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1.
Int J Nanomedicine ; 16: 2477-2486, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33824586

RESUMEN

Purpose: Sensitive and selective point-of-care biosensor is an urgent pursuit of serological antibody detection to control parasite pathogen. For specific, quantitative and on-site screening of Trichinella spiralis infection in livestock, a quantum dot nanobead-monoclonal antibody (QB-mAb) probe-based immunochromatographic assay (ICA) was developed by introducing a competitive sandwich strategy (QB-CICA). Methods: In the QB-CICA, QB-mAb probes competed with serum antibody for a particular epitope, followed by immunocomplexes binding to capture antibody on the test line. With the accumulation of target antibody, captured probes served as signal elements for fluorescent readout in a "turn off" mode, along with the fluorescence gradually weakened. The sensitivity and standard calibration curve of the QB-CICA were quantified using swine sera as negative control (n = 200) and artificial infected swine sera (n = 80) compared with a commercial ELISA kit. Besides, Trichinella spiralis-antibody targeting test ability of the QB-CICA, instead of other parasites or viruses antibodies (n = 10), was evaluated. Results: The QB-CICA exhibited a good linear range, a low detection limit of 189.92 ng mL-1 and 100% selectivity that was higher than commercial ELISA kit (90%), as well as the same serological positive rate (100%) with commercial ELISA kit in different infection dose models. Conclusion: Taking advantage of its simplicity, short response time (25 min), sensitivity and specificity, the proposed QB-CICA has potential applications for parasite-related antibody monitoring in food safety and clinical diagnosis fields.


Asunto(s)
Anticuerpos Antihelmínticos/análisis , Anticuerpos Monoclonales/inmunología , Cromatografía de Afinidad/métodos , Nanopartículas/química , Puntos Cuánticos/química , Trichinella spiralis/inmunología , Triquinelosis/diagnóstico , Triquinelosis/inmunología , Animales , Anticuerpos Antihelmínticos/sangre , Anticuerpos Antihelmínticos/inmunología , Nanopartículas/ultraestructura , Puntos Cuánticos/ultraestructura , Porcinos , Triquinelosis/parasitología
2.
Science ; 372(6537)2021 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-33795432

RESUMEN

Multivalent display of receptor-engaging antibodies or ligands can enhance their activity. Instead of achieving multivalency by attachment to preexisting scaffolds, here we unite form and function by the computational design of nanocages in which one structural component is an antibody or Fc-ligand fusion and the second is a designed antibody-binding homo-oligomer that drives nanocage assembly. Structures of eight nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage, respectively, closely match the corresponding computational models. Antibody nanocages targeting cell surface receptors enhance signaling compared with free antibodies or Fc-fusions in death receptor 5 (DR5)-mediated apoptosis, angiopoietin-1 receptor (Tie2)-mediated angiogenesis, CD40 activation, and T cell proliferation. Nanocage assembly also increases severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus neutralization by α-SARS-CoV-2 monoclonal antibodies and Fc-angiotensin-converting enzyme 2 (ACE2) fusion proteins.


Asunto(s)
Anticuerpos/química , Anticuerpos/inmunología , Nanoestructuras , Ingeniería de Proteínas , Transducción de Señal , Angiopoyetinas/química , Angiopoyetinas/inmunología , Angiopoyetinas/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Antígenos CD40/química , Antígenos CD40/inmunología , Antígenos CD40/metabolismo , Línea Celular Tumoral , Proliferación Celular , Simulación por Computador , Genes Sintéticos , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Activación de Linfocitos , Modelos Moleculares , Unión Proteica , Receptor TIE-2/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Linfocitos T/inmunología , Linfocitos T/fisiología
3.
Monoclon Antib Immunodiagn Immunother ; 40(2): 36-49, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33900819

RESUMEN

The dawn of the 20th century saw the formative years of developments in immunology. In particular, immunochemistry, specifically pertaining to antibodies, was extensively studied. These studies laid the foundations for employing antibodies in a variety of ways. Not surprisingly, antibodies have been used for applications ranging from biomedical research to disease diagnostics and therapeutics to evaluation of immune responses during natural infection and those elicited by vaccines. Despite recent advancements in cellular immunology and the excitement of T cell therapy, use of antibodies represents a large proportion of immunotherapeutic approaches as well as clinical interventions. Polyclonal antibodies in the form of plasma or sera continue to be used to treat a number of diseases, including autoimmune disorders, cancers, and infectious diseases. Historically, antisera to toxins have been the longest serving biotherapeutics. In addition, intravenous immunoglobulins (IVIg) have been extensively used to treat not only immunodeficiency conditions but also autoimmune disorders. Beyond the simplistic suppositions of their action, the IVIg have also unraveled the immune regulatory and homeostatic ramifications of their use. The advent of monoclonal antibodies (MAbs), on the other hand, has provided a clear pathway for their development as drug molecules. MAbs have found a clear place in the treatment of cancers and extending lives and have been used in a variety of other conditions. In this review, we capture the important developments in the therapeutic applications of antibodies to alleviate disease, with a focus on some of the recent developments.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Difteria/terapia , Neoplasias/terapia , Animales , Anticuerpos Monoclonales/inmunología , /virología , Tratamiento Basado en Trasplante de Células y Tejidos , Difteria/inmunología , Humanos , Inmunoglobulinas/inmunología , Inmunoglobulinas/uso terapéutico , Neoplasias/inmunología , /patogenicidad , Linfocitos T/inmunología
4.
MAbs ; 13(1): 1905978, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33843452

RESUMEN

Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NPNTD) from SARS-CoV-2 using small-angle X-ray scattering and transmission electron microscopy. Our solution-based results distinguished the mAbs' flexibility and how this flexibility affects the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NPNTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the antigen-binding fragments is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich enzyme-linked immunosorbent assay, we show that rigid mAb-pairings with linear polymerization led to increased NPNTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices, which are critical for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Epítopos/inmunología , Proteínas de la Nucleocápside/inmunología , /enzimología , Animales , Humanos
5.
Nat Commun ; 12(1): 1538, 2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33750786

RESUMEN

Plasmodium vivax preferentially invades reticulocytes and recognition of these cells is mediated by P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) binding to human Transferrin receptor 1 (TfR1) and Transferrin (Tf). Longitudinal cohort studies in Papua New Guinea, Thailand and Brazil show that PvRBP2b antibodies are correlated with protection against P. vivax infection and disease. Here, we isolate and characterize anti-PvRBP2b human monoclonal antibodies from two individuals in Cambodia with natural P. vivax infection. These antibodies bind with high affinities and map to different regions of PvRBP2b. Several human antibodies block PvRBP2b binding to reticulocytes and inhibit complex formation with human TfR1-Tf. We describe different structural mechanisms for functional inhibition, including either steric hindrance with TfR1-Tf or the reticulocyte membrane. These results show that naturally acquired human antibodies against PvRBP2b can inhibit its function which is important for P. vivax invasion.


Asunto(s)
Anticuerpos Bloqueadores , Anticuerpos Monoclonales/inmunología , Proteínas de la Membrana/metabolismo , Plasmodium vivax/metabolismo , Proteínas Protozoarias/metabolismo , Reticulocitos/metabolismo , Anticuerpos Antiprotozoarios/inmunología , Antígenos CD , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Cambodia , Cristalografía por Rayos X , Humanos , Estudios Longitudinales , Malaria Vivax/inmunología , Malaria Vivax/parasitología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Plasmodium vivax/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Receptores de Transferrina
6.
Nat Commun ; 12(1): 1750, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741942

RESUMEN

Malaria elimination requires tools that interrupt parasite transmission. Here, we characterize B cell receptor responses among Malian adults vaccinated against the first domain of the cysteine-rich 230 kDa gamete surface protein Pfs230, a key protein in sexual stage development of P. falciparum parasites. Among nine Pfs230 human monoclonal antibodies (mAbs) that we generated, one potently blocks transmission to mosquitoes in a complement-dependent manner and reacts to the gamete surface; the other eight show only low or no blocking activity. The structure of the transmission-blocking mAb in complex with vaccine antigen reveals a large discontinuous conformational epitope, specific to domain 1 of Pfs230 and comprising six structural elements in the protein. The epitope is conserved, suggesting the transmission-blocking mAb is broadly functional. This study provides a rational basis to improve malaria vaccines and develop therapeutic antibodies for malaria elimination.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Antiprotozoarios/farmacología , Epítopos/inmunología , Células Germinativas/inmunología , Malaria Falciparum/prevención & control , Plasmodium falciparum/efectos de los fármacos , Adulto , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Sitios de Unión , Células Cultivadas , Epítopos/química , Interacciones Huésped-Parásitos/efectos de los fármacos , Interacciones Huésped-Parásitos/inmunología , Humanos , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/inmunología , Malaria Falciparum/parasitología , Malaria Falciparum/transmisión , Mosquitos Vectores/parasitología , Plasmodium falciparum/inmunología , Plasmodium falciparum/fisiología , Conformación Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología
7.
Nat Commun ; 12(1): 1951, 2021 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-33782398

RESUMEN

Serological detection of antibodies to SARS-CoV-2 is essential for establishing rates of seroconversion in populations, and for seeking evidence for a level of antibody that may be protective against COVID-19 disease. Several high-performance commercial tests have been described, but these require centralised laboratory facilities that are comparatively expensive, and therefore not available universally. Red cell agglutination tests do not require special equipment, are read by eye, have short development times, low cost and can be applied at the Point of Care. Here we describe a quantitative Haemagglutination test (HAT) for the detection of antibodies to the receptor binding domain of the SARS-CoV-2 spike protein. The HAT has a sensitivity of 90% and specificity of 99% for detection of antibodies after a PCR diagnosed infection. We will supply aliquots of the test reagent sufficient for ten thousand test wells free of charge to qualified research groups anywhere in the world.


Asunto(s)
Anticuerpos Antivirales/análisis , /diagnóstico , Pruebas de Hemaglutinación/métodos , Glicoproteína de la Espiga del Coronavirus/inmunología , Pruebas de Aglutinación/métodos , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , /inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Sistemas de Atención de Punto , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Seroconversión
8.
Int J Mol Sci ; 22(4)2021 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-33670003

RESUMEN

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359-394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1-0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Péptidos/inmunología , alfa 1-Antitripsina/inmunología , Secuencia de Aminoácidos , Especificidad de Anticuerpos/inmunología , Trampas Extracelulares , Humanos , Lipopolisacáridos/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Péptidos/sangre , Péptidos/química , Desnaturalización Proteica
9.
Virology ; 558: 28-37, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33714753

RESUMEN

To help fight COVID-19, new molecular tools specifically targeting critical components of the causative agent of COVID-19, SARS-Coronavirus-2 (SARS-CoV-2), are desperately needed. The SARS-CoV-2 nucleocapsid protein is critical for viral replication, integral to viral particle assembly, and a major diagnostic marker for infection and immune protection. Currently the limited available antibody reagents targeting the nucleocapsid protein are not specific to SARS-CoV-2 nucleocapsid protein, and sequences for these antibodies are not publicly available. In this work we developed and characterized a series of new mouse monoclonal antibodies against the SARS-CoV-2 nucleocapsid protein, with a specific clone, mBG86, targeting only SARS-CoV-2 nucleocapsid protein. The monoclonal antibodies were validated in ELISA, Western blot, and immunofluorescence analyses. The variable regions from six select clones were cloned and sequenced, and preliminary epitope mapping of the sequenced clones was performed. Overall, these new antibody reagents will be of significant value in the fight against COVID-19.


Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antivirales , /inmunología , /inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Clonación Molecular , Escherichia coli , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Fosfoproteínas/inmunología , Proteínas Recombinantes/inmunología
10.
Cell ; 184(7): 1804-1820.e16, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33691139

RESUMEN

SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes and CD8+ T cells for optimal clinical and virological benefit. Thus, potently neutralizing mAbs utilize Fc effector functions during therapy to mitigate lung infection and disease.


Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Linfocitos T CD8-positivos , Fragmentos Fc de Inmunoglobulinas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/uso terapéutico , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Células CHO , /terapia , Chlorocebus aethiops , Cricetulus , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Células Vero , Carga Viral
11.
Int J Mol Sci ; 22(4)2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-33671877

RESUMEN

Since it was first reported in Wuhan, China, in 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic outbreak resulting in a tremendous global threat due to its unprecedented rapid spread and an absence of a prophylactic vaccine or therapeutic drugs treating the virus. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a key player in the viral entry into cells through its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor protein, and the RBD has therefore been crucial as a drug target. In this study, we used phage display to develop human monoclonal antibodies (mAbs) that neutralize SARS-CoV-2. A human synthetic Fab phage display library was panned against the RBD of the SARS-CoV-2 spike protein (SARS-2 RBD), yielding ten unique Fabs with moderate apparent affinities (EC50 = 19-663 nM) for the SARS-2 RBD. All of the Fabs showed no cross-reactivity to the MERS-CoV spike protein, while three Fabs cross-reacted with the SARS-CoV spike protein. Five Fabs showed neutralizing activities in in vitro assays based on the Fabs' activities antagonizing the interaction between the SARS-2 RBD and ACE2. Reformatting the five Fabs into immunoglobulin Gs (IgGs) greatly increased their apparent affinities (KD = 0.08-1.0 nM), presumably due to the effects of avidity, without compromising their non-aggregating properties and thermal stability. Furthermore, two of the mAbs (D12 and C2) significantly showed neutralizing activities on pseudo-typed and authentic SARS-CoV-2. Given their desirable properties and neutralizing activities, we anticipate that these human anti-SARS-CoV-2 mAbs would be suitable reagents to be further developed as antibody therapeutics to treat COVID-19, as well as for diagnostics and research tools.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Monoclonales/inmunología , Sitios de Unión , Humanos , Inmunoglobulina G/inmunología , Biblioteca de Péptidos , Dominios Proteicos , Glicoproteína de la Espiga del Coronavirus/química
12.
J Virol Methods ; 292: 114141, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33753172

RESUMEN

In this study, we developed and evaluated a luciferase immunosorbent assay (LISA) for quantitative detection of IgG antibody against SARS-CoV-2 nucleoprotein (NP). Anti-SARS-CoV-2 NP antibody in serum or plasma samples was captured by protein G-coated microtiter plate and detected using the crude cell lysates expressing Nanoluc luciferase (Nluc) enzyme fused with SARS-CoV-2 NP. After the addition of furimazine substrate, the levels of anti-SARS-CoV-2 NP IgG antibody were quantitatively measured as luciferase light units. As expected, SARS-CoV-2 NP showed cross-reactivity with the monoclonal antibodies against SARS-CoV NP, but not MERS-CoV NP-specific monoclonal antibodies or the monoclonal antibodies against SARS-CoV Spike protein. LISA for detecting murine monoclonal antibody against SARS-CoV NP showed a low limit of detection of 0.4 pg/µl and linear detection range from 0.4 pg/µl to 75 pg/µl. Furthermore, LISA had a sensitivity of 71 % when testing COVID-19 patients at the second week post onset and a specificity of 100 % when testing healthy blood donors.


Asunto(s)
Anticuerpos Antivirales/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina G/sangre , Proteínas de la Nucleocápside/inmunología , /inmunología , Anticuerpos Monoclonales/inmunología , Reacciones Cruzadas , Humanos , Luciferasas
13.
Nat Med ; 27(4): 717-726, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33664494

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global COVID-19 pandemic. Rapidly spreading SARS-CoV-2 variants may jeopardize newly introduced antibody and vaccine countermeasures. Here, using monoclonal antibodies (mAbs), animal immune sera, human convalescent sera and human sera from recipients of the BNT162b2 mRNA vaccine, we report the impact on antibody neutralization of a panel of authentic SARS-CoV-2 variants including a B.1.1.7 isolate, chimeric strains with South African or Brazilian spike genes and isogenic recombinant viral variants. Many highly neutralizing mAbs engaging the receptor-binding domain or N-terminal domain and most convalescent sera and mRNA vaccine-induced immune sera showed reduced inhibitory activity against viruses containing an E484K spike mutation. As antibodies binding to spike receptor-binding domain and N-terminal domain demonstrate diminished neutralization potency in vitro against some emerging variants, updated mAb cocktails targeting highly conserved regions, enhancement of mAb potency or adjustments to the spike sequences of vaccines may be needed to prevent loss of protection in vivo.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , /inmunología , Animales , /inmunología , Chlorocebus aethiops , Cricetinae , Humanos , Ratones , Mutación , Pruebas de Neutralización , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Células Vero
14.
Cell Rep ; 34(13): 108915, 2021 03 30.
Artículo en Inglés | MEDLINE | ID: mdl-33761319

RESUMEN

To fully decipher the immunogenicity of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein, it is essential to assess which part is highly immunogenic in a systematic way. We generate a linear epitope landscape of the Spike protein by analyzing the serum immunoglobulin G (IgG) response of 1,051 coronavirus disease 2019 (COVID-19) patients with a peptide microarray. We reveal two regions rich in linear epitopes, i.e., C-terminal domain (CTD) and a region close to the S2' cleavage site and fusion peptide. Unexpectedly, we find that the receptor binding domain (RBD) lacks linear epitope. We reveal that the number of responsive peptides is highly variable among patients and correlates with disease severity. Some peptides are moderately associated with severity and clinical outcome. By immunizing mice, we obtain linear-epitope-specific antibodies; however, no significant neutralizing activity against the authentic virus is observed for these antibodies. This landscape will facilitate our understanding of SARS-CoV-2-specific humoral responses and might be useful for vaccine refinement.


Asunto(s)
/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , /genética , China/epidemiología , Modelos Animales de Enfermedad , Mapeo Epitopo/métodos , Epítopos/inmunología , Femenino , Humanos , Inmunoglobulina G/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo
15.
Front Immunol ; 12: 635677, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33777026

RESUMEN

The outbreak and worldwide pandemic of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have a significant impact on global economy and human health. In order to reduce the disease spread, 16 monoclonal antibodies (McAbs) again SARS-CoV-2 were generated by immunized mice with the spike protein receptor binding domain (RBD), which was expressed in Chinese hamster ovary cell (CHO). A colloidal gold-based immunochromatographic strip was developed with two McAbs to detect SARS-CoV-2 spike protein, which can play a potential role in monitoring vaccine quality. The strip is highly specific, detecting only SARS-CoV-2 spike protein, and does not show any non-specific reactions with syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and other coronavirus and influenza viruses. The strip detected subunit vaccine in our laboratory with a detection limit of spike protein of 62.5 ng/mL. This strip provides an effective method in monitoring vaccine quality by detecting the antigen content of spike protein.


Asunto(s)
Antígenos Virales/análisis , /diagnóstico , Oro Coloide , Inmunoensayo/instrumentación , Tiras Reactivas , Glicoproteína de la Espiga del Coronavirus/análisis , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Antígenos Virales/inmunología , /virología , Humanos , Límite de Detección , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Glicoproteína de la Espiga del Coronavirus/inmunología
16.
Cell Rep ; 34(12): 108890, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33713594

RESUMEN

Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines show protective efficacy, which is most likely mediated by neutralizing antibodies recognizing the viral entry protein, spike. Because new SARS-CoV-2 variants are emerging rapidly, as exemplified by the B.1.1.7, B.1.351, and P.1 lineages, it is critical to understand whether antibody responses induced by infection with the original SARS-CoV-2 virus or current vaccines remain effective. In this study, we evaluate neutralization of a series of mutated spike pseudotypes based on divergence from SARS-CoV and then compare neutralization of the B.1.1.7 spike pseudotype and individual mutations. Spike-specific monoclonal antibody neutralization is reduced dramatically; in contrast, polyclonal antibodies from individuals infected in early 2020 remain active against most mutated spike pseudotypes, but potency is reduced in a minority of samples. This work highlights that changes in SARS-CoV-2 spike can alter neutralization sensitivity and underlines the need for effective real-time monitoring of emerging mutations and their effect on vaccine efficacy.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , /genética , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos , /metabolismo , Células HEK293 , Humanos , Pruebas de Neutralización/métodos , Mutación Puntual , Receptores Virales/genética , Receptores Virales/metabolismo , Glicoproteína de la Espiga del Coronavirus/inmunología
17.
BMC Infect Dis ; 21(1): 300, 2021 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-33761869

RESUMEN

BACKGROUND: Pseudomonas aeruginosa is the leading cause of nosocomial infections, especially in people with a compromised immune system. Targeting virulence factors by neutralizing antibodies is a novel paradigm for the treatment of antibiotic-resistant pseudomonas infections. In this respect, exotoxin A is one of the most potent virulence factors in P. aeruginosa. The present study was carried out to identify a novel human scFv antibody against the P. aeruginosa exotoxin A domain I (ExoA-DI) from a human scFv phage library. METHODS: The recombinant ExoA-DI of P. aeruginosa was expressed in E. coli, purified by Ni-NTA column, and used for screening of human antibody phage library. A novel screening procedure was conducted to prevent the elimination of rare specific clones. The phage clone with high reactivity was evaluated by ELISA and western blot. RESULTS: Based on the results of polyclonal phage ELISA, the fifth round of biopanning leads to the isolation of several ExoA-DI reactive clones. One positive clone with high affinity was selected by monoclonal phage ELISA and used for antibody expression. The purified scFv showed high reactivity with the recombinant domain I and full-length native exotoxin A. CONCLUSIONS: The purified anti-exotoxin A scFv displayed high specificity against exotoxin A. The human scFv identified in this study could be the groundwork for developing a novel therapeutic agent to control P. aeruginosa infections.


Asunto(s)
ADP Ribosa Transferasas/inmunología , Toxinas Bacterianas/inmunología , Exotoxinas/inmunología , Pseudomonas aeruginosa/inmunología , Anticuerpos de Cadena Única/inmunología , Factores de Virulencia/inmunología , ADP Ribosa Transferasas/genética , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos , Toxinas Bacterianas/genética , Escherichia coli/genética , Exotoxinas/genética , Humanos , Biblioteca de Péptidos , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/aislamiento & purificación , Factores de Virulencia/genética
19.
Int J Mol Sci ; 22(4)2021 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-33670450

RESUMEN

Hemophilia is an X-linked recessive bleeding disorder. In pregnant women carrier of hemophilia, the fetal sex can be determined by non-invasive analysis of fetal DNA circulating in the maternal blood. However, in case of a male fetus, conventional invasive procedures are required for the diagnosis of hemophilia. Fetal cells, circulating in the maternal bloodstream, are an ideal target for a safe non-invasive prenatal diagnosis. Nevertheless, the small number of cells and the lack of specific fetal markers have been the most limiting factors for their isolation. We aimed to develop monoclonal antibodies (mAbs) against the ribosomal protein RPS4Y1 expressed in male cells. By Western blotting, immunoprecipitation and immunofluorescence analyses performed on cell lysates from male human hepatoma (HepG2) and female human embryonic kidney (HEK293) we developed and characterized a specific monoclonal antibody against the native form of the male RPS4Y1 protein that can distinguish male from female cells. The availability of the RPS4Y1-targeting monoclonal antibody should facilitate the development of novel methods for the reliable isolation of male fetal cells from the maternal blood and their future use for non-invasive prenatal diagnosis of X-linked inherited disease such as hemophilia.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Ácidos Nucleicos Libres de Células/inmunología , Enfermedades Fetales/inmunología , Hemofilia A/inmunología , Diagnóstico Prenatal/métodos , Proteínas Ribosómicas/inmunología , Especificidad de Anticuerpos/inmunología , Ácidos Nucleicos Libres de Células/genética , Ácidos Nucleicos Libres de Células/metabolismo , Femenino , Enfermedades Fetales/sangre , Enfermedades Fetales/diagnóstico , Células HEK293 , Hemofilia A/sangre , Hemofilia A/diagnóstico , Células Hep G2 , Humanos , Masculino , Embarazo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Sensibilidad y Especificidad
20.
Cell Host Microbe ; 29(4): 529-539.e3, 2021 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-33705729

RESUMEN

All current vaccines for COVID-19 utilize ancestral SARS-CoV-2 spike with the goal of generating protective neutralizing antibodies. The recent emergence and rapid spread of several SARS-CoV-2 variants carrying multiple spike mutations raise concerns about possible immune escape. One variant, first identified in the United Kingdom (B.1.1.7, also called 20I/501Y.V1), contains eight spike mutations with potential to impact antibody therapy, vaccine efficacy, and risk of reinfection. Here, we show that B.1.1.7 remains sensitive to neutralization, albeit at moderately reduced levels (∼sim;2-fold), by serum samples from convalescent individuals and recipients of an mRNA vaccine (mRNA-1273, Moderna) and a protein nanoparticle vaccine (NVX-CoV2373, Novavax). A subset of monoclonal antibodies to the receptor binding domain (RBD) of spike are less effective against the variant, while others are largely unaffected. These findings indicate that variant B.1.1.7 is unlikely to be a major concern for current vaccines or for an increased risk of reinfection.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , /inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Pruebas de Neutralización , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto Joven
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