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1.
Front Immunol ; 12: 637651, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33767706

RESUMEN

As COVID-19 cases continue to rise, it is imperative to learn more about antibodies and T-cells produced against the causative virus, SARS-CoV-2, in order to guide the rapid development of therapies and vaccines. While much of the current antibody and vaccine research focuses on the receptor-binding domain of S1, a less-recognized opportunity is to harness the potential benefits of the more conserved S2 subunit. Similarities between the spike proteins of both SARS-CoV-2 and HIV-1 warrant exploring S2. Possible benefits of employing S2 in therapies and vaccines include the structural conservation of S2, extant cross-reactive neutralizing antibodies in populations (due to prior exposure to common cold coronaviruses), the steric neutralization potential of antibodies against S2, and the stronger memory B-cell and T-cell responses. More research is necessary on the effect of glycans on the accessibility and stability of S2, SARS-CoV-2 mutants that may affect infectivity, the neutralization potential of antibodies produced by memory B-cells, cross-reactive T-cell responses, antibody-dependent enhancement, and antigen competition. This perspective aims to highlight the evidence for the potential advantages of using S2 as a target of therapy or vaccine design.


Asunto(s)
/uso terapéutico , /inmunología , Glicoproteína de la Espiga del Coronavirus/uso terapéutico , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Especificidad de Anticuerpos , /virología , Reacciones Cruzadas , Epítopos , Interacciones Huésped-Patógeno , Humanos , Inmunogenicidad Vacunal , Subunidades de Proteína , Glicoproteína de la Espiga del Coronavirus/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/virología , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/uso terapéutico
2.
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
3.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33688035

RESUMEN

Modified vaccinia virus Ankara (MVA) is a replication-restricted smallpox vaccine, and numerous clinical studies of recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases, including COVID-19, are in progress. Here, we characterize rMVAs expressing the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Modifications of full-length S individually or in combination included two proline substitutions, mutations of the furin recognition site, and deletion of the endoplasmic retrieval signal. Another rMVA in which the receptor binding domain (RBD) is flanked by the signal peptide and transmembrane domains of S was also constructed. Each modified S protein was displayed on the surface of rMVA-infected cells and was recognized by anti-RBD antibody and soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced antibodies, which neutralized a pseudovirus in vitro and, upon passive transfer, protected hACE2 transgenic mice from lethal infection with SARS-CoV-2, as well as S-specific CD3+CD8+IFNγ+ T cells. Antibody boosting occurred following a second rMVA or adjuvanted purified RBD protein. Immunity conferred by a single vaccination of hACE2 mice prevented morbidity and weight loss upon intranasal infection with SARS-CoV-2 3 wk or 7 wk later. One or two rMVA vaccinations also prevented detection of infectious SARS-CoV-2 and subgenomic viral mRNAs in the lungs and greatly reduced induction of cytokine and chemokine mRNAs. A low amount of virus was found in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of low levels of subgenomic mRNAs in turbinates indicated that replication was aborted in immunized animals.


Asunto(s)
/inmunología , Vectores Genéticos/genética , Vacunas de ADN/inmunología , Virus Vaccinia/genética , /genética , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos/inmunología , /genética , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Inmunización , Inmunización Pasiva , Inmunoglobulina G/inmunología , Ratones , Ratones Transgénicos , Glicoproteína de la Espiga del Coronavirus/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética
4.
N Biotechnol ; 62: 79-85, 2021 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-33556628

RESUMEN

A phage library displaying 1010 variants of the fibronectin type III (FN3) domain was affinity selected with the biotinylated form of the receptor binding domain (RBD, residues 319-541) of the SARS-CoV-2 virus spike protein. Nine binding FN3 variants (i.e. monobodies) were recovered, representing four different primary structures. Soluble forms of the monobodies bound to several different preparations of the RBD and the S1 spike subunit, with affinities ranging from 3 to 14 nM as measured by bio-layer interferometry. Three of the four monobodies bound selectively to the RBD of SARS-CoV-2, with the fourth monobody showing slight cross-reactivity to the RBD of SARS-CoV-1 virus. Examination of binding to the spike fragments and its trimeric form revealed that the monobodies recognise at least three overlapping epitopes on the RBD of SARS-CoV-2. While pairwise tests failed to identify a monobody pair that could bind simultaneously to the RBD, one monobody could simultaneously bind to the RBD with the ectodomain of the cellular receptor angiotensin converting enzyme 2 (ACE2). All four monobodies successfully bound the RBD after overexpression in Chinese hamster ovary (CHO) cells as fusions to the Fc domain of human IgG1.


Asunto(s)
/inmunología , Especificidad de Anticuerpos , Epítopos/inmunología , Anticuerpos de Cadena Única/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Línea Celular , Reacciones Cruzadas , Humanos , Dominios Proteicos
5.
Nat Commun ; 12(1): 1102, 2021 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-33597521

RESUMEN

The four-dengue virus (DENV) serotypes infect several hundred million people annually. For the greatest safety and efficacy, tetravalent DENV vaccines are designed to stimulate balanced protective immunity to all four serotypes. However, this has been difficult to achieve. Clinical trials with a leading vaccine demonstrated that unbalanced replication and immunodominance of one vaccine component over others can lead to low efficacy and vaccine enhanced severe disease. The Laboratory of Infectious Diseases at the National Institutes of Health has developed a live attenuated tetravalent DENV vaccine (TV003), which is currently being tested in phase 3 clinical trials. Here we report, our study to determine if TV003 stimulate balanced and serotype-specific (TS) neutralizing antibody (nAb) responses to each serotype. Serum samples from twenty-one dengue-naive individuals participated under study protocol CIR287 (ClinicalTrials.gov NCT02021968) are analyzed 6 months after vaccination. Most subjects (76%) develop TS nAbs to 3 or 4 DENV serotypes, indicating immunity is induced by each vaccine component. Vaccine-induced TS nAbs map to epitopes known to be targets of nAbs in people infected with wild type DENVs. Following challenge with a partially attenuated strain of DENV2, all 21 subjects are protected from the efficacy endpoints. However, some vaccinated individuals develop post challenge nAb boost, while others mount post-challenge antibody responses that are consistent with sterilizing immunity. TV003 vaccine induced DENV2 TS nAbs are associated with sterilizing immunity. Our results indicate that nAbs to TS epitopes on each serotype may be a better correlate than total levels of nAbs currently used for guiding DENV vaccine development.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Dengue/inmunología , Formación de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Dengue/prevención & control , Dengue/virología , Vacunas contra el Dengue/administración & dosificación , Virus del Dengue/clasificación , Epítopos/inmunología , Humanos , Serotipificación , Especificidad de la Especie , Resultado del Tratamiento , Vacunación/métodos , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología
6.
Sci Rep ; 11(1): 2776, 2021 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-33531605

RESUMEN

The accurate and prompt diagnosis of SARS-CoV-2 infection is required for the control and treatment of the coronavirus infection disease 2019 (COVID-19). In this study, we aimed to investigate the time courses of the anti-severe acute corona respiratory syndrome coronavirus 2 (SARS-CoV-2) IgM and IgG titers and to evaluate the sensitivity and specificity of such tests according to the specific day after the onset of COVID-19 among a patient population in Japan. We measured the titers of SARS-CoV-2 IgM and IgG in sera from 105 subjects, including 26 symptomatic COVID-19 patients, using chemiluminescent immunoassay (CLIA) methods utilizing magnetic beads coated with SARS-CoV-2 nucleocapsid protein and spike protein. The results of a ROC analysis suggested the possibility that the cutoff values in Japan might be lower than the manufacturer's reported cutoff (10 AU/mL): 1  AU/mL for IgM and 5  AU/mL for IgG. The sensitivity of the test before Day 8 after symptom onset was less than 50%; at Days 9-10, however, we obtained a much higher sensitivity of 81.8% for both IgM and IgG. At 15 days or later after symptom onset, the SARS-CoV-2 IgG test had a sensitivity of 100%. These results suggest that if the number of days since disease onset is taken into consideration, these antibody tests could be very useful for the diagnosis of COVID-19 and similar diseases.


Asunto(s)
Especificidad de Anticuerpos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , /inmunología , /virología , Ensayo de Inmunoadsorción Enzimática , Humanos , Japón
7.
Int J Nanomedicine ; 16: 715-724, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33542626

RESUMEN

Objective: The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is now rapidly spreading globally. Serological tests are an important method to assist in the diagnosis of COVID-19, used for epidemiological investigations. In this study, we aimed to investigate the impact of different types of vacuum collection tubes on the detection of SARS-CoV-2 IgM and IgG antibodies, using the colloidal gold immunochromatographic assay (GICA). Patients and Methods: A total of 112 patients with COVID-19 and 200 healthy control subjects with no infection were enrolled in this study. Their serum and plasma were collected into four different types of vacuum blood collection tubes. SARS-CoV-2 IgM and IgG specific antibodies in the plasma and serum were then detected by GICA and chemiluminescence assay (CA), respectively. In addition, the particle sizes of different colloidal gold solutions in the presence of different anticoagulants and coagulants were evaluated by both laser diffraction (Malvern) and confocal laser microscope, respectively. Results: Our results revealed that anticoagulated plasma with EDTA-K2 improved the positive detection rate of SARS-CoV-2 IgM antibodies. Furthermore, our results shown that the detection results by GICA and CA were highly consistent, especially, the results of EDTA-K2 anticoagulated plasma detected by GICA was more consistent with CA results. We confirmed that EDTA-K2 could improve the detection sensitivity of SARS-CoV-2 IgG antibodies by chelating excessive colloidal gold compared with sodium citrate or lithium heparin, these methodologies did not appear to cause false positives. Colloidal gold particles could be chelated and aggregated by EDTA-K2, but not by sodium citrate, lithium heparin and coagulants. Conclusion: GICA is widely used to detect antibodies for the advantages of convenient, fast, low cost, suitable for screening large sample and require minimal equipment. In this study, we found that EDTA-K2 amplified the positive antibody signal by chelating colloidal gold and improved the detection sensitivity of SARS-CoV-2 IgM and IgG antibodies when using the GICA. Therefore, we suggested that EDTA-K2 anticoagulated plasma was more suitable for the detection of SARS-CoV-2 antibodies.


Asunto(s)
Anticuerpos Antivirales/aislamiento & purificación , Quelantes/química , Ácido Edético/química , Oro Coloide/química , Inmunoensayo/métodos , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina M/aislamiento & purificación , /inmunología , Adulto , Anticuerpos Antivirales/sangre , Especificidad de Anticuerpos/inmunología , /inmunología , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , Peso Molecular , Tamaño de la Partícula , Polímeros/química , Sensibilidad y Especificidad
8.
ACS Synth Biol ; 10(2): 379-390, 2021 02 19.
Artículo en Inglés | MEDLINE | ID: mdl-33534552

RESUMEN

Generating and characterizing immunoreagents to enable studies of novel emerging viruses is an area where ensembles of synthetic genes, recombinant antibody pipelines, and modular antibody-reporter fusion proteins can respond rapidly. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread through the global population causing widespread morbidity, mortality, and socioeconomic chaos. Using SARS-CoV-2 as our model and starting with a gBlocks encoded nucleocapsid (N) gene, we purified recombinant protein from E. coli, to serve as bait for selecting semisynthetic nanobodies from our Nomad single-pot library. Clones were isolated in days and first fused to Gaussia luciferase to determine EC50 in the tens of nM range, and second fused to the ascorbate peroxidase derivative APEX2 for sensitive detection of SARS-CoV-2 infected cells. To generate inherently fluorescent immunoreagents, we introduce novel periplasmic sdAb fusions made with mNeonGreen and mScarlet-I, which were produced at milligram amounts. The fluorescent fusion proteins enabled concise visualization of SARS-CoV-2 N in the cytoplasm but not in the nucleus 24 h post infection, akin to the distribution of SARS-CoV N, thereby validating these useful imaging tools. SdAb reactivity appeared specific to SARS-CoV-2 with very much weaker binding to SARS-CoV, and no noticeable cross-reactivity to a panel of overexpressed human codon optimized N proteins from other CoV. High periplasmic expression levels and in silico immortalization of the nanobody constructs guarantees a cost-effective and reliable source of SARS-CoV-2 immunoreagents. Our proof-of-principle study should be applicable to known and newly emerging CoV to broaden the tools available for their analysis and help safeguard human health in a more proactive than reactive manner.


Asunto(s)
/epidemiología , /genética , Sondas Moleculares/genética , Pandemias , /inmunología , Anticuerpos Antivirales/genética , Especificidad de Anticuerpos/genética , Enfermedades Transmisibles Emergentes/virología , Escherichia coli/genética , Técnica del Anticuerpo Fluorescente , Genes Sintéticos , Genes Virales , Células HEK293 , Humanos , Sondas Moleculares/inmunología , Pandemias/prevención & control , Biblioteca de Péptidos , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos de Dominio Único/genética , Biología Sintética
9.
Methods Mol Biol ; 2261: 291-306, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33420997

RESUMEN

Sensitive and reproducible pharmacokinetic (PK) assays and immunogenicity assessment are required as part of the complex and lengthy development process for biotherapeutic proteins. Ligand binding assays (LBAs) are included in a range of approaches applied to understand the nature and properties of the drug as well as the induction of anti-drug antibodies (ADA) against the therapeutic, which can cause adverse events and loss of efficacy. Currently, most biotherapeutics are monoclonal human or humanized antibodies. Anti-idiotypic antibodies, targeting the idiotopic determinants of individual antibody drugs are recognized as perfect reagents for such LBAs. Here we describe the typical setups for these assays and how different types of anti-biotherapeutic antibodies can be used to establish selective and sensitive assays.


Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Productos Biológicos/inmunología , Desarrollo de Medicamentos , Monitoreo de Drogas , Epítopos , Inmunoensayo , Proteínas/inmunología , Anticuerpos Monoclonales Humanizados/farmacocinética , Especificidad de Anticuerpos , Productos Biológicos/farmacocinética , Humanos , Idiotipos de Inmunoglobulinas , Ligandos , Unión Proteica , Proteínas/farmacocinética
10.
Methods Mol Biol ; 2261: 457-479, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33421008

RESUMEN

Western blotting continues to be a workhorse assay in laboratories throughout the world. The utility, low cost and accessibility of western blotting have allowed the technique to remain in practice, despite being developed over 40 years ago. Advances in antibody specificity, chemiluminescent formulations, properties of fluorescent molecules and imaging techniques provide gains in sensitivity, dynamic range, and ease of use. Here we discuss such aspects for the users' consideration when planning and executing western blots, to take full advantage of contemporary practices.


Asunto(s)
Western Blotting , Proteínas/análisis , Animales , Especificidad de Anticuerpos , Western Blotting/normas , Calibración , Técnica del Anticuerpo Fluorescente , Humanos , Estándares de Referencia , Coloración y Etiquetado
11.
JCI Insight ; 6(4)2021 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-33497357

RESUMEN

Four endemic human coronaviruses (HCoVs) are commonly associated with acute respiratory infection in humans. B cell responses to these "common cold" viruses remain incompletely understood. Here we report a comprehensive analysis of CoV-specific antibody repertoires in 231 children and 1168 adults using phage immunoprecipitation sequencing. Seroprevalence of antibodies against endemic HCoVs ranged between approximately 4% and 27% depending on the species and cohort. We identified at least 136 novel linear B cell epitopes. Antibody repertoires against endemic HCoVs were qualitatively different between children and adults in that anti-HCoV IgG specificities more frequently found among children targeted functionally important and structurally conserved regions of the spike, nucleocapsid, and matrix proteins. Moreover, antibody specificities targeting the highly conserved fusion peptide region and S2' cleavage site of the spike protein were broadly cross-reactive with peptides of epidemic human and nonhuman coronaviruses. In contrast, an acidic tandem repeat in the N-terminal region of the Nsp3 subdomain of the HCoV-HKU1 polyprotein was the predominant target of antibody responses in adult donors. Our findings shed light on the dominant species-specific and pan-CoV target sites of human antibody responses to coronavirus infection, thereby providing important insights for the development of prophylactic or therapeutic monoclonal antibodies and vaccine design.


Asunto(s)
Anticuerpos Antivirales/aislamiento & purificación , Resfriado Común/virología , Infecciones por Coronavirus/inmunología , Coronavirus/inmunología , Enfermedades Endémicas , Adulto , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Antígenos Virales/sangre , Antígenos Virales/inmunología , Niño , Preescolar , Resfriado Común/sangre , Resfriado Común/epidemiología , Resfriado Común/inmunología , Coronavirus/aislamiento & purificación , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/virología , Reacciones Cruzadas , Epítopos de Linfocito B/sangre , Epítopos de Linfocito B/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Dominios Proteicos/inmunología , Estudios Retrospectivos , Estudios Seroepidemiológicos , Proteínas Virales/inmunología
12.
Int Immunopharmacol ; 92: 107330, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33412393

RESUMEN

In addition to molecular testing, there is evolving interest for anti-SARS-CoV-2 antibodies serologic assays. Majority of them focus on IgM/IgG despite IgA important role in mucosal immunity. A simultaneous anti-SARS-CoV-2 IgA/IgG/IgM immunoassay, performed on an automated instrument by ELISA kit coated with native inactivated SARS-CoV-2, was detected on two control groups (negative swab healthcare workers; pre-pandemic healthy or with other viral infections individuals) and on two COVID-19 patient groups (early and late infection). Specificities were 100% in all groups, indicating no cross-reactivity with other infectious or pre-pandemic sera. Sensitivities were 94% in early infection group and 97% in total positive patient group, reaching 100% in late infection group. To our knowledge, this is the first technique based on native SARS-CoV-2. It is able to identify more positive samples than kits using recombinant antigens, therefore virus native epitopes as well as simultaneous anti-SARS-CoV-2 IgA/IgM/IgG detection could help to contain COVID-19 spreading.


Asunto(s)
Anticuerpos Antivirales/sangre , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , /virología , Humanos , Inmunoensayo/métodos , Sensibilidad y Especificidad , Pruebas Serológicas/métodos
13.
Methods Mol Biol ; 2248: 91-107, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33185870

RESUMEN

Systemic cytokine inhibition may be an effective therapeutic strategy for several autoimmune diseases. However, recent studies suggest that tissue or cell type-specific targeting of certain cytokines, including TNF, may have distinct advantages and show fewer side effects. Here we describe protocols for generating and testing bispecific cytokine inhibitors using variable domain of single-chain antibodies from Camelidae (VHH) with a focus on cell-specific TNF inhibitors.


Asunto(s)
Cadenas Pesadas de Inmunoglobulina , Región Variable de Inmunoglobulina , Anticuerpos de Dominio Único/biosíntesis , Inhibidores del factor de Necrosis Tumorales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Citocinas/biosíntesis , Citometría de Flujo , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/aislamiento & purificación , Anticuerpos de Dominio Único/farmacología , Inhibidores del factor de Necrosis Tumorales/química , Inhibidores del factor de Necrosis Tumorales/aislamiento & purificación , Inhibidores del factor de Necrosis Tumorales/farmacología , Factores de Necrosis Tumoral/química , Factores de Necrosis Tumoral/metabolismo
14.
Biosens Bioelectron ; 172: 112765, 2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-33126179

RESUMEN

To accurately diagnose COVID-19 infection and its time-dependent progression, the rapid, sensitive, and noninvasive determination of immunoglobulins A specific to SARS-CoV-2 (IgA) in saliva and serum is needed to complement tests that detect immunoglobulins G and M. We have developed a dual optical/chemiluminescence format of a lateral flow immunoassay (LFIA) immunosensor for IgA in serum and saliva. A recombinant nucleocapsid antigen specifically captures SARS-CoV-2 antibodies in patient specimens. A labelled anti-human IgA reveals the bound IgA fraction. A dual colorimetric and chemiluminescence detection enables the affordable and ultrasensitive determination of IgA to SARS-CoV-2. Specifically, a simple smartphone-camera-based device measures the colour signal provided by nanogold-labelled anti-human IgA. For the ultrasensitive chemiluminescence transduction, we used a contact imaging portable device based on cooled CCD, and measured the light signal resulting from the reaction of the HRP-labelled anti-human IgA with a H2O2/luminol/enhancers substrate. A total of 25 serum and 9 saliva samples from infected and/or recovered individuals were analysed by the colorimetric LFIA, which was sensitive and reproducible enough for the semi-quantification of IgA in subjects with a strong serological response and in the early stage of COVID-19 infection. Switching to CL detection, the same immunosensor exhibited higher detection capability, revealing the presence of salivary IgA in infected individuals. For the patients included in the study (n = 4), the level of salivary IgA correlated with the time elapsed from diagnosis and with the severity of the disease. This IgA-LFIA immunosensor could be useful for noninvasively monitoring early immune responses to COVID-19 and for investigating the diagnostic/prognostic utility of salivary IgA in the context of large-scale screening to assess the efficacy of SARS-CoV-2 vaccines.


Asunto(s)
Anticuerpos Antivirales/análisis , Técnicas Biosensibles/instrumentación , /diagnóstico , /inmunología , Especificidad de Anticuerpos , Técnicas Biosensibles/métodos , /virología , Colorimetría/instrumentación , Colorimetría/métodos , Diseño de Equipo , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina A Secretora/análisis , Mediciones Luminiscentes/instrumentación , Mediciones Luminiscentes/métodos , Saliva/inmunología
15.
Talanta ; 223(Pt 1): 121737, 2021 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-33303174

RESUMEN

A rapid test for detecting total immunoglobulins directed towards the nucleocapsid protein (N) of severe acute syndrome coronavirus 2 (SARS CoV-2) was developed, based on a multi-target lateral flow immunoassay comprising two test lines. Both test lines bound to several classes of immunoglobulins (G, M, and A). Specific anti-SARS immunoglobulins were revealed by a colorimetric probe formed by N and gold nanoparticles. Targeting the total antibodies response to infection enabled achieving 100% diagnostic specificity (95.75-100, C.I. 95%, n = 85 healthy and with other infections individuals) and 94.6% sensitivity (84.9-98.9, C.I. 95%, n = 62 SARS CoV-2 infected subjects) as early as 7 days post confirmation of positivity. Agreeing results with a reference serological ELISA were achieved, except for the earlier detection capability of the rapid test. Follow up of the three seroconverting patients endorsed the hypothesis of the random rise of the different immunoglobulins and strengthened the 'total antibodies' approach for the trustworthy detection of serological response to SARS CoV-2 infection.


Asunto(s)
/métodos , /inmunología , Inmunoensayo/métodos , Adulto , Especificidad de Anticuerpos , Colorimetría , Diagnóstico Precoz , Diseño de Equipo , Oro , Humanos , Inmunoglobulinas/análisis , Masculino , Nanopartículas del Metal , Persona de Mediana Edad , Nucleocápside/química , Sensibilidad y Especificidad
16.
Methods Mol Biol ; 2183: 9-18, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32959237

RESUMEN

The immunoglobulin capture assay (ICA) enables the enrichment for pathogen-specific plasmablasts from individuals with a confirmed adaptive immune response to vaccination or disseminated infection. Only single recombinant antigens have been used previously as probes in this ICA and it was unclear whether the method was applicable to complex probes such as whole bacterial cells. Here, we describe the enrichment of plasmablasts specific for polysaccharide and protein antigens of both Streptococcus pneumoniae and Neisseria meningitidis using whole formalin-fixed bacterial cells as probes. The modified ICA protocol described here allowed for a pathogen-specific hmAb cloning efficiency of >80%.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos/inmunología , Bacterias/inmunología , Inmunoensayo/métodos , Sondas Moleculares , Anticuerpos Antibacterianos/inmunología , Afinidad de Anticuerpos , Formación de Anticuerpos/inmunología , Antígenos Bacterianos/inmunología , Interacciones Huésped-Patógeno , Humanos , Inmunoglobulina G/inmunología , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Streptococcus pneumoniae/inmunología
17.
Methods Mol Biol ; 2183: 19-28, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32959238

RESUMEN

Chlamydia trachomatis is one of the most prevalent sexually transmitted infectious agents in the world and the leading cause of infectious blindness. The role of antibodies in the prevention and clearance of infection is still not fully understood, but the analysis of the immunoglobulin response to novel vaccine candidates is an important part of many of these studies. In this chapter, we describe a novel method to identify and isolate Chlamydia-specific memory B cells by fluorescence-activated cell sorting (FACS) using fluorescently labeled whole bacteria from cryopreserved human PBMC samples. This method allows for live single cells to be sorted for cell culture, in vitro assays, single-cell RNA sequencing, and cloning of paired heavy and light chains for recombinant monoclonal antibody production.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Especificidad de Anticuerpos/inmunología , Antígenos Bacterianos/inmunología , Chlamydia/inmunología , Sondas Moleculares , Anticuerpos Antibacterianos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Vacunas Bacterianas/inmunología , Criopreservación , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Memoria Inmunológica , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo
18.
Methods Mol Biol ; 2183: 537-547, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32959266

RESUMEN

Direct ELISA allows for the measurement of antibody levels to a particular antigen. Serum or plasma from the vaccinated subject are incubated on high-binding capacity microplates precoated with the antigen of interest and detected utilizing an enzyme-linked secondary antibody. Herein, using influenza hemagglutinin as model antigen, we describe the quantification of antigen-specific IgG titers in mouse serum to measure vaccine-induced humoral responses.


Asunto(s)
Especificidad de Anticuerpos/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Inmunoglobulina G/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Ratones , Vacunas/inmunología
19.
Protein Eng Des Sel ; 332020 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-33341882

RESUMEN

Single-domain antibody fragments known as VHH have emerged in the pharmaceutical industry as useful biotherapeutics. These molecules, which are naturally produced by camelids, share the characteristics of high affinity and specificity with traditional human immunoglobulins, while consisting of only a single heavy chain. Currently, the most common method for generating VHH is via animal immunization, which can be costly and time-consuming. Here we describe the development of a synthetic VHH library for in vitro selection of single domain binders. We combine structure-based design and next-generation sequencing analysis to build a library with characteristics that closely mimic the natural repertoire. To validate the performance of our synthetic library, we isolated VHH against three model antigens (soluble mouse PD-1 ectodomain, amyloid-ß peptide, and MrgX1 GPCR) of different sizes and characteristics. We were able to isolate diverse binders targeting different epitopes with high affinity (as high as 5 nM) against all three targets. We then show that anti-mPD-1 binders have functional activity in a receptor blocking assay.


Asunto(s)
Especificidad de Anticuerpos , Antígenos/química , Epítopos/química , Biblioteca de Péptidos , Ingeniería de Proteínas , Anticuerpos de Dominio Único , Animales , Antígenos/inmunología , Camélidos del Nuevo Mundo/genética , Camélidos del Nuevo Mundo/inmunología , Camelus/genética , Camelus/inmunología , Epítopos/inmunología , Ratones , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunología , Relación Estructura-Actividad
20.
PLoS Pathog ; 16(12): e1009103, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33315937

RESUMEN

The antibody molecule comprises a variable domain conferring antigen specificity and affinity distinct from the heavy chain constant (CH) domains dictating effector functions. We here interrogate this paradigm by evaluating the unique influence of the CH1α domain on epitope specificity and functions using two mucosal gp41-specific Fab-IgAs (FabA) derived from HIV-1 highly-exposed but persistently seronegative individuals (HESN). These HESN develop selectively affinity-matured HIV-1-specific mucosal IgA that target the gp41 viral envelope and might provide protection although by unclear mechanisms. Isotype-switching FabAs into Fab-IgGs (FabGs) results in a >10-fold loss in affinity for HIV-1 clade A, B, and C gp41, together with reduced neutralization of HIV-1 cross-clade. The FabA conformational epitopes map selectively on gp41 in 6-Helix bundle and pre-fusion conformations cross-clade, unlike FabGs. Finally, we designed in silico, a 12 amino-acid peptide recapitulating one FabA conformational epitope that inhibits the FabA binding to gp41 cross-clade and its neutralizing activity. Altogether, our results reveal that the CH1α domain shapes the antibody paratope through an allosteric effect, thereby strengthening the antibody specificity and functional activities. Further, they clarify the mechanisms by which these HESN IgAs might confer protection against HIV-1-sexual acquisition. The IgA-specific epitope we characterized by reverse vaccinology could help designing a mucosal HIV-1 vaccine.


Asunto(s)
Especificidad de Anticuerpos/inmunología , Sitios de Unión de Anticuerpos/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Inmunoglobulina A/inmunología , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Proteína gp41 de Envoltorio del VIH/inmunología , Seronegatividad para VIH/inmunología , Humanos , Inmunoglobulina A/química , Inmunoglobulina G/inmunología , Dominios Proteicos/inmunología
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