpos Monoclonais/farmacologia , COVID-19 , Desenho de Fármacos , Descoberta de Drogas , Humanos , Proteínas Recombinantes , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/efeitos dos fármacosRESUMO
Background: Laboratory diagnosis of SARS-CoV-2 infection (the cause of COVID-19) uses PCR to detect viral RNA (vRNA) in respiratory samples. SARS-CoV-2 RNA has also been detected in other sample types, but there is limited understanding of the clinical or laboratory significance of its detection in blood. Methods: We undertook a systematic literature review to assimilate the evidence for the frequency of vRNA in blood, and to identify associated clinical characteristics. We performed RT-PCR in serum samples from a UK clinical cohort of acute and convalescent COVID-19 cases (n=212), together with convalescent plasma samples collected by NHS Blood and Transplant (NHSBT) (n=462 additional samples). To determine whether PCR-positive blood samples could pose an infection risk, we attempted virus isolation from a subset of RNA-positive samples. Results: We identified 28 relevant studies, reporting SARS-CoV-2 RNA in 0-76% of blood samples; pooled estimate 10% (95%CI 5-18%). Among serum samples from our clinical cohort, 27/212 (12.7%) had SARS-CoV-2 RNA detected by RT-PCR. RNA detection occurred in samples up to day 20 post symptom onset, and was associated with more severe disease (multivariable odds ratio 7.5). Across all samples collected ≥28 days post symptom onset, 0/494 (0%, 95%CI 0-0.7%) had vRNA detected. Among our PCR-positive samples, cycle threshold (ct) values were high (range 33.5-44.8), suggesting low vRNA copy numbers. PCR-positive sera inoculated into cell culture did not produce any cytopathic effect or yield an increase in detectable SARS-CoV-2 RNA. Conclusions: vRNA was detectable at low viral loads in a minority of serum samples collected in acute infection, but was not associated with infectious SARS-CoV-2 (within the limitations of the assays used). This work helps to inform biosafety precautions for handling blood products from patients with current or previous COVID-19.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral , Receptores Virais/metabolismo , Anticorpos de Domínio Único/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes/metabolismo , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/ultraestrutura , Afinidade de Anticorpos , Reações Antígeno-Anticorpo/imunologia , Betacoronavirus/metabolismo , Ligação Competitiva , COVID-19 , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Modelos Moleculares , Biblioteca de Peptídeos , Peptidil Dipeptidase A/ultraestrutura , Ligação Proteica , Conformação Proteica , Receptores Virais/ultraestrutura , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , SARS-CoV-2 , Homologia de Sequência de Aminoácidos , Anticorpos de Domínio Único/metabolismo , Anticorpos de Domínio Único/ultraestrutura , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/ultraestruturaRESUMO
The COVID-19 pandemic has had an unprecedented health and economic impact and there are currently no approved therapies. We have isolated an antibody, EY6A, from an individual convalescing from COVID-19 and have shown that it neutralizes SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds the receptor binding domain (RBD) of the viral spike glycoprotein tightly (KD of 2 nM), and a 2.6-Å-resolution crystal structure of an RBD-EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues within this footprint are key to stabilizing the pre-fusion spike. Cryo-EM analyses of the pre-fusion spike incubated with EY6A Fab reveal a complex of the intact spike trimer with three Fabs bound and two further multimeric forms comprising the destabilized spike attached to Fab. EY6A binds what is probably a major neutralizing epitope, making it a candidate therapeutic for COVID-19.