RESUMEN
The biosafety level 3 (BSL-3) requirement to culture severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a bottleneck for research. Here, we report a trans-complementation system that produces single-round infectious SARS-CoV-2 that recapitulates authentic viral replication. We demonstrate that the single-round infectious SARS-CoV-2 can be used at BSL-2 laboratories for high-throughput neutralization and antiviral testing. The trans-complementation system consists of two components: a genomic viral RNA containing ORF3 and envelope gene deletions, as well as mutated transcriptional regulator sequences, and a producer cell line expressing the two deleted genes. Trans-complementation of the two components generates virions that can infect naive cells for only one round but does not produce wild-type SARS-CoV-2. Hamsters and K18-hACE2 transgenic mice inoculated with the complementation-derived virions exhibited no detectable disease, even after intracranial inoculation with the highest possible dose. Thus, the trans-complementation platform can be safely used at BSL-2 laboratories for research and countermeasure development.
Asunto(s)
COVID-19/virología , Contención de Riesgos Biológicos/métodos , SARS-CoV-2 , Células A549 , Animales , Chlorocebus aethiops , Cricetinae , Prueba de Complementación Genética/métodos , Genoma Viral , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , ARN Viral , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , Células Vero , Virulencia , Replicación ViralRESUMEN
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 VeroRESUMEN
The emergence of Zika virus (ZIKV) and its association with congenital malformations has prompted the rapid development of vaccines. Although efficacy with multiple viral vaccine platforms has been established in animals, no study has addressed protection during pregnancy. We tested in mice two vaccine platforms, a lipid nanoparticle-encapsulated modified mRNA vaccine encoding ZIKV prM and E genes and a live-attenuated ZIKV strain encoding an NS1 protein without glycosylation, for their ability to protect against transmission to the fetus. Vaccinated dams challenged with a heterologous ZIKV strain at embryo day 6 (E6) and evaluated at E13 showed markedly diminished levels of viral RNA in maternal, placental, and fetal tissues, which resulted in protection against placental damage and fetal demise. As modified mRNA and live-attenuated vaccine platforms can restrict in utero transmission of ZIKV in mice, their further development in humans to prevent congenital ZIKV syndrome is warranted.
Asunto(s)
Vacunas Virales/administración & dosificación , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/prevención & control , Virus Zika/fisiología , Aedes/virología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Células Sanguíneas/virología , Embrión de Mamíferos/virología , Femenino , Feto/virología , Humanos , Lípidos/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , ARN Mensajero/genética , ARN Mensajero/inmunología , Organismos Libres de Patógenos Específicos , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Vacunas Virales/inmunología , Infección por el Virus Zika/virologíaRESUMEN
Cerebral organoids, three-dimensional cultures that model organogenesis, provide a new platform to investigate human brain development. High cost, variability, and tissue heterogeneity limit their broad applications. Here, we developed a miniaturized spinning bioreactor (SpinΩ) to generate forebrain-specific organoids from human iPSCs. These organoids recapitulate key features of human cortical development, including progenitor zone organization, neurogenesis, gene expression, and, notably, a distinct human-specific outer radial glia cell layer. We also developed protocols for midbrain and hypothalamic organoids. Finally, we employed the forebrain organoid platform to model Zika virus (ZIKV) exposure. Quantitative analyses revealed preferential, productive infection of neural progenitors with either African or Asian ZIKV strains. ZIKV infection leads to increased cell death and reduced proliferation, resulting in decreased neuronal cell-layer volume resembling microcephaly. Together, our brain-region-specific organoids and SpinΩ provide an accessible and versatile platform for modeling human brain development and disease and for compound testing, including potential ZIKV antiviral drugs.
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Encéfalo/citología , Técnicas de Cultivo de Célula , Modelos Biológicos , Organoides , Virus Zika/fisiología , Reactores Biológicos , Técnicas de Cultivo de Célula/economía , Embrión de Mamíferos , Desarrollo Embrionario , Humanos , Células Madre Pluripotentes Inducidas , Neurogénesis , Neuronas/citología , Organoides/virología , Infección por el Virus Zika/fisiopatología , Infección por el Virus Zika/virologíaRESUMEN
With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility and potential resistance, antibodies and vaccines with broadly inhibitory activity are needed. Here, we developed a panel of neutralizing anti-SARS-CoV-2 monoclonal antibodies (mAbs) that bound the receptor binding domain of the spike protein at distinct epitopes and blocked virus attachment to its host receptor, human angiotensin converting enzyme-2 (hACE2). Although several potently neutralizing mAbs protected K18-hACE2 transgenic mice against infection caused by ancestral SARS-CoV-2 strains, others induced escape variants in vivo or lost neutralizing activity against emerging strains. One mAb, SARS2-38, potently neutralized all tested SARS-CoV-2 variants of concern and protected mice against challenge by multiple SARS-CoV-2 strains. Structural analysis showed that SARS2-38 engaged a conserved epitope proximal to the receptor binding motif. Thus, treatment with or induction of neutralizing antibodies that bind conserved spike epitopes may limit the loss of potency of therapies or vaccines against emerging SARS-CoV-2 variants.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Epítopos/inmunología , SARS-CoV-2/inmunología , Secuencias de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/uso terapéutico , COVID-19/prevención & control , COVID-19/virología , Epítopos/química , Epítopos/metabolismo , Humanos , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/metabolismo , Ratones , Pruebas de Neutralización , Dominios Proteicos , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
The emergence of SARS-CoV-2 antigenic variants with increased transmissibility is a public health threat. Some variants show substantial resistance to neutralization by SARS-CoV-2 infection- or vaccination-induced antibodies. Here, we analyzed receptor binding domain-binding monoclonal antibodies derived from SARS-CoV-2 mRNA vaccine-elicited germinal center B cells for neutralizing activity against the WA1/2020 D614G SARS-CoV-2 strain and variants of concern. Of five monoclonal antibodies that potently neutralized the WA1/2020 D614G strain, all retained neutralizing capacity against the B.1.617.2 variant, four also neutralized the B.1.1.7 variant, and only one, 2C08, also neutralized the B.1.351 and B.1.1.28 variants. 2C08 reduced lung viral load and morbidity in hamsters challenged with the WA1/2020 D614G, B.1.351, or B.1.617.2 strains. Clonal analysis identified 2C08-like public clonotypes among B cells responding to SARS-CoV-2 infection or vaccination in 41 out of 181 individuals. Thus, 2C08-like antibodies can be induced by SARS-CoV-2 vaccines and mitigate resistance by circulating variants of concern.
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Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/metabolismo , Linfocitos B/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , Centro Germinal/inmunología , Pulmón/virología , SARS-CoV-2/fisiología , Animales , Células Cultivadas , Células Clonales , Cricetinae , Modelos Animales de Enfermedad , Humanos , Pruebas de Neutralización , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunación , Carga ViralRESUMEN
A combination of vaccination approaches will likely be necessary to fully control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane-anchored pre-fusion stabilized spike (MVA/S) but not secreted S1 induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and conferred protection from SARS-CoV-2 infection and virus replication in the lungs as early as day 2 following intranasal and intratracheal challenge. Single-cell RNA sequencing analysis of lung cells on day 4 after infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities and lowered induction of interferon-stimulated genes. These results demonstrate that MVA/S vaccination induces neutralizing antibodies and CD8+ T cells in the blood and lungs and is a potential vaccine candidate for SARS-CoV-2.
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Vacunas contra la COVID-19/inmunología , COVID-19/prevención & control , Vectores Genéticos/genética , SARS-CoV-2/inmunología , Vacunas de ADN/inmunología , Virus Vaccinia/genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/genética , Antígenos Virales/inmunología , COVID-19/inmunología , COVID-19/patología , COVID-19/virología , Vacunas contra la COVID-19/genética , Modelos Animales de Enfermedad , Expresión Génica , Orden Génico , Inmunofenotipificación , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Macaca , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patología , Ratones , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Vacunación/métodos , Vacunas de ADN/genéticaRESUMEN
Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation. We found that the radical SAM activity of viperin is required for translation inhibition and that this is mediated by viperin's enzymatic product, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Viperin triggers ribosome collisions and activates the MAPKKK ZAK pathway that in turn activates the GCN2 arm of the integrated stress response pathway to inhibit translation. The study illustrates the importance of translational repression in the antiviral response and identifies viperin as a translation regulator in innate immunity.
Asunto(s)
Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Proteínas , Antivirales/farmacología , Inmunidad Innata , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Proteínas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , S-Adenosilmetionina , Replicación ViralRESUMEN
Conventional thermochemical syntheses by continuous heating under near-equilibrium conditions face critical challenges in improving the synthesis rate, selectivity, catalyst stability and energy efficiency, owing to the lack of temporal control over the reaction temperature and time, and thus the reaction pathways1-3. As an alternative, we present a non-equilibrium, continuous synthesis technique that uses pulsed heating and quenching (for example, 0.02 s on, 1.08 s off) using a programmable electric current to rapidly switch the reaction between high (for example, up to 2,400 K) and low temperatures. The rapid quenching ensures high selectivity and good catalyst stability, as well as lowers the average temperature to reduce the energy cost. Using CH4 pyrolysis as a model reaction, our programmable heating and quenching technique leads to high selectivity to value-added C2 products (>75% versus <35% by the conventional non-catalytic method and versus <60% by most conventional methods using optimized catalysts). Our technique can be extended to a range of thermochemical reactions, such as NH3 synthesis, for which we achieve a stable and high synthesis rate of about 6,000 µmol gFe-1 h-1 at ambient pressure for >100 h using a non-optimized catalyst. This study establishes a new model towards highly efficient non-equilibrium thermochemical synthesis.
RESUMEN
The B.1.1.7 variant (also known as Alpha) of SARS-CoV-2, the cause of the COVID-19 pandemic, emerged in the UK in the summer of 2020. The prevalence of this variant increased rapidly owing to an increase in infection and/or transmission efficiency1. The Alpha variant contains 19 nonsynonymous mutations across its viral genome, including 8 substitutions or deletions in the spike protein that interacts with cellular receptors to mediate infection and tropism. Here, using a reverse genetics approach, we show that of the 8 individual spike protein substitutions, only N501Y resulted in consistent fitness gains for replication in the upper airway in a hamster model as well as in primary human airway epithelial cells. The N501Y substitution recapitulated the enhanced viral transmission phenotype of the eight mutations in the Alpha spike protein, suggesting that it is a major determinant of the increased transmission of the Alpha variant. Mechanistically, the N501Y substitution increased the affinity of the viral spike protein for cellular receptors. As suggested by its convergent evolution in Brazil, South Africa and elsewhere2,3, our results indicate that N501Y substitution is an adaptive spike mutation of major concern.
Asunto(s)
Sustitución de Aminoácidos , COVID-19/transmisión , COVID-19/virología , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Unión Competitiva , Bronquios/citología , Células Cultivadas , Cricetinae , Humanos , Masculino , Mesocricetus , Modelos Moleculares , Mutación , Unión Proteica , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Replicación ViralRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and caused the devastating global pandemic of coronavirus disease 2019 (COVID-19), in part because of its ability to effectively suppress host cell responses1-3. In rare cases, viral proteins dampen antiviral responses by mimicking critical regions of human histone proteins4-8, particularly those containing post-translational modifications required for transcriptional regulation9-11. Recent work has demonstrated that SARS-CoV-2 markedly disrupts host cell epigenetic regulation12-14. However, how SARS-CoV-2 controls the host cell epigenome and whether it uses histone mimicry to do so remain unclear. Here we show that the SARS-CoV-2 protein encoded by ORF8 (ORF8) functions as a histone mimic of the ARKS motifs in histone H3 to disrupt host cell epigenetic regulation. ORF8 is associated with chromatin, disrupts regulation of critical histone post-translational modifications and promotes chromatin compaction. Deletion of either the ORF8 gene or the histone mimic site attenuates the ability of SARS-CoV-2 to disrupt host cell chromatin, affects the transcriptional response to infection and attenuates viral genome copy number. These findings demonstrate a new function of ORF8 and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation. Further, this work provides a molecular basis for the finding that SARS-CoV-2 lacking ORF8 is associated with decreased severity of COVID-19.
Asunto(s)
COVID-19 , Epigénesis Genética , Histonas , Interacciones Microbiota-Huesped , Imitación Molecular , SARS-CoV-2 , Proteínas Virales , COVID-19/genética , COVID-19/metabolismo , COVID-19/virología , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Epigenoma/genética , Histonas/química , Histonas/metabolismo , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Although interferon-induced proteins with tetratricopeptide repeats (IFIT proteins) inhibit infection of many viruses by recognizing their RNA, the regulatory mechanisms involved remain unclear. Here we report a crystal structure of cap 0 (m7GpppN) RNA bound to human IFIT1 in complex with the C-terminal domain of human IFIT3. Structural, biochemical, and genetic studies suggest that IFIT3 binding to IFIT1 has dual regulatory functions: (1) extending the half-life of IFIT1 and thereby increasing its steady-state amounts in cells; and (2) allosterically regulating the IFIT1 RNA-binding channel, thereby enhancing the specificity of recognition for cap 0 but not cap 1 (m7GpppNm) or 5'-ppp RNA. Mouse Ifit3 lacks this key C-terminal domain and does not bind mouse Ifit1. The IFIT3 interaction with IFIT1 is important for restricting infection of viruses lacking 2'-O methylation in their RNA cap structures. Our experiments establish differences in the regulation of IFIT1 orthologs and define targets for modulation of human IFIT protein activity.
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Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Metilación , Ratones , Modelos Moleculares , Conformación Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , ARN/química , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Especificidad de la Especie , Relación Estructura-ActividadRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuing to evolve around the world, generating new variants that are of concern on the basis of their potential for altered transmissibility, pathogenicity, and coverage by vaccines and therapeutic agents1-5. Here we show that serum samples taken from twenty human volunteers, two or four weeks after their second dose of the BNT162b2 vaccine, neutralize engineered SARS-CoV-2 with a USA-WA1/2020 genetic background (a virus strain isolated in January 2020) and spike glycoproteins from the recently identified B.1.617.1, B.1.617.2, B.1.618 (all of which were first identified in India) or B.1.525 (first identified in Nigeria) lineages. Geometric mean plaque reduction neutralization titres against the variant viruses-particularly the B.1.617.1 variant-seemed to be lower than the titre against the USA-WA1/2020 virus, but all sera tested neutralized the variant viruses at titres of at least 1:40. The susceptibility of the variant strains to neutralization elicited by the BNT162b2 vaccine supports mass immunization as a central strategy to end the coronavirus disease 2019 (COVID-19) pandemic globally.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , COVID-19/virología , Pruebas de Neutralización , SARS-CoV-2/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vacuna BNT162 , COVID-19/prevención & control , Vacunas contra la COVID-19/genética , Chlorocebus aethiops , Humanos , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Sintéticas/genética , Células Vero , Vacunas de ARNmRESUMEN
SARS-CoV-2 mRNA-based vaccines are about 95% effective in preventing COVID-191-5. The dynamics of antibody-secreting plasmablasts and germinal centre B cells induced by these vaccines in humans remain unclear. Here we examined antigen-specific B cell responses in peripheral blood (n = 41) and draining lymph nodes in 14 individuals who had received 2 doses of BNT162b2, an mRNA-based vaccine that encodes the full-length SARS-CoV-2 spike (S) gene1. Circulating IgG- and IgA-secreting plasmablasts that target the S protein peaked one week after the second immunization and then declined, becoming undetectable three weeks later. These plasmablast responses preceded maximal levels of serum anti-S binding and neutralizing antibodies to an early circulating SARS-CoV-2 strain as well as emerging variants, especially in individuals who had previously been infected with SARS-CoV-2 (who produced the most robust serological responses). By examining fine needle aspirates of draining axillary lymph nodes, we identified germinal centre B cells that bound S protein in all participants who were sampled after primary immunization. High frequencies of S-binding germinal centre B cells and plasmablasts were sustained in these draining lymph nodes for at least 12 weeks after the booster immunization. S-binding monoclonal antibodies derived from germinal centre B cells predominantly targeted the receptor-binding domain of the S protein, and fewer clones bound to the N-terminal domain or to epitopes shared with the S proteins of the human betacoronaviruses OC43 and HKU1. These latter cross-reactive B cell clones had higher levels of somatic hypermutation as compared to those that recognized only the SARS-CoV-2 S protein, which suggests a memory B cell origin. Our studies demonstrate that SARS-CoV-2 mRNA-based vaccination of humans induces a persistent germinal centre B cell response, which enables the generation of robust humoral immunity.
Asunto(s)
Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , Centro Germinal/inmunología , Células Plasmáticas/inmunología , Vacunas Sintéticas/inmunología , Adulto , Anciano , Animales , Anticuerpos Antivirales/inmunología , Vacuna BNT162 , COVID-19/prevención & control , Chlorocebus aethiops , Células Clonales/citología , Células Clonales/inmunología , Centro Germinal/citología , Voluntarios Sanos , Humanos , Persona de Mediana Edad , Células Plasmáticas/citología , SARS-CoV-2/inmunología , Factores de Tiempo , Células Vero , Vacunas de ARNmRESUMEN
Resistance represents a major challenge for antibody-based therapy for COVID-191-4. Here we engineered an immunoglobulin M (IgM) neutralizing antibody (IgM-14) to overcome the resistance encountered by immunoglobulin G (IgG)-based therapeutics. IgM-14 is over 230-fold more potent than its parental IgG-14 in neutralizing SARS-CoV-2. IgM-14 potently neutralizes the resistant virus raised by its corresponding IgG-14, three variants of concern-B.1.1.7 (Alpha, which first emerged in the UK), P.1 (Gamma, which first emerged in Brazil) and B.1.351 (Beta, which first emerged in South Africa)-and 21 other receptor-binding domain mutants, many of which are resistant to the IgG antibodies that have been authorized for emergency use. Although engineering IgG into IgM enhances antibody potency in general, selection of an optimal epitope is critical for identifying the most effective IgM that can overcome resistance. In mice, a single intranasal dose of IgM-14 at 0.044 mg per kg body weight confers prophylactic efficacy and a single dose at 0.4 mg per kg confers therapeutic efficacy against SARS-CoV-2. IgM-14, but not IgG-14, also confers potent therapeutic protection against the P.1 and B.1.351 variants. IgM-14 exhibits desirable pharmacokinetics and safety profiles when administered intranasally in rodents. Our results show that intranasal administration of an engineered IgM can improve efficacy, reduce resistance and simplify the prophylactic and therapeutic treatment of COVID-19.
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COVID-19/prevención & control , COVID-19/virología , Inmunoglobulina M/administración & dosificación , Inmunoglobulina M/inmunología , SARS-CoV-2/clasificación , SARS-CoV-2/inmunología , Administración Intranasal , Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/efectos adversos , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/metabolismo , COVID-19/inmunología , Relación Dosis-Respuesta Inmunológica , Femenino , Humanos , Inmunoglobulina A/genética , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/efectos adversos , Inmunoglobulina M/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ingeniería de Proteínas , Receptores Virales/antagonistas & inhibidores , Receptores Virales/metabolismo , SARS-CoV-2/genética , Tratamiento Farmacológico de COVID-19RESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein substitution D614G became dominant during the coronavirus disease 2019 (COVID-19) pandemic1,2. However, the effect of this variant on viral spread and vaccine efficacy remains to be defined. Here we engineered the spike D614G substitution in the USA-WA1/2020 SARS-CoV-2 strain, and found that it enhances viral replication in human lung epithelial cells and primary human airway tissues by increasing the infectivity and stability of virions. Hamsters infected with SARS-CoV-2 expressing spike(D614G) (G614 virus) produced higher infectious titres in nasal washes and the trachea, but not in the lungs, supporting clinical evidence showing that the mutation enhances viral loads in the upper respiratory tract of COVID-19 patients and may increase transmission. Sera from hamsters infected with D614 virus exhibit modestly higher neutralization titres against G614 virus than against D614 virus, suggesting that the mutation is unlikely to reduce the ability of vaccines in clinical trials to protect against COVID-19, and that therapeutic antibodies should be tested against the circulating G614 virus. Together with clinical findings, our work underscores the importance of this variant in viral spread and its implications for vaccine efficacy and antibody therapy.
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COVID-19/transmisión , COVID-19/virología , Aptitud Genética , Mutación , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Cricetinae , Modelos Animales de Enfermedad , Humanos , Pulmón/virología , Masculino , Mesocricetus/virología , Modelos Biológicos , Mucosa Nasal/virología , Pruebas de Neutralización , Estabilidad Proteica , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Técnicas de Cultivo de Tejidos , Tráquea/virología , Carga Viral , Virión/química , Virión/patogenicidad , Virión/fisiología , Replicación Viral/genéticaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.
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COVID-19/virología , Furina/metabolismo , Mutación , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Neutralizantes/inmunología , COVID-19/patología , COVID-19/fisiopatología , Línea Celular , Chlorocebus aethiops , Cricetinae , Femenino , Humanos , Enfermedades Pulmonares/patología , Enfermedades Pulmonares/fisiopatología , Enfermedades Pulmonares/virología , Masculino , Ratones , Ratones Transgénicos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteolisis , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero , Replicación Viral/genéticaRESUMEN
Rapidly emerging SARS-CoV-2 variants jeopardize antibody-based countermeasures. Although cell culture experiments have demonstrated a loss of potency of several anti-spike neutralizing antibodies against variant strains of SARS-CoV-21-3, the in vivo importance of these results remains uncertain. Here we report the in vitro and in vivo activity of a panel of monoclonal antibodies (mAbs), which correspond to many in advanced clinical development by Vir Biotechnology, AbbVie, AstraZeneca, Regeneron and Lilly, against SARS-CoV-2 variant viruses. Although some individual mAbs showed reduced or abrogated neutralizing activity in cell culture against B.1.351, B.1.1.28, B.1.617.1 and B.1.526 viruses with mutations at residue E484 of the spike protein, low prophylactic doses of mAb combinations protected against infection by many variants in K18-hACE2 transgenic mice, 129S2 immunocompetent mice and hamsters, without the emergence of resistance. Exceptions were LY-CoV555 monotherapy and LY-CoV555 and LY-CoV016 combination therapy, both of which lost all protective activity, and the combination of AbbVie 2B04 and 47D11, which showed a partial loss of activity. When administered after infection, higher doses of several mAb cocktails protected in vivo against viruses with a B.1.351 spike gene. Therefore, many-but not all-of the antibody products with Emergency Use Authorization should retain substantial efficacy against the prevailing variant strains of SARS-CoV-2.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/farmacología , Anticuerpos Antivirales/uso terapéutico , COVID-19/virología , Pruebas de Neutralización , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/inmunología , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/inmunología , COVID-19/genética , COVID-19/inmunología , COVID-19/prevención & control , Chlorocebus aethiops , Femenino , Humanos , Masculino , Mesocricetus/inmunología , Mesocricetus/virología , Ratones , Ratones Transgénicos , Profilaxis Posexposición , Profilaxis Pre-Exposición , SARS-CoV-2/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Células VeroRESUMEN
BNT162b2, a nucleoside-modified mRNA formulated in lipid nanoparticles that encodes the SARS-CoV-2 spike glycoprotein (S) stabilized in its prefusion conformation, has demonstrated 95% efficacy in preventing COVID-191. Here we extend a previous phase-I/II trial report2 by presenting data on the immune response induced by BNT162b2 prime-boost vaccination from an additional phase-I/II trial in healthy adults (18-55 years old). BNT162b2 elicited strong antibody responses: at one week after the boost, SARS-CoV-2 serum geometric mean 50% neutralizing titres were up to 3.3-fold above those observed in samples from individuals who had recovered from COVID-19. Sera elicited by BNT162b2 neutralized 22 pseudoviruses bearing the S of different SARS-CoV-2 variants. Most participants had a strong response of IFNγ+ or IL-2+ CD8+ and CD4+ T helper type 1 cells, which was detectable throughout the full observation period of nine weeks following the boost. Using peptide-MHC multimer technology, we identified several BNT162b2-induced epitopes that were presented by frequent MHC alleles and conserved in mutant strains. One week after the boost, epitope-specific CD8+ T cells of the early-differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating CD8+ T cells and were detectable (0.01-0.28%) eight weeks later. In summary, BNT162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes that are conserved in a broad range of variants, at well-tolerated doses.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Vacuna BNT162 , Linfocitos T CD8-positivos/inmunología , COVID-19/virología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/efectos adversos , Epítopos de Linfocito T/inmunología , Femenino , Humanos , Inmunoglobulina G/inmunología , Memoria Inmunológica , Interferón gamma/inmunología , Interleucina-2/inmunología , Masculino , Persona de Mediana Edad , SARS-CoV-2/química , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Células TH1/inmunología , Adulto JovenRESUMEN
Iron-based hexacyanoferrate (Fe-HCF) are promising cathode materials for sodium-ion batteries (SIBs) due to their unique open-channel structure that facilitates fast ion transport and framework stability. However, practical implementation of SIBs has been hindered by low initial Coulombic efficiency (ICE), poor rate performance, and short lifespan. Herein, we report a coordination engineering to synthesize sodium-rich Fe-HCF as cathodes for SIBs through a uniquely designed 10-kg-scale chemical reactor. Our study systematically investigated the relationship between coordination surroundings and the electrochemical behavior. Building on this understanding, the cathode delivered a reversible capacity of 99.3 mAh g-1 at 5 C (1 C = 100 mA g-1), exceptional rate capability (51 mAh g-1 even at 100 C), long lifespan (over 15,000 times at 50 C), and a high ICE of 92.7%. A full cell comprising the Fe-HCF cathode and hard carbon (HC) anode exhibited an impressive cyclic stability with a high-capacity retention rate of 98.3% over 1,000 cycles. Meanwhile, this material can be readily scaled to the practical levels of yield. The findings underscore the potential of Fe-HCF as cathodes for SIBs and highlight the significance of controlling nucleation and morphology through coordination engineering for a sustainable energy storage system.