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1.
Cell ; 184(8): 2183-2200.e22, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33756110

RESUMEN

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.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Sitios de Unión de Anticuerpos , Células CHO , Chlorocebus aethiops , Cricetulus , Epítopos , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , SARS-CoV-2/inmunología , Células Vero
2.
Nat Immunol ; 23(1): 50-61, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34853448

RESUMEN

NP105-113-B*07:02-specific CD8+ T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105-113-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP105-113-B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP105-113-B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP105-113-B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design.


Asunto(s)
Antígeno HLA-B7/inmunología , Epítopos Inmunodominantes/inmunología , Proteínas de la Nucleocápside/inmunología , SARS-CoV-2/inmunología , Linfocitos T Citotóxicos/inmunología , Anciano , Secuencia de Aminoácidos , Anticuerpos Antivirales/inmunología , Afinidad de Anticuerpos/inmunología , COVID-19/inmunología , COVID-19/patología , Línea Celular Transformada , Femenino , Perfilación de la Expresión Génica , Humanos , Memoria Inmunológica/inmunología , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T/inmunología , Índice de Severidad de la Enfermedad , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Virus Vaccinia/metabolismo
3.
Immunity ; 55(4): 718-733.e8, 2022 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-35349789

RESUMEN

Resident memory B (BRM) cells develop and persist in the lungs of influenza-infected mice and humans; however, their contribution to recall responses has not been defined. Here, we used two-photon microscopy to visualize BRM cells within the lungs of influenza -virus immune and reinfected mice. Prior to re-exposure, BRM cells were sparsely scattered throughout the tissue, displaying limited motility. Within 24 h of rechallenge, these cells increased their migratory capacity, localized to infected sites, and subsequently differentiated into plasma cells. Alveolar macrophages mediated this process, in part by inducing expression of chemokines CXCL9 and CXCL10 from infiltrating inflammatory cells. This led to the recruitment of chemokine receptor CXCR3-expressing BRM cells to infected regions and increased local antibody concentrations. Our study uncovers spatiotemporal mechanisms that regulate lung BRM cell reactivation and demonstrates their capacity to rapidly deliver antibodies in a highly localized manner to sites of viral replication.


Asunto(s)
Gripe Humana , Infecciones por Orthomyxoviridae , Orthomyxoviridae , Animales , Anticuerpos , Humanos , Memoria Inmunológica , Células B de Memoria , Ratones
4.
Immunity ; 54(9): 1961-1975.e5, 2021 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-34525337

RESUMEN

Nucleic acids are powerful triggers of innate immunity and can adopt the Z-conformation, an unusual left-handed double helix. Here, we studied the biological function(s) of Z-RNA recognition by the adenosine deaminase ADAR1, mutations in which cause Aicardi-Goutières syndrome. Adar1mZα/mZα mice, bearing two point mutations in the Z-nucleic acid binding (Zα) domain that abolish Z-RNA binding, displayed spontaneous induction of type I interferons (IFNs) in multiple organs, including in the lung, where both stromal and hematopoietic cells showed IFN-stimulated gene (ISG) induction. Lung neutrophils expressed ISGs induced by the transcription factor IRF3, indicating an initiating role for neutrophils in this IFN response. The IFN response in Adar1mZα/mZα mice required the adaptor MAVS, implicating cytosolic RNA sensing. Adenosine-to-inosine changes were enriched in transposable elements and revealed a specific requirement of ADAR1's Zα domain in editing of a subset of RNAs. Thus, endogenous RNAs in Z-conformation have immunostimulatory potential curtailed by ADAR1, with relevance to autoinflammatory disease in humans.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Adenosina Desaminasa/genética , Interferón Tipo I/inmunología , ARN Bicatenario/genética , Adenosina/genética , Adenosina/metabolismo , Animales , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Inosina/genética , Inosina/metabolismo , Interferón Tipo I/genética , Ratones , Mutación , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/inmunología , Edición de ARN/genética , ARN Bicatenario/metabolismo
5.
J Immunol ; 209(10): 1832-1836, 2022 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-36426954

RESUMEN

In this study, we investigated how pre-existing Ab immunity to influenza virus established from prior immunizations affects the development of CD8+ T cell responses evoked after vaccination with a live attenuated vaccine. Using a mouse model and a panel of live attenuated influenza virus vaccine candidates (cold adapted and single cycle), we show that pre-existing influenza-specific Abs directed against the vaccine backbone attenuate the size and quality of the vaccine-induced CD8+ T cell response. Importantly, we show that increasing the vaccine dose can overcome this impediment, resulting in improved vaccine-induced circulating and tissue-resident memory CD8+ T cell responses, which were protective against heterologous influenza challenge. Thus, the reduced size and quality of the T cell response elicited by a live attenuated influenza virus vaccine imparted by the influenza-specific Ab landscape of the vaccinee can be overcome by increasing vaccine dose.


Asunto(s)
Vacunas contra la Influenza , Gripe Humana , Humanos , Vacunas Atenuadas , Inmunidad Humoral , Linfocitos T CD8-positivos
6.
Avian Pathol ; : 1-9, 2024 Sep 25.
Artículo en Inglés | MEDLINE | ID: mdl-39318350

RESUMEN

RESEARCH HIGHLIGHTS: Development of nr-NDV.Reverse transfection was applied for the recovery of nr-NDV.Propagation of nr-NDV was done by sub-passaging transfected BSR T7/5 cells.Safety profile was done to prove that the nr-NDV is non-replicating.

7.
PLoS Pathog ; 17(2): e1009352, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33635919

RESUMEN

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.


Asunto(s)
Anticuerpos Antivirales/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Adulto , Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/genética , Células Productoras de Anticuerpos/inmunología , Sitios de Unión , Epítopos , Humanos , Inmunoglobulina G/inmunología , Nucleocápside/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología
9.
J Gen Virol ; 102(1)2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33054904

RESUMEN

Although enveloped viruses canonically mediate particle entry through virus-cell fusion, certain viruses can spread by cell-cell fusion, brought about by receptor engagement and triggering of membrane-bound, viral-encoded fusion proteins on the surface of cells. The formation of pathogenic syncytia or multinucleated cells is seen in vivo, but their contribution to viral pathogenesis is poorly understood. For the negative-strand paramyxoviruses respiratory syncytial virus (RSV) and Nipah virus (NiV), cell-cell spread is highly efficient because their oligomeric fusion protein complexes are active at neutral pH. The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also been reported to induce syncytia formation in infected cells, with the spike protein initiating cell-cell fusion. Whilst it is well established that fusion protein-specific antibodies can block particle attachment and/or entry into the cell (canonical virus neutralization), their capacity to inhibit cell-cell fusion and the consequences of this neutralization for the control of infection are not well characterized, in part because of the lack of specific tools to assay and quantify this activity. Using an adapted bimolecular fluorescence complementation assay, based on a split GFP-Renilla luciferase reporter, we have established a micro-fusion inhibition test (mFIT) that allows the identification and quantification of these neutralizing antibodies. This assay has been optimized for high-throughput use and its applicability has been demonstrated by screening monoclonal antibody (mAb)-mediated inhibition of RSV and NiV fusion and, separately, the development of fusion-inhibitory antibodies following NiV vaccine immunization in pigs. In light of the recent emergence of coronavirus disease 2019 (COVID-19), a similar assay was developed for SARS-CoV-2 and used to screen mAbs and convalescent patient plasma for fusion-inhibitory antibodies. Using mFITs to assess antibody responses following natural infection or vaccination is favourable, as this assay can be performed entirely at low biocontainment, without the need for live virus. In addition, the repertoire of antibodies that inhibit cell-cell fusion may be different to those that inhibit particle entry, shedding light on the mechanisms underpinning antibody-mediated neutralization of viral spread.


Asunto(s)
Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , COVID-19/diagnóstico , Infecciones por Henipavirus/diagnóstico , Ensayos Analíticos de Alto Rendimiento , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Proteínas Virales de Fusión/antagonistas & inhibidores , Animales , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/aislamiento & purificación , Anticuerpos Antivirales/metabolismo , COVID-19/inmunología , COVID-19/virología , Fusión Celular , Convalecencia , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Infecciones por Henipavirus/inmunología , Infecciones por Henipavirus/virología , Humanos , Sueros Inmunes/química , Luciferasas/genética , Luciferasas/metabolismo , Modelos Moleculares , Virus Nipah/inmunología , Virus Nipah/patogenicidad , Conformación Proteica , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/inmunología , Virus Sincitial Respiratorio Humano/patogenicidad , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Porcinos , Inhibidores de Proteínas Virales de Fusión/química , Inhibidores de Proteínas Virales de Fusión/metabolismo , Inhibidores de Proteínas Virales de Fusión/farmacología , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/inmunología
10.
J Virol ; 94(4)2020 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-31748388

RESUMEN

The majority of antibodies induced by influenza neuraminidase (NA), like those against hemagglutinin (HA), are relatively specific to viruses isolated within a limited time window, as seen in serological studies and the analysis of many murine monoclonal antibodies (MAbs). We report three broadly reactive human MAbs targeting N1 NA. Two were isolated from a young adult vaccinated with trivalent influenza vaccine (TIV), which inhibited N1 NA from viruses isolated from humans over a period of a hundred years. The third antibody, isolated from a child with acute mild H7N9 infection, inhibited both group 1 N1 and group 2 N9 NAs. In addition, the antibodies cross-inhibited the N1 NAs of highly pathogenic avian H5N1 influenza viruses. These antibodies are protective in prophylaxis against seasonal H1N1 viruses in mice. This study demonstrates that human antibodies to N1 NA with exceptional cross-reactivity can be recalled by vaccination and highlights the importance of standardizing the NA antigen in seasonal vaccines to offer optimal protection.IMPORTANCE Antibodies to the influenza virus NA can provide protection against influenza disease. Analysis of human antibodies to NA lags behind that of antibodies to HA. We show that human monoclonal antibodies against NA induced by vaccination and infection can be very broadly reactive, with the ability to inhibit a wide spectrum of N1 NAs on viruses isolated between 1918 and 2018. This suggests that antibodies to NA may be a useful therapy and that the efficacy of influenza vaccines could be enhanced by ensuring the appropriate content of NA antigen.


Asunto(s)
Protección Cruzada/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Neuraminidasa/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Niño , Reacciones Cruzadas/inmunología , Perros , Femenino , Células HEK293 , Hemaglutininas/inmunología , Humanos , Inmunización Pasiva , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Subtipo H7N9 del Virus de la Influenza A/inmunología , Células de Riñón Canino Madin Darby , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Neuraminidasa/metabolismo , Infecciones por Orthomyxoviridae/virología , Vacunación , Adulto Joven
11.
Angew Chem Int Ed Engl ; 60(1): 321-330, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-32886840

RESUMEN

Matching of symmetry at interfaces is a fundamental obstacle in molecular assembly. Virus-like particles (VLPs) are important vaccine platforms against pathogenic threats, including Covid-19. However, symmetry mismatch can prohibit vaccine nanoassembly. We established an approach for coupling VLPs to diverse antigen symmetries. SpyCatcher003 enabled efficient VLP conjugation and extreme thermal resilience. Many people had pre-existing antibodies to SpyTag:SpyCatcher but less to the 003 variants. We coupled the computer-designed VLP not only to monomers (SARS-CoV-2) but also to cyclic dimers (Newcastle disease, Lyme disease), trimers (influenza hemagglutinins), and tetramers (influenza neuraminidases). Even an antigen with dihedral symmetry could be displayed. For the global challenge of influenza, SpyTag-mediated display of trimer and tetramer antigens strongly induced neutralizing antibodies. SpyCatcher003 conjugation enables nanodisplay of diverse symmetries towards generation of potent vaccines.


Asunto(s)
Vacunas contra la COVID-19/química , Nanoestructuras/química , Vacunas de Partículas Similares a Virus/química , Anticuerpos Neutralizantes/análisis , Anticuerpos Antivirales , Antígenos Virales/química , Antígenos Virales/inmunología , Congelación , Humanos , Modelos Moleculares
12.
Thorax ; 75(12): 1112-1115, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32883885

RESUMEN

When recombinant simian immunodeficiency virus (SIV) is pseudotyped with the F and HN glycoproteins from murine respiratory Sendai virus (rSIV.F/HN), it provides efficient lung cell targeting and lifelong transgene expression in the murine airways. We have shown that a single dose of rSIV.F/HN can direct stable expression of neutralising antibody against influenza in the murine airways and systemic circulation, and protects mice against two different influenza strains in lethal challenge experiments. These data suggest that rSIV.F/HN could be used as a vector for passive immunisation against influenza and other respiratory pathogens.


Asunto(s)
Anticuerpos Neutralizantes/genética , Vectores Genéticos/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Pulmón/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Virus de la Inmunodeficiencia de los Simios/genética , Animales , Anticuerpos Neutralizantes/inmunología , Expresión Génica , Vectores Genéticos/administración & dosificación , Glicoproteínas Hemaglutininas del Virus de la Influenza , Inmunización Pasiva , Inmunoglobulina G , Ratones , Virus Sendai/genética , Transgenes , Pérdida de Peso
13.
Nat Nanotechnol ; 19(8): 1216-1223, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38710880

RESUMEN

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.


Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacunas contra la COVID-19 , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , Anticuerpos Neutralizantes/inmunología , SARS-CoV-2/inmunología , Anticuerpos Antivirales/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/química , COVID-19/prevención & control , COVID-19/inmunología , COVID-19/virología , Humanos , Vacunación/métodos , Ratones , Nanopartículas/química , Femenino
14.
Elife ; 122023 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-36626205

RESUMEN

Virus-based tumour vaccines offer many advantages compared to other antigen-delivering systems. They generate concerted innate and adaptive immune response, and robust CD8+ T cell responses. We engineered a non-replicating pseudotyped influenza virus (S-FLU) to deliver the well-known cancer testis antigen, NY-ESO-1 (NY-ESO-1 S-FLU). Intranasal or intramuscular immunization of NY-ESO-1 S-FLU virus in mice elicited a strong NY-ESO-1-specific CD8+ T cell response in lungs and spleen that resulted in the regression of NY-ESO-1-expressing lung tumour and subcutaneous tumour, respectively. Combined administration with anti-PD-1 antibody, NY-ESO-1 S-FLU virus augmented the tumour protection by reducing the tumour metastasis. We propose that the antigen delivery through S-FLU is highly efficient in inducing antigen-specific CD8+ T cell response and protection against tumour development in combination with PD-1 blockade.


Asunto(s)
Inhibidores de Puntos de Control Inmunológico , Orthomyxoviridae , Masculino , Ratones , Animales , Antígenos de Neoplasias , Proteínas de la Membrana , Inmunización , Anticuerpos , Linfocitos T CD8-positivos
15.
Sci Adv ; 9(36): eadg3469, 2023 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-37683004

RESUMEN

Influenza virus-specific tissue-resident memory (Trm) CD8+ T cells located along the respiratory tract provide cross-strain protection against a breadth of influenza viruses. We show that immunization with a single-cycle influenza virus vaccine candidate (S-FLU) results in the deposition of influenza virus nucleoprotein (NP)-specific CD8+ Trm along the respiratory tract that were more cross-reactive against viral variants and less likely to drive the development of cytotoxic T lymphocyte (CTL) escape mutants, as compared to the lung memory NP-specific CD8+ T cell pool established following influenza infection. This immune profile was linked to the limited inflammatory response evoked by S-FLU vaccination, which increased TCR repertoire diversity within the memory CD8+ T cell compartment. Cumulatively, this work shows that S-FLU vaccination evokes a clonally diverse, cross-reactive memory CD8+ T cell pool, which protects against severe disease without driving the virus to rapidly evolve and escape, and thus represents an attractive vaccine for use against rapidly mutating influenza viruses.


Asunto(s)
Vacunas contra la Influenza , Gripe Humana , Humanos , Linfocitos T CD8-positivos , Gripe Humana/prevención & control , Inmunización , Levonorgestrel , Nucleoproteínas/genética , Pulmón
16.
Mol Ther Nucleic Acids ; 31: 370-382, 2023 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-36714461

RESUMEN

Since its discovery, COVID-19 has rapidly spread across the globe and has had a massive toll on human health, with infection mortality rates as high as 10%, and a crippling impact on the world economy. Despite numerous advances, there remains an urgent need for accurate and rapid point-of-care diagnostic tests and better therapeutic treatment options. To contribute chemically distinct, non-protein-based affinity reagents, we report here the identification of modified DNA-based aptamers that selectively bind to the S1, S2, or receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Several aptamers inhibit the binding of the spike protein to its cell-surface receptor angiotensin-converting enzyme 2 (ACE2) and neutralize authentic SARS-CoV-2 virus in vitro, including all variants of concern. With a high degree of nuclease resistance imparted by the base modifications, these reagents represent a new class of molecules with potential for further development as diagnostics or therapeutics.

17.
Nat Commun ; 14(1): 311, 2023 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-36658148

RESUMEN

Antibody-mediated immunity plays a crucial role in protection against SARS-CoV-2 infection. We isolated a panel of neutralizing anti-receptor-binding domain (RBD) antibodies elicited upon natural infection and vaccination and showed that they recognize an immunogenic patch on the internal surface of the core RBD, which faces inwards and is hidden in the "down" state. These antibodies broadly neutralize wild type (Wuhan-Hu-1) SARS-CoV-2, Beta and Delta variants and some are effective against other sarbecoviruses. We observed a continuum of partially overlapping antibody epitopes from lower to upper part of the inner face of the RBD and some antibodies extend towards the receptor-binding motif. The majority of antibodies are substantially compromised by three mutational hotspots (S371L/F, S373P and S375F) in the lower part of the Omicron BA.1, BA.2 and BA.4/5 RBD. By contrast, antibody IY-2A induces a partial unfolding of this variable region and interacts with a conserved conformational epitope to tolerate all antigenic variations and neutralize diverse sarbecoviruses as well. This finding establishes that antibody recognition is not limited to the normal surface structures on the RBD. In conclusion, the delineation of functionally and structurally conserved RBD epitopes highlights potential vaccine and therapeutic candidates for COVID-19.


Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , Glicoproteína de la Espiga del Coronavirus , Humanos , Epítopos , SARS-CoV-2 , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología
18.
Nat Commun ; 13(1): 3714, 2022 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-35764623

RESUMEN

Proteins can be empowered via SpyTag for anchoring and nanoassembly, through covalent bonding to SpyCatcher partners. Here we generate a switchable version of SpyCatcher, allowing gentle purification of SpyTagged proteins. We introduce numerous histidines adjacent to SpyTag's binding site, giving moderate pH-dependent release. After phage-based selection, our final SpySwitch allows purification of SpyTag- and SpyTag003-fusions from bacterial or mammalian culture by capture at neutral pH and release at pH 5, with purity far beyond His-tag methods. SpySwitch is also thermosensitive, capturing at 4 °C and releasing at 37 °C. With flexible choice of eluent, SpySwitch-purified proteins can directly assemble onto multimeric scaffolds. 60-mer multimerization enhances immunogenicity and we use SpySwitch to purify receptor-binding domains from SARS-CoV-2 and 11 other sarbecoviruses. For these receptor-binding domains we determine thermal resilience (for mosaic vaccine development) and cross-recognition by antibodies. Antibody EY6A reacts across all tested sarbecoviruses, towards potential application against new coronavirus pandemic threats.


Asunto(s)
COVID-19 , Calor , Animales , Anticuerpos , Concentración de Iones de Hidrógeno , Mamíferos , SARS-CoV-2
19.
Theranostics ; 12(1): 1-17, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-34987630

RESUMEN

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.


Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Tratamiento Farmacológico de COVID-19 , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Sitios de Unión , Unión Competitiva , COVID-19/virología , Cricetinae , Microscopía por Crioelectrón , Cristalografía por Rayos X , Perros , Epítopos , Femenino , Humanos , Células de Riñón Canino Madin Darby , Pruebas de Neutralización , Dominios Proteicos , SARS-CoV-2/genética , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo
20.
Commun Med (Lond) ; 2: 36, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35603265

RESUMEN

Background: Evaluation of susceptibility to emerging SARS-CoV-2 variants of concern (VOC) requires rapid screening tests for neutralising antibodies which provide protection. Methods: Firstly, we developed a receptor-binding domain-specific haemagglutination test (HAT) to Wuhan and VOC (alpha, beta, gamma and delta) and compared to pseudotype, microneutralisation and virus neutralisation assays in 835 convalescent sera. Secondly, we investigated the antibody response using the HAT after two doses of mRNA (BNT162b2) vaccination. Sera were collected at baseline, three weeks after the first and second vaccinations from older (80-99 years, n = 89) and younger adults (23-77 years, n = 310) and compared to convalescent sera from naturally infected individuals (1-89 years, n = 307). Results: Here we show that HAT antibodies highly correlated with neutralising antibodies (R = 0.72-0.88) in convalescent sera. Home-dwelling older individuals have significantly lower antibodies to the Wuhan strain after one and two doses of BNT162b2 vaccine than younger adult vaccinees and naturally infected individuals. Moverover, a second vaccine dose boosts and broadens the antibody repertoire to VOC in naïve, not previously infected older and younger adults. Most (72-76%) older adults respond after two vaccinations to alpha and delta, but only 58-62% to beta and gamma, compared to 96-97% of younger vaccinees and 68-76% of infected individuals. Previously infected older individuals have, similarly to younger adults, high antibody titres after one vaccination. Conclusions: Overall, HAT provides a surrogate marker for neutralising antibodies, which can be used as a simple inexpensive, rapid test. HAT can be rapidly adaptable to emerging VOC for large-scale evaluation of potentially decreasing vaccine effectiveness.

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