RESUMEN
Spike (S) glycoprotein is the largest structural protein of SARS-CoV-2 virus and the main one involved in anchoring of the host receptor ACE2 through the receptor binding domain (RBD). S protein secondary structure is of great interest for shedding light on various aspects, from functionality to pathogenesis, finally to spectral fingerprint for the design of optical biosensors. In this paper, the secondary structure of SARS-CoV-2 S protein and its constituting components, namely RBD, S1 and S2 regions, are investigated at serological pH by measuring their amide I infrared absorption bands through Attenuated Total Reflection Infrared (ATR-IR) spectroscopy. Experimental data in combination with MultiFOLD predictions, Define Secondary Structure of Proteins (DSSP) web server and Gravy value calculations, provide a comprehensive understanding of RBD, S1, S2, and S proteins in terms of their secondary structure content, conformational order, and interaction with the solvent.
RESUMEN
While an important part of the world's population is vaccinated against SARS-CoV-2, new variants continue to emerge. We observe that even after a fifth dose of the mRNA bivalent vaccine, most vaccinated individuals have antibodies that poorly neutralize several Omicron subvariants, including BQ.1.1, XBB, XBB.1.5, FD.1.1, and CH.1.1. However, Fc-effector functions remain strong and stable over time against new variants, which may partially explain why vaccines continue to be effective. We also observe that donors who have been recently infected have stronger antibody functional activities, including neutralization and Fc-effector functions, supporting the observations that hybrid immunity leads to better humoral responses.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/prevención & control , Anticuerpos , Vacunas Combinadas , ARN Mensajero/genéticaRESUMEN
All coronaviruses are characterized by spike glycoproteins whose S1 subunits contain the receptor binding domain (RBD). The RBD anchors the virus to the host cellular membrane to regulate the virus transmissibility and infectious process. Although the protein/receptor interaction mainly depends on the spike's conformation, particularly on its S1 unit, their secondary structures are poorly known. In this paper, the S1 conformation was investigated for MERS-CoV, SARS-CoV, and SARS-CoV-2 at serological pH by measuring their Amide I infrared absorption bands. The SARS-CoV-2 S1 secondary structure revealed a strong difference compared to those of MERS-CoV and SARS-CoV, with a significant presence of extended ß-sheets. Furthermore, the conformation of the SARS-CoV-2 S1 showed a significant change by moving from serological pH to mild acidic and alkaline pH conditions. Both results suggest the capability of infrared spectroscopy to follow the secondary structure adaptation of the SARS-CoV-2 S1 to different environments.
Asunto(s)
COVID-19 , Coronavirus del Síndrome Respiratorio de Oriente Medio , Humanos , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Análisis EspectralRESUMEN
Since the beginning of the SARS-CoV-2 pandemic, several variants of concern (VOCs), such as the Alpha, Beta, Gamma, Delta and Omicron variants, have arisen and spread worldwide. Today, the predominant circulating subvariants are sublineages of the Omicron variant, which have more than 30 mutations in their Spike glycoprotein compared to the ancestral strain. The Omicron subvariants were significantly less recognized and neutralized by antibodies from vaccinated individuals. This resulted in a surge in the number of infections, and booster shots were recommended to improve responses against these variants. While most studies mainly measured the neutralizing activity against variants, we and others previously reported that Fc-effector functions, including antibody-dependent cellular cytotoxicity (ADCC), play an important role in humoral responses against SARS-CoV-2. In this study, we analyzed Spike recognition and ADCC activity against several Omicron subvariants by generating cell lines expressing different Omicron subvariant Spikes. We tested these responses in a cohort of donors, who were recently infected or not, before and after a fourth dose of mRNA vaccine. We showed that ADCC activity is less affected than neutralization by the antigenic shift of the tested Omicron subvariant Spikes. Moreover, we found that individuals with a history of recent infection have higher antibody binding and ADCC activity against all Omicron subvariants than people who were not recently infected. With an increase in the number of reinfections, this study helps better understand Fc-effector responses in the context of hybrid immunity.
Asunto(s)
COVID-19 , Humanos , COVID-19/prevención & control , SARS-CoV-2/genética , Citotoxicidad Celular Dependiente de Anticuerpos , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacunas de ARNmRESUMEN
Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants have recently emerged, becoming the dominant circulating strains in many countries. These variants contain a large number of mutations in their spike glycoprotein, raising concerns about vaccine efficacy. In this study, we evaluate the ability of plasma from a cohort of individuals that received three doses of mRNA vaccine to recognize and neutralize these Omicron subvariant spikes. We observed that BA.4/5 and BQ.1.1 spikes are markedly less recognized and neutralized compared with the D614G and other Omicron subvariant spikes tested. Also, individuals who have been infected before or after vaccination present better humoral responses than SARS-CoV-2-naive vaccinated individuals, thus indicating that hybrid immunity generates better humoral responses against these subvariants.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/prevención & control , Vacunas Sintéticas , Mutación , Anticuerpos Antivirales , Anticuerpos Neutralizantes , Vacunas de ARNmRESUMEN
Due to the recrudescence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections worldwide, mainly caused by the Omicron variant of concern (VOC) and its sub-lineages, several jurisdictions are administering an mRNA vaccine boost. Here, we analyze humoral responses induced after the second and third doses of an mRNA vaccine in naive and previously infected donors who received their second dose with an extended 16-week interval. We observe that the extended interval elicits robust humoral responses against VOCs, but this response is significantly diminished 4 months after the second dose. Administering a boost to these individuals brings back the humoral responses to the same levels obtained after the extended second dose. Interestingly, we observe that administering a boost to individuals that initially received a short 3- to 4-week regimen elicits humoral responses similar to those observed in the long interval regimen. Nevertheless, humoral responses elicited by the boost in naive individuals do not reach those present in previously infected vaccinated individuals.
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COVID-19 , Vacunas Virales , Humanos , SARS-CoV-2 , Vacuna BNT162 , COVID-19/prevención & control , Anticuerpos Antivirales , Vacunas contra la COVID-19 , Vacunación , Vacunas de ARNmRESUMEN
We have used NMR experiments to explore the binding of selected glycans and glycomimetics to the SARS CoV-2 spike glycoprotein (S-protein) and to its receptor binding domain (RBD). STD NMR experiments confirm the binding of sialoglycans to the S-protein of the prototypic Wuhan strain virus and yield dissociation constants in the millimolar range. The absence of STD effects for sialoglycans in the presence of the Omicron/BA.1 S-protein reflects a loss of binding as a result of S-protein evolution. Likewise, no STD effects are observed for the deletion mutant Δ143-145 of the Wuhan S-protein, thus supporting localization of the binding site in the N-terminal domain (NTD). The glycomimetics Oseltamivir and Zanamivir bind weakly to the S-protein of both virus strains. Binding of blood group antigens to the Wuhan S-protein cannot be confirmed by STD NMR. Using 1 H,15 N TROSY HSQC-based chemical shift perturbation (CSP) experiments, we excluded binding of any of the ligands studied to the RBD of the Wuhan S-protein. Our results put reported data on glycan binding into perspective and shed new light on the potential role of glycan-binding to the S-protein.
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COVID-19 , Síndrome Respiratorio Agudo Grave , Humanos , Glicoproteína de la Espiga del Coronavirus , Sitios de Unión , Polisacáridos , Espectroscopía de Resonancia Magnética , Unión ProteicaRESUMEN
Spacing of BNT162b2 mRNA doses beyond 3 weeks raises concerns about vaccine efficacy. We longitudinally analyze B cell, T cell, and humoral responses to two BNT162b2 mRNA doses administered 16 weeks apart in 53 SARS-CoV-2 naive and previously infected donors. This regimen elicits robust RBD-specific B cell responses whose kinetics differs between cohorts, the second dose leading to increased magnitude in naive participants only. While boosting does not increase magnitude of CD4+ T cell responses further compared with the first dose, unsupervised clustering of single-cell features reveals phenotypic and functional shifts over time and between cohorts. Integrated analysis shows longitudinal immune component-specific associations, with early T helper responses post first dose correlating with B cell responses after the second dose, and memory T helper generated between doses correlating with CD8 T cell responses after boosting. Therefore, boosting elicits a robust cellular recall response after the 16-week interval, indicating functional immune memory.
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COVID-19 , Vacunas Virales , Anticuerpos Antivirales , Vacuna BNT162 , Humanos , Inmunidad Humoral , ARN Mensajero , SARS-CoV-2RESUMEN
Continuous emergence of SARS-CoV-2 variants of concern (VOCs) is fueling the COVID-19 pandemic. Omicron (B.1.1.529) rapidly spread worldwide. The large number of mutations in its Spike raise concerns about a major antigenic drift that could significantly decrease vaccine efficacy and infection-induced immunity. A long interval between BNT162b2 mRNA doses elicits antibodies that efficiently recognize Spikes from different VOCs. Here, we evaluate the recognition of Omicron Spike by plasma from a cohort of SARS-CoV-2 naive and previously infected individuals who received their BNT162b2 mRNA vaccine 16 weeks apart. Omicron Spike is recognized less efficiently than D614G, Alpha, Beta, Gamma, and Delta Spikes. We compare with plasma activity from participants receiving a short (4 weeks) interval regimen. Plasma from individuals of the long-interval cohort recognize and neutralize better the Omicron Spike compared with those who received a short interval. Whether this difference confers any clinical benefit against Omicron remains unknown.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Vacuna BNT162/administración & dosificación , Esquemas de Inmunización , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto , Anciano , Anticuerpos Neutralizantes/análisis , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Vacuna BNT162/inmunología , Estudios de Cohortes , Femenino , Células HEK293 , Humanos , Inmunización Secundaria/métodos , Masculino , Persona de Mediana Edad , Quebec , SARS-CoV-2/patogenicidad , Factores de Tiempo , Vacunación/métodos , Potencia de la Vacuna , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Adulto Joven , Vacunas de ARNm/administración & dosificación , Vacunas de ARNm/inmunologíaRESUMEN
The rapid emergence of SARS-CoV-2 variants is fueling the recent waves of the COVID-19 pandemic. Here, we assessed ACE2 binding and antigenicity of Mu (B.1.621) and A.2.5 Spikes. Both these variants carry some mutations shared by other emerging variants. Some of the pivotal mutations such as N501Y and E484K in the receptor-binding domain (RBD) detected in B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) are now present within the Mu variant. Similarly, the L452R mutation of B.1.617.2 (Delta) variant is present in A.2.5. In this study, we observed that these Spike variants bound better to the ACE2 receptor in a temperature-dependent manner. Pseudoviral particles bearing the Spike of Mu were similarly neutralized by plasma from vaccinated individuals than those carrying the Beta (B.1.351) and Delta (B.1.617.2) Spikes. Altogether, our results indicate the importance of measuring critical parameters such as ACE2 interaction, plasma recognition and neutralization ability of each emerging variant.
Asunto(s)
SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Neutralizantes/inmunología , COVID-19/inmunología , COVID-19/virología , Vacunas contra la COVID-19/inmunología , Células HEK293 , Humanos , Mutación , Pruebas de Neutralización , Unión Proteica , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , TemperaturaRESUMEN
The standard regimen of the BNT162b2 mRNA vaccine for SARS-CoV-2 includes two doses administered three weeks apart. However, some public health authorities spaced these doses, raising questions about efficacy. We analyzed longitudinal humoral responses against the D614G strain and variants of concern for SARS-CoV-2 in a cohort of SARS-CoV-2-naive and previously infected individuals who received the BNT162b2 mRNA vaccine with sixteen weeks between doses. While administering a second dose to previously infected individuals did not significantly improve humoral responses, these responses significantly increased in naive individuals after a 16-week spaced second dose, achieving similar levels as in previously infected individuals. Comparing these responses to those elicited in individuals receiving a short (4-week) dose interval showed that a 16-week interval induced more robust responses among naive vaccinees. These findings suggest that a longer interval between vaccine doses does not compromise efficacy and may allow greater flexibility in vaccine administration.
Asunto(s)
Vacuna BNT162/inmunología , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , Inmunidad Humoral/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Sintéticas/inmunología , Vacunas de ARNm/inmunología , Adulto , Anciano , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/virología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Vacunación/métodos , Adulto JovenRESUMEN
The seasonal nature of outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. Accordingly, temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. The receptor-binding domain (RBD) of the Spike glycoprotein is known to bind to its host receptor angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Using biochemical, biophysical, and functional assays to dissect the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike glycoprotein with the ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide (including the B.1.1.7 (α) lineage), bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/química , COVID-19/patología , COVID-19/virología , Calorimetría , Humanos , Interferometría , Polimorfismo de Nucleótido Simple , Unión Proteica , Estructura Cuaternaria de Proteína , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/química , Temperatura , TermodinámicaRESUMEN
Towards the end of 2020, multiple variants of concern (VOCs) and variants of interest (VOIs) have arisen from the original SARS-CoV-2 Wuhan-Hu-1 strain. Mutations in the Spike protein are highly scrutinized for their impact on transmissibility, pathogenesis and vaccine efficacy. Here, we contribute to the growing body of literature on emerging variants by evaluating the impact of single mutations on the overall antigenicity of selected variants and their binding to the ACE2 receptor. We observe a differential contribution of single mutants to the global variants phenotype related to ACE2 interaction and antigenicity. Using biolayer interferometry, we observe that enhanced ACE2 interaction is mostly modulated by a decrease in off-rate. Finally, we made the interesting observation that the Spikes from tested emerging variants bind better to ACE2 at 37°C compared to the D614G variant. Whether improved ACE2 binding at higher temperature facilitates emerging variants transmission remain to be demonstrated.
Asunto(s)
Enzima Convertidora de Angiotensina 2/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Sitios de Unión , Células HEK293 , Humanos , Mutación , Unión Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
While the standard regimen of the BNT162b2 mRNA vaccine for SARS-CoV-2 includes two doses administered 3 weeks apart, some public health authorities are spacing these doses, raising concerns about efficacy. However, data indicate that a single dose can be up to 90% effective starting 14 days post-administration. To assess the mechanisms contributing to protection, we analyzed humoral and T cell responses three weeks after a single BNT162b2 dose. We observed weak neutralizing activity elicited in SARS-CoV-2 naive individuals but strong anti-receptor binding domain and spike antibodies with Fc-mediated effector functions and cellular CD4+ T cell responses. In previously infected individuals, a single dose boosted all humoral and T cell responses, with strong correlations between T helper and antibody immunity. Our results highlight the potential role of Fc-mediated effector functions and T cell responses in vaccine efficacy. They also provide support for spacing doses to vaccinate more individuals in conditions of vaccine scarcity.
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Anticuerpos Antivirales/inmunología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , SARS-CoV-2/inmunología , Linfocitos T/inmunología , Adulto , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/química , Vacuna BNT162 , Betacoronavirus , COVID-19/prevención & control , Proteínas Portadoras , Femenino , Humanos , Inmunidad , Fragmentos Fc de Inmunoglobulinas , Masculino , Persona de Mediana Edad , Vacunación , Vacunas Sintéticas/inmunología , Adulto Joven , Vacunas de ARNmRESUMEN
With the recent approval of highly effective coronavirus disease 2019 (COVID-19) vaccines, functional and lasting immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently under investigation as antibody levels in plasma were shown to decline during convalescence. Since the absence of antibodies does not equate to absence of immune memory, we evaluate the presence of SARS-CoV-2-specific memory B cells in convalescent individuals. Here, we report a longitudinal assessment of humoral immune responses on 32 donors up to 8 months post-symptom onset. Our observations indicate that anti-Spike and anti-receptor binding domain (RBD) immunoglobulin M (IgM) in plasma decay rapidly, whereas the reduction of IgG is less prominent. Neutralizing activity also declines rapidly when compared to Fc-effector functions. Concomitantly, the frequencies of RBD-specific IgM+ B cells wane significantly when compared to RBD-specific IgG+ B cells, which remain stable. Our results add to the current understanding of immune memory following SARS-CoV-2 infection, which is critical for secondary infection prevention and vaccine efficacy.
RESUMEN
SARS-CoV-2 virus was first detected in late 2019 and circulated globally, causing COVID-19, which is characterised by sub-clinical to severe disease in humans. Here, we investigate the serological antibody responses to SARS-CoV-2 infection during acute and convalescent infection using a cohort of (i) COVID-19 patients admitted to hospital, (ii) healthy individuals who had experienced 'COVID-19 like-illness', and (iii) a cohort of healthy individuals prior to the emergence of SARS-CoV-2. We compare SARS-CoV-2 specific antibody detection rates from four different serological methods, virus neutralisation test (VNT), ID Screen® SARS-CoV-2-N IgG ELISA, Whole Antigen ELISA, and lentivirus-based SARS-CoV-2 pseudotype virus neutralisation tests (pVNT). All methods were able to detect prior infection with COVID-19, albeit with different relative sensitivities. The VNT and SARS-CoV-2-N ELISA methods showed a strong correlation yet provided increased detection rates when used in combination. A pVNT correlated strongly with SARS-CoV-2 VNT and was able to effectively discriminate SARS-CoV-2 antibody positive and negative serum with the same efficiency as the VNT. Moreover, the pVNT was performed with the same level of discrimination across multiple separate institutions. Therefore, the pVNT is a sensitive, specific, and reproducible lower biosafety level alternative to VNT for detecting SARS-CoV-2 antibodies for diagnostic and research applications. Our data illustrate the potential utility of applying VNT or pVNT and ELISA antibody tests in parallel to enhance the sensitivity of exposure to infection.
Asunto(s)
Anticuerpos Antivirales/sangre , Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/inmunología , Anciano , Anticuerpos Neutralizantes/sangre , COVID-19/sangre , COVID-19/inmunología , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lentivirus/genética , Masculino , Persona de Mediana Edad , Pruebas de Neutralización , Reproducibilidad de los Resultados , SARS-CoV-2/aislamiento & purificación , Sensibilidad y Especificidad , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
COVID-19, a global-pandemic binds human-lung-ACE2. ACE2 causes vasodilatation. ACE2 works in balance with ACE1. The vaso-status maintains blood-pressure/vascular-health which is demolished in Covid-19 manifesting aldosterone/salt-deregulations/inflammations/endothelial-dysfunctions/hyper-hypo- tension, sepsis/hypovolemic-shock and vessel-thrombosis/coagulations. Here, nigellidine, an indazole-alkaloid was analyzed by molecular-docking for binding to different Angiotensin-binding-proteins (enzymes, ACE1(6en5)/ACE2(4aph)/receptors, AT1(6os1)/AT2(5xjm)) and COVID-19 spike-glycoprotein(6vsb). Nigellidine strongly binds to the spike-protein at the hinge-region/active-site-opening which may hamper proper-binding of nCoV2-ACE2 surface. Nigellidine effectively binds in the Angiotensin- II binding-site/entry-pocket (-7.54 kcal/mol, -211.76, Atomic-Contact-Energy; ACE-value) of ACE2 (Ki 8.68 and 8.3 µmol) in comparison to known-binder EGCG (-4.53) and Theaflavin-di-gallate (-2.85). Nigellidine showed strong-binding (Ki, 50.93 µmol/binding-energy -5.48 kcal/mol) to mono/multi-meric ACE1. Moreover, it binds Angiotensin-receptors, AT1/AT2 (Ki, 42.79/14.22 µmol, binding-energy, -5.96/-6.61 kcal/mol) at active-sites, respectively. This article reports the novel binding of nigellidine and subsequent blockage of angiotensin-binding proteins. The ACEs-blocking could restore Angiotensin-level, restrict vaso-turbulence in Covid patients and receptor-blocking might stop inflammatory/vascular impairment. Nigellidine may slowdown the vaso-fluctuations due to Angiotensin-deregulations in Covid patients. Angiotensin II-ACE2 binding (ACE-value -294.81) is more favorable than nigellidine-ACE2. Conversely, nigellidine-ACE1 binding-energy/Ki is lower than nigellidine-ACE2 values indicating a balanced-state between constriction-dilatation. Moreover, nigellidine binds to the viral-spike, closer-proximity to its ACE2 binding-domain. Taken together, Covid patients/elderly-patients, comorbid-patients (with hypertensive/diabetic/cardiac/renal-impairment, counting >80% of non-survivors) could be greatly benefited.
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Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/metabolismo , Nigella sativa , Peptidil-Dipeptidasa A/metabolismo , Extractos Vegetales/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , COVID-19/patología , COVID-19/prevención & control , Comorbilidad , Simulación por Computador/tendencias , Evaluación Preclínica de Medicamentos/métodos , Humanos , Simulación del Acoplamiento Molecular/métodos , Peptidil-Dipeptidasa A/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/uso terapéutico , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptor de Angiotensina Tipo 1/química , Receptor de Angiotensina Tipo 2/química , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/metabolismoRESUMEN
BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing more than two million deaths. The SARS-CoV-2 Spike glycoproteins mediate viral entry and represent the main target for antibody responses. Humoral responses were shown to be important for preventing and controlling infection by coronaviruses. A promising approach to reduce the severity of COVID-19 is the transfusion of convalescent plasma. However, longitudinal studies revealed that the level of antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike declines rapidly after the resolution of the infection. STUDY DESIGN AND METHODS: To extend this observation beyond the RBD domain, we performed a longitudinal analysis of the persistence of antibodies targeting the full-length SARS-CoV-2 Spike in the plasma from 15 convalescent donors. We generated a 293T cell line constitutively expressing the SARS-CoV-2 Spike and used it to develop a high-throughput flow cytometry-based assay to detect SARS-CoV-2 Spike-specific antibodies in the plasma of convalescent donors. RESULTS AND CONCLUSION: We found that the level of antibodies targeting the full-length SARS-CoV-2 Spike declines gradually after the resolution of the infection. This decline was not related to the number of donations but strongly correlated with the decline of RBD-specific antibodies and the number of days post-symptom onset. These findings help to better understand the decline of humoral responses against the SARS-CoV-2 Spike and provide important information on when to collect plasma after recovery from active infection for convalescent plasma transfusion.
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Anticuerpos Antivirales/sangre , COVID-19/sangre , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/sangre , COVID-19/terapia , Femenino , Células HEK293 , Humanos , Inmunización Pasiva , Estudios Longitudinales , Masculino , Sueroterapia para COVID-19RESUMEN
Different serological assays were rapidly generated to study humoral responses against the SARS-CoV-2 Spike glycoprotein. Due to the intrinsic difficulty of working with SARS-CoV-2 authentic virus, most serological assays use recombinant forms of the Spike glycoprotein or its receptor binding domain (RBD). Cell-based assays expressing different forms of the Spike, as well as pseudoviral assays, are also widely used. To evaluate whether these assays recapitulate findings generated when the Spike is expressed in its physiological context (at the surface of the infected primary cells), we developed an intracellular staining against the SARS-CoV-2 nucleocapsid (N) to distinguish infected from uninfected cells. Human airway epithelial cells (pAECs) were infected with authentic SARS-CoV-2 D614G or Alpha variants. We observed robust cell-surface expression of the SARS-CoV-2 Spike at the surface of the infected pAECs using the conformational-independent anti-S2 CV3-25 antibody. The infected cells were also readily recognized by plasma from convalescent and vaccinated individuals and correlated with several serological assays. This suggests that the antigenicity of the Spike present at the surface of the infected primary cells is maintained in serological assays involving expression of the native full-length Spike.
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Membrana Celular/metabolismo , Células Epiteliales/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Anticuerpos Antivirales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Bronquiolos/citología , Células Cultivadas , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Células Epiteliales/virología , Células HEK293 , Humanos , Pruebas de Neutralización , Fosfoproteínas/metabolismo , SARS-CoV-2/inmunología , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is responsible for the current global coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The viral entry of SARS-CoV-2 depends on an interaction between the receptor-binding domain of its trimeric spike glycoprotein and the human angiotensin-converting enzyme 2 (ACE2) receptor. A better understanding of the spike/ACE2 interaction is still required to design anti-SARS-CoV-2 therapeutics. Here, we investigated the degree of cooperativity of ACE2 within both the SARS-CoV-2 and the closely related SARS-CoV-1 membrane-bound S glycoproteins. We show that there exist differential inter-protomer conformational transitions between both spike trimers. Interestingly, the SARS-CoV-2 spike exhibits a positive cooperativity for monomeric soluble ACE2 binding when compared to the SARS-CoV-1 spike, which might have more structural restraints. Our findings can be of importance in the development of therapeutics that block the spike/ACE2 interaction.