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1.
Arch Immunol Ther Exp (Warsz) ; 69(1): 5, 2021 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-33677719

RESUMEN

Coronaviruses share conservative spike protein (S) on their enveloped membrane surface, where S1 subunit recognizes and binds the cellular receptor, and the S2 subunit mediates membrane fusion. This similarity raises the question: does coronaviral infection by one create protection to others? Convalescent SARS-CoV-2 (COVID-19) sera were tested for cross reactivity with peptides from Middle East respiratory syndrome coronavirus (MERS-CoV) which shares 74% homology. Our results showed significant cross-reactivity with a peptide of the heptad repeat 2 (HR2) domain of the MERS-CoV spike protein. Sera samples of 47 validated seropositive convalescent COVID-19 patients and 40 sera samples of control patients, collected in pre-COVID time were used to establish cross-bind reactivity with the MERS-CoV peptide. Significantly stronger binding (p < 0.0001) was observed for IgG antibodies in convalescent COVID-19 patients compared to the control group. In ELISA, MERS-CoV peptide helps to discriminate post-COVID-19 populations and non-infected ones by the presence of antibodies in blood samples. This suggests that polyclonal antibodies established during SARS-CoV-2 infection can recognize and probably decrease severity of MERS-CoV and other coronaviral infections. The high homology of the spike protein domain also suggests that the opposite effect can be true: coronaviral infections produce cross-reactive antibodies effective against SARS-CoV-2. The collected data prove that despite the core HR2 region is hidden in the native viral conformation, its exposure during cell entry makes it highly immunogenic. Since inhibitory peptides to this region were previously described, this opens new possibilities in fighting coronaviral infections and developing vaccines effective even after possible viral mutations.


Asunto(s)
Anticuerpos Antivirales/inmunología , Convalecencia , Glicoproteína de la Espiga del Coronavirus/inmunología , Reacciones Cruzadas , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Virus del SRAS/inmunología
2.
Front Immunol ; 12: 627285, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33679770

RESUMEN

Introduction: Cross-reactivity to SARS-CoV-2 antigenic peptides has been detected on T-cells from pre-pandemic donors due to recognition of conserved protein fragments within members of the coronavirus's family. Further, preexisting antibodies recognizing SARS-CoV-2 with conserved epitopes in the spike region have been now seen in uninfected individuals. High-dose Intravenous Immunoglobulin (IVIg), derived from thousands of healthy donors, contains natural IgG antibodies against various antigens which can be detected both within the IVIg preparations and in the serum of IVIg-receiving patients. Whether IVIg preparations from pre-pandemic donors also contain antibodies against pre-pandemic coronaviruses or autoreactive antibodies that cross-react with SARS-CoV-2 antigenic epitopes, is unknown. Methods: 13 samples from 5 commercial IVIg preparations from pre-pandemic donors (HyQvia (Baxalta Innovations GmbH); Privigen (CSL Behring); Intratect (Biotest AG); IgVena (Kedrion S.p.A); and Flebogamma (Grifols S.A.) were blindly screened using a semi-quantitative FDA-approved and validated enzyme-linked immunosorbent assay (ELISA) (Euroimmun, Lubeck, Germany). Results: Nine of thirteen preparations (69.2%), all from two different manufactures, were antibody-positive based on the defined cut-off positivity (index of sample OD to calibrator OD > 1.1). From one manufacturer, 7/7 lots (100%) and from another 2/3 lots (67%), tested positive for cross-reacting antibodies. 7/9 of the positive preparations (77%) had titers as seen in asymptomatically infected individuals or recent COVID19-recovered patients, while 2/9 (23%) had higher titers, comparable to those seen in patients with active symptomatic COVID-19 infection (index > 2.2). Conclusion: Pre-pandemic IVIg donors have either natural autoantibodies or pre-pandemic cross-reactive antibodies against antigenic protein fragments conserved among the "common cold" - related coronaviruses. The findings are important in: (a) assessing true anti-SARS-CoV-2-IgG seroprevalence avoiding false positivity in IVIg-receiving patients; (b) exploring potential protective benefits in patients with immune-mediated conditions and immunodeficiencies receiving acute or chronic maintenance IVIg therapy, and (c) validating data from a recent controlled study that showed significantly lower in-hospital mortality in the IVIg- treated group.


Asunto(s)
Anticuerpos Antivirales/inmunología , Autoinmunidad , Inmunoglobulinas Intravenosas/inmunología , Estaciones del Año , /epidemiología , Reacciones Cruzadas , Epítopos/inmunología , Humanos , Glicoproteína de la Espiga del Coronavirus/inmunología
3.
Nat Commun ; 12(1): 1577, 2021 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-33707427

RESUMEN

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.


Asunto(s)
/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/genética , /inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , /química , Animales , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Afinidad de Anticuerpos , Línea Celular , Chlorocebus aethiops , Biblioteca de Genes , Voluntarios Sanos , Interacciones Microbiota-Huesped/inmunología , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/aislamiento & purificación , Modelos Moleculares , Mutación , Pruebas de Neutralización , Pandemias , Biblioteca de Péptidos , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Células Vero
4.
Drugs ; 81(4): 495-501, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33683637

RESUMEN

BNT162b2 (Comirnaty®; BioNTech and Pfizer) is a lipid nanoparticle-formulated, nucleoside-modified mRNA vaccine for the prevention of the novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. BNT162b2 encodes the SARS-CoV-2 spike protein, the expression of which elicits immune responses against the antigen in recipients. In early December 2020, BNT162b2 received a temporary emergency use authorization (EUA) in the UK and, subsequently, a series of approvals or authorizations for emergency use in Bahrain, Canada, Mexico, Saudi Arabia and the USA. Soon after, BNT162b2 received conditional marketing authorizations in Switzerland (19 December 2020) and the EU (21 December 2020) for active immunization to prevent COVID-19 caused by SARS-CoV-2 in individuals 16 years of age and older. BNT162b2 is administered intramuscularly in a two-dose regimen. This article summarizes the milestones in the development of BNT162b2 leading to these first approvals for the prevention of COVID-19.


Asunto(s)
/uso terapéutico , Aprobación de Drogas , Bahrein , Canadá , Ensayos Clínicos como Asunto , Desarrollo de Medicamentos , Unión Europea , Humanos , México , /inmunología , Arabia Saudita , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Suiza , Reino Unido , Estados Unidos , Vacunas Sintéticas , Organización Mundial de la Salud
5.
PLoS One ; 16(3): e0247640, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33661923

RESUMEN

BACKGROUND: Neutralizing-antibody (nAb) is the major focus of most ongoing COVID-19 vaccine trials. However, nAb response against SARS-CoV-2, when present, decays rapidly. Given the myriad roles of antibodies in immune responses, it is possible that antibodies could also mediate protection against SARS-CoV-2 via effector mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), which we sought to explore here. METHODS: Plasma of 3 uninfected controls and 20 subjects exposed to, or recovering from, SARS-CoV-2 infection were collected from U.S. and sub-Saharan Africa. Immunofluorescence assay was used to detect the presence of SARS-CoV-2 specific IgG antibodies in the plasma samples. SARS-CoV-2 specific neutralizing capability of these plasmas was assessed with SARS-CoV-2 spike pseudotyped virus. ADCC activity was assessed with a calcein release assay. RESULTS: SARS-CoV-2 specific IgG antibodies were detected in all COVID-19 subjects studied. All but three COVID-19 subjects contained nAb at high potency (>80% neutralization). Plasma from 19/20 of COVID-19 subjects also demonstrated strong ADCC activity against SARS-CoV-2 spike glycoprotein, including two individuals without nAb against SARS-CoV-2. CONCLUSION: Both neutralizing and non-neutralizing COVID-19 plasmas can mediate ADCC. Our findings argue that evaluation of potential vaccines against SARS-CoV-2 should include investigation of the magnitude and durability of ADCC, in addition to nAb.


Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , /inmunología , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Femenino , Células HEK293 , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto Joven
6.
PLoS One ; 16(3): e0247711, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33661990

RESUMEN

PCR methods are presently the standard for the diagnosis of Coronavirus disease 2019 (COVID-19), but additional methodologies are needed to complement PCR methods, which have some limitations. Here, we validated and investigated the usefulness of measuring serum antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using the iFlash3000 CLIA analyzer. We measured IgM and IgG titers against SARS-CoV-2 in sera collected from 26 PCR-positive COVID-19 patients, 53 COVID-19-suspected but PCR-negative patients, and 20 and 100 randomly selected non-COVID-19 patients who visited our hospital in 2020 and 2017, respectively. The repeatability and within-laboratory precision were obviously good in validations, following to the CLSI document EP15-A3. Linearity was also considered good between 0.6 AU/mL and 112.7 AU/mL for SARS-CoV-2 IgM and between 3.2 AU/mL and 55.3 AU/mL for SARS-CoV-2 IgG, while the linearity curves plateaued above the upper measurement range. We also confirmed that the seroconversion and no-antibody titers were over the cutoff values in all 100 serum samples collected in 2017. These results indicate that this measurement system successfully detects SARS-CoV-2 IgM/IgG. We observed four false-positive cases in the IgM assay and no false-positive cases in the IgG assay when 111 serum samples known to contain autoantibodies were evaluated. The concordance rates of the antibody test with the PCR test were 98.1% for SARS-CoV-2 IgM and 100% for IgG among PCR-negative cases and 30.8% for SARS-CoV-2 IgM and 73.1% for SARS-CoV-2 IgG among PCR-positive cases. In conclusion, the performance of this new automated method for detecting antibody against both N and S proteins of SARS-CoV-2 is sufficient for use in laboratory testing.


Asunto(s)
Anticuerpos Antivirales/sangre , /diagnóstico , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , /aislamiento & purificación , Anticuerpos Antivirales/inmunología , /epidemiología , /inmunología , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Japón/epidemiología , Mediciones Luminiscentes/métodos , Fosfoproteínas/inmunología , Fosfoproteínas/aislamiento & purificación , Sensibilidad y Especificidad , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/aislamiento & purificación
7.
Dokl Biochem Biophys ; 496(1): 44-47, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33689074

RESUMEN

The high efficiency of using thermoheliox (inhalation with a high-temperature mixture of helium and oxygen) in the treatment of patients affected by COVID-19 was shown. The dynamics of accumulation of IgG, IgM, and C-reactive protein (CRP) in patients with coronavirus infection in the "working" and control groups was studied experimentally. It was shown that thermoheliox intensifies the synthesis of IgG, IgM, and CRP antibodies, while eliminating the induction period on the kinetic curves of the synthesis of specific antibodies in the IgG form and transfers the synthesis of CRP to a fast phase. The results of experiments confirm the previously obtained data based on the analysis of the kinetic model of the development of coronaviral infection in the human body.


Asunto(s)
Anticuerpos Antivirales/inmunología , Proteína C-Reactiva/biosíntesis , /prevención & control , Inmunidad/inmunología , Vacunación/métodos , /inmunología , Humanos , Cinética , Glicoproteína de la Espiga del Coronavirus/inmunología
8.
Sci Adv ; 7(10)2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33674317

RESUMEN

Limited knowledge exists on immune markers associated with disease severity or recovery in patients with coronavirus disease 2019 (COVID-19). Here, we elucidated longitudinal evolution of SARS-CoV-2 antibody repertoire in patients with acute COVID-19. Differential kinetics was observed for immunoglobulin M (IgM)/IgG/IgA epitope diversity, antibody binding, and affinity maturation in "severe" versus "mild" COVID-19 patients. IgG profile demonstrated immunodominant antigenic sequences encompassing fusion peptide and receptor binding domain (RBD) in patients with mild COVID-19 who recovered early compared with "fatal" COVID-19 patients. In patients with severe COVID-19, high-titer IgA were observed, primarily against RBD, especially in patients who succumbed to SARS-CoV-2 infection. The patients with mild COVID-19 showed marked increase in antibody affinity maturation to prefusion SARS-CoV-2 spike that associated with faster recovery from COVID-19. This study revealed antibody markers associated with disease severity and resolution of clinical disease that could inform development and evaluation of effective immune-based countermeasures against COVID-19.


Asunto(s)
Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Biomarcadores/sangre , /patología , Índice de Severidad de la Enfermedad , Afinidad de Anticuerpos/inmunología , Formación de Anticuerpos/inmunología , /virología , Citocinas/sangre , Células HEK293 , Hospitalización , Humanos , Cambio de Clase de Inmunoglobulina , Cinética , Pruebas de Neutralización , Unión Proteica , Dominios Proteicos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Carga Viral
9.
MAbs ; 13(1): 1893426, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33666135

RESUMEN

Numerous neutralizing antibodies that target SARS-CoV-2 have been reported, and most directly block binding of the viral Spike receptor-binding domain (RBD) to angiotensin-converting enzyme II (ACE2). Here, we deliberately exploit non-neutralizing RBD antibodies, showing they can dramatically assist in neutralization when linked to neutralizing binders. We identified antigen-binding fragments (Fabs) by phage display that bind RBD, but do not block ACE2 or neutralize virus as IgGs. When these non-neutralizing Fabs were assembled into bispecific VH/Fab IgGs with a neutralizing VH domain, we observed a ~ 25-fold potency improvement in neutralizing SARS-CoV-2 compared to the mono-specific bi-valent VH-Fc alone or the cocktail of the VH-Fc and IgG. This effect was epitope-dependent, reflecting the unique geometry of the bispecific antibody toward Spike. Our results show that a bispecific antibody that combines both neutralizing and non-neutralizing epitopes on Spike-RBD is a promising and rapid engineering strategy to improve the potency of SARS-CoV-2 antibodies.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Epítopos/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/uso terapéutico , /genética , Epítopos/genética , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Glicoproteína de la Espiga del Coronavirus/genética
10.
Viruses ; 13(2)2021 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-33669922

RESUMEN

As a major surface glycoprotein of enveloped viruses, the virus spike protein is a primary target for vaccines and anti-viral treatments. Current vaccines aiming at controlling the COVID-19 pandemic are mostly directed against the SARS-CoV-2 spike protein. To promote virus entry and facilitate immune evasion, spikes must be dynamic. Interactions with host receptors and coreceptors trigger a cascade of conformational changes/structural rearrangements in spikes, which bring virus and host membranes in proximity for membrane fusion required for virus entry. Spike-mediated viral membrane fusion is a dynamic, multi-step process, and understanding the structure-function-dynamics paradigm of virus spikes is essential to elucidate viral membrane fusion, with the ultimate goal of interventions. However, our understanding of this process primarily relies on individual structural snapshots of endpoints. How these endpoints are connected in a time-resolved manner, and the order and frequency of conformational events underlying virus entry, remain largely elusive. Single-molecule Förster resonance energy transfer (smFRET) has provided a powerful platform to connect structure-function in motion, revealing dynamic aspects of spikes for several viruses: SARS-CoV-2, HIV-1, influenza, and Ebola. This review focuses on how smFRET imaging has advanced our understanding of virus spikes' dynamic nature, receptor-binding events, and mechanism of antibody neutralization, thereby informing therapeutic interventions.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Interacciones Microbiota-Huesped/inmunología , Fusión de Membrana , Receptores Virales/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Internalización del Virus , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Humanos , Unión Proteica
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.
Viruses ; 13(2)2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33671997

RESUMEN

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes serious and highly contagious enteric disease in swine worldwide. In this study, we constructed a recombinant baculovirus (S-Bac) expressing full-length spike protein of the virulent epidemic genotype 2b (G2b) PEDV strain for serological studies of infected pigs. We found that most spike-specific antibodies produced upon PEDV infection in pigs are conformation-specific and they could be detected on S-Bac-infected insect cells by immunofluorescent assay, but they were insensitive to Western blot analysis, the typical method for antiserum analysis. These results indicated that spike conformation is crucial for serum recognition. Since it is difficult to purify trimeric spike membrane protein for conventional enzyme-linked immunosorbent assay (ELISA), we used S-Bac to generate a novel cell-based ELISA for convenient PEDV detection. We analyzed 100 pig serum samples, and our cell-based ELISA exhibited a sensitivity of 100%, a specificity of 97%, and almost perfect agreement [Cohen's kappa coefficient value (κ) = 0.98] with immunocytochemical staining results. Our cell-based ELISA rapidly presented antigen for proper detection of conformation-specific antibodies, making PEDV detection more convenient, and it will be useful for detecting many viral diseases in the future.


Asunto(s)
Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Infecciones por Coronavirus/veterinaria , Ensayo de Inmunoadsorción Enzimática , Virus de la Diarrea Epidémica Porcina/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Baculoviridae/inmunología , Chlorocebus aethiops , Infecciones por Coronavirus/inmunología , Proteínas Recombinantes/inmunología , Spodoptera , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/virología , Células Vero
13.
J Med Microbiol ; 70(3)2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33734961

RESUMEN

In this work, we studied the profile of IgM and IgG antibody responses to SARS-CoV-2 in 32 patients with COVID-19 from day 1 to day 24. IgM remained measurable for a much shorter period than IgG, suggesting that IgG antibody may represent the primary immune response.


Asunto(s)
Anticuerpos Antivirales/sangre , /inmunología , Adulto , Anciano , /inmunología , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Cinética , Masculino , Persona de Mediana Edad , Fosfoproteínas/inmunología , ARN Viral/análisis , /aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/inmunología
14.
Nat Commun ; 12(1): 1806, 2021 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-33753733

RESUMEN

Better diagnostic tools are needed to combat the ongoing COVID-19 pandemic. Here, to meet this urgent demand, we report a homogeneous immunoassay to detect IgG antibodies against SARS-CoV-2. This serological assay, called SATiN, is based on a tri-part Nanoluciferase (tNLuc) approach, in which the spike protein of SARS-CoV-2 and protein G, fused respectively to two different tNLuc tags, are used as antibody probes. Target engagement of the probes allows reconstitution of a functional luciferase in the presence of the third tNLuc component. The assay is performed directly in the liquid phase of patient sera and enables rapid, quantitative and low-cost detection. We show that SATiN has a similar sensitivity to ELISA, and its readouts are consistent with various neutralizing antibody assays. This proof-of-principle study suggests potential applications in diagnostics, as well as disease and vaccination management.


Asunto(s)
Anticuerpos Antivirales/sangre , /diagnóstico , Inmunoensayo/métodos , Luciferasas/metabolismo , /aislamiento & purificación , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/inmunología , /virología , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Glicoproteína de la Espiga del Coronavirus/inmunología
15.
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
17.
PLoS One ; 16(3): e0248061, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33730022

RESUMEN

Developing an efficacious vaccine for SARS-CoV-2 infection is critical to stemming COVID-19 fatalities and providing the global community with immune protection. We have used a bioinformatic approach to aid in designing an epitope peptide-based vaccine against the spike protein of the virus. Five antigenic B cell epitopes with viable antigenicity and a total of 27 discontinuous B cell epitopes were mapped out structurally in the spike protein for antibody recognition. We identified eight CD8+ T cell 9-mers and 12 CD4+ T cell 14-15-mer as promising candidate epitopes putatively restricted by a large number of MHC I and II alleles, respectively. We used this information to construct an in silico chimeric peptide vaccine whose translational rate was highly expressed when cloned in pET28a (+) vector. With our In silico test, the vaccine construct was predicted to elicit high antigenicity and cell-mediated immunity when given as a homologous prime-boost, triggering of toll-like receptor 5 by the adjuvant linker. The vaccine was also characterized by an increase in IgM and IgG and an array of Th1 and Th2 cytokines. Upon in silico challenge with SARS-CoV-2, there was a decrease in antigen levels using our immune simulations. We, therefore, propose that potential vaccine designs consider this approach.


Asunto(s)
/inmunología , Epítopos de Linfocito T/inmunología , Inmunogenicidad Vacunal/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas de Subunidad/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Biología Computacional/métodos , Citocinas/inmunología , Epítopos de Linfocito B/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunidad Celular/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Simulación del Acoplamiento Molecular , Péptidos/inmunología
18.
Nat Commun ; 12(1): 1715, 2021 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-33731724

RESUMEN

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry and the focus for development of protective antibodies and vaccines. Structural studies show exposed sites on the spike trimer that might be targeted by antibodies with cross-species specificity. Here we isolated two human monoclonal antibodies from immunized humanized mice that display a remarkable cross-reactivity against distinct spike proteins of betacoronaviruses including SARS-CoV, SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both cross-reactive antibodies target the stem helix in the spike S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle. Both antibodies block MERS-CoV infection in cells and provide protection to mice from lethal MERS-CoV challenge in prophylactic and/or therapeutic models. Our work highlights an immunogenic and vulnerable site on the betacoronavirus spike protein enabling elicitation of antibodies with unusual binding breadth.


Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Betacoronavirus/inmunología , Epítopos/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/inmunología , Betacoronavirus/clasificación , Camelus , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Reacciones Cruzadas , Epítopos/química , Epítopos/genética , Humanos , Ratones , Conformación Proteica , Subunidades de Proteína , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética
19.
Sci Adv ; 7(12)2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33741598

RESUMEN

Vaccination against SARS-CoV-2 provides an effective tool to combat the COVID-19 pandemic. Here, we combined antigen optimization and nanoparticle display to develop vaccine candidates for SARS-CoV-2. We first displayed the receptor-binding domain (RBD) on three self-assembling protein nanoparticle (SApNP) platforms using the SpyTag/SpyCatcher system. We then identified heptad repeat 2 (HR2) in S2 as the cause of spike metastability, designed an HR2-deleted glycine-capped spike (S2GΔHR2), and displayed S2GΔHR2 on SApNPs. An antibody column specific for the RBD enabled tag-free vaccine purification. In mice, the 24-meric RBD-ferritin SApNP elicited a more potent neutralizing antibody (NAb) response than the RBD alone and the spike with two stabilizing proline mutations in S2 (S2P). S2GΔHR2 elicited twofold higher NAb titers than S2P, while S2GΔHR2 SApNPs derived from multilayered E2p and I3-01v9 60-mers elicited up to 10-fold higher NAb titers. The S2GΔHR2-presenting I3-01v9 SApNP also induced critically needed T cell immunity, thereby providing a promising vaccine candidate.


Asunto(s)
/inmunología , Nanopartículas , Glicoproteína de la Espiga del Coronavirus , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , /química , /farmacología , Células HEK293 , Humanos , Inmunogenicidad Vacunal , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Nanopartículas/uso terapéutico , Dominios Proteicos , /inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/farmacología
20.
Sci Rep ; 11(1): 6614, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758278

RESUMEN

There is a plethora of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) serological tests based either on nucleocapsid phosphoprotein (N), S1-subunit of spike glycoprotein (S1) or receptor binding domain (RBD). Although these single-antigen based tests demonstrate high clinical performance, there is growing evidence regarding their limitations in epidemiological serosurveys. To address this, we developed a Luminex-based multiplex immunoassay that detects total antibodies (IgG/IgM/IgA) against the N, S1 and RBD antigens and used it to compare antibody responses in 1225 blood donors across Greece. Seroprevalence based on single-antigen readouts was strongly influenced by both the antigen type and cut-off value and ranged widely [0.8% (95% CI 0.4-1.5%)-7.5% (95% CI 6.0-8.9%)]. A multi-antigen approach requiring partial agreement between RBD and N or S1 readouts (RBD&N|S1 rule) was less affected by cut-off selection, resulting in robust seroprevalence estimation [0.6% (95% CI 0.3-1.1%)-1.2% (95% CI 0.7-2.0%)] and accurate identification of seroconverted individuals.


Asunto(s)
Antígenos/inmunología , Pruebas Serológicas/métodos , Adolescente , Adulto , Anciano , Anticuerpos Antivirales/sangre , /inmunología , Femenino , Humanos , Inmunoensayo , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , Fosfoproteínas/inmunología , Sensibilidad y Especificidad , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto Joven
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