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1.
Adverse Drug Reactions Journal ; 24(4):197-202, 2022.
Artículo en Chino | EMBASE | ID: covidwho-2302140

RESUMEN

Coronavirus disease 2019 (COVID-19) can increase the risk of thrombosis and arterial embolism events in patients. The more serious the condition, the higher the risk. Therefore, many academic groups at home and abroad have successively issued guidelines on the prevention and treatment of thrombosis in patients with COVID-19. Among them, American Society of Hematology 2021 guidelines on the use of anticoagulation for thromboprophylaxis in patients with COVID-19 (ASH guidelines) and its updates are newer and have more detailed recommendations on the application of anticoagulant drugs to prevent venous thromboembolism in acutely and critically ill patients with COVID-19. This review aims to provide reference for clinic through general viewing the ASH guidelines as well as other relevant guidelines at home and abroad.Copyright © 2022 Adverse Drug Reactions Journal.

2.
Curr Protoc ; 2(10): e521, 2022 Oct.
Artículo en Inglés | MEDLINE | ID: covidwho-2047527

RESUMEN

Antibody detection assays are essential for evaluating immunity of individuals against a given virus, and this has been particularly relevant during the COVID-19 pandemic. Current serology assays either require a laboratory setting and take >1 hr (i.e., enzyme-linked immunosorbent assay [ELISA]) or are rapid but only qualitative in nature and cannot accurately track antibody levels over time (i.e., lateral flow assay [LFA]). Therefore, there is a need for development of a rapid and simple but also quantitative assay that can evaluate antibody levels in patients accurately over time. We have developed an assay that uses a split nanoluciferase fused to the spike or nucleocapsid proteins of the SARS-CoV-2 virus to enable luminescent-based detection of spike- or nucleocapsid-binding antibodies in serum, plasma, and whole blood samples. The resulting approach is simple, rapid, and quantitative and is highly amenable to low-/medium-throughput scale using plate-based assays, high-throughput scale using robotics, and point-of-care applications. In this article, we describe how to perform the assay in a laboratory setting using a plate reader or liquid-handling robotics and in a point-of-care setting using a handheld, battery-powered luminometer. Together, these assays allow antibody detection to be easily performed in multiple settings by simplifying and reducing assay time in a laboratory or clinical environment and by allowing for antibody detection in point-of-care, nonlaboratory settings. © 2022 Wiley Periodicals LLC. Basic Protocol: SARS-CoV-2 antibody detection using the split-luciferase assay on a medium-throughput scale with a laboratory luminometer Alternate Protocol 1: High-throughput-based protocol for SARS-CoV-2 antibody detection using a robotic platform Alternate Protocol 2: Point-of-care-based protocol for SARS-CoV-2 antibody detection using a handheld luminometer Support Protocol: Determining positive/negative cutoffs for test samples and standardizing the assay between days.


Asunto(s)
Técnicas Biosensibles , COVID-19 , Anticuerpos Antivirales/análisis , COVID-19/diagnóstico , Técnicas de Laboratorio Clínico/métodos , Humanos , Luciferasas , Proteínas de la Nucleocápside , Pandemias , SARS-CoV-2 , Sensibilidad y Especificidad
3.
J Hosp Infect ; 130: 95-103, 2022 Dec.
Artículo en Inglés | MEDLINE | ID: covidwho-2031452

RESUMEN

BACKGROUND: In the wake of the coronavirus disease 2019 (COVID-19) pandemic, demand for deep cleaning and environmental services workers grew exponentially. Although there is extant literature examining the impact of the COVID-19 pandemic on healthcare workers, less emphasis has been placed on environmental services workers, who play an equally important front-line role. AIM: To examine the impact of the COVID-19 pandemic on environmental services workers employed in healthcare settings. METHODS: Scoping review methodology. A search strategy was developed, in consultation with a medical information specialist, employing various combinations of the keywords [(environmental services worker OR health attendant OR housekeeping) AND (COVID OR coronavirus OR pandemic OR epidemic)]. Four bibliographical databases were searched from inception to 5th July 2022: OVID Medline, Embase, Cumulative Index to Nursing and Allied Health Literature (CINAHL) and Cochrane Database. RESULTS: In total, 24 studies were included in this review. The studies were generally cross-sectional in design. Seroprevalence studies highlighted significantly higher rates of COVID-19 among environmental services workers (housekeeping, cleaning and janitorial staff) compared with other clinical and non-clinical staff in the same institutions. In addition, based on qualitative interviews, environmental services workers experienced greater psychological stress working during the pandemic. CONCLUSIONS: Environmental services workers were particularly vulnerable to increased work stress and COVID-19 during the pandemic. Health systems need to do more to support these workers. Further research could investigate specific policy and procedural changes to benefit this under-recognized group in the greater healthcare workforce.


Asunto(s)
COVID-19 , Humanos , COVID-19/epidemiología , Pandemias , Estudios Seroepidemiológicos , Estudios Transversales , Personal de Salud/psicología , Atención a la Salud
4.
Adverse Drug Reactions Journal ; 24(4):197-202, 2022.
Artículo en Chino | Scopus | ID: covidwho-1875841

RESUMEN

Coronavirus disease 2019 (COVID‐19) can increase the risk of thrombosis and arterial embolism events in patients. The more serious the condition, the higher the risk. Therefore, many academic groups at home and abroad have successively issued guidelines on the prevention and treatment of thrombosis in patients with COVID‐19. Among them, American Society of Hematology 2021 guidelines on the use of anticoagulation for thromboprophylaxis in patients with COVID‐19 (ASH guidelines) and its updates are newer and have more detailed recommendations on the application of anticoagulant drugs to prevent venous thromboembolism in acutely and critically ill patients with COVID‐19. This review aims to provide reference for clinic through general viewing the ASH guidelines as well as other relevant guidelines at home and abroad. © 2022 Adverse Drug Reactions Journal.

5.
Journal of Gastroenterology and Hepatology ; 36(SUPPL 2):48, 2021.
Artículo en Inglés | EMBASE | ID: covidwho-1409944

RESUMEN

Background and Aim: The coronavirus disease-2019 (COVID-19) pandemic has placed significant stress on gastroenterologists worldwide. However, its toll on the mental health of gastroenterologists within Southeast Asia was unknown. A mixed methods, multi-national study was conducted to elucidate the prevalence of burnout and its stressors within the region. Methods: A survey was disseminated electronically to 1761 gastroenterologists via the gastroenterology and endoscopy societies of Brunei, the Philippines, Indonesia, Malaysia, Singapore, and Thailand from September 1 to December 7, 2020. This included the 22-item Maslach Burnout Inventory to detect burnout. Ethical approval was granted. Quantitative and qualitative data were collected. Logistic regression identified associations between variables and burnout. Qualitative data were analyzed by content analysis method. Results: The response rate was 38.8%;66.6% reported significant stress. The regional prevalence of burnout was 17.1% although inter-country variation existed (Fig. 1A). Depression, being a trainee, public sector work, and the lack of awareness or access to mental health support services increased burnout risk significantly (Fig. 1B). The 50.1% of gastroenterologists were unaware of or did not have access to support services. The onset of depression intra-pandemic was 2.1%;the pre-pandemic prevalence was 2.2%. Stressors commonly involved service requirements (53.2%), difficult relationships with patients and relatives (23.0%), and difficult relationships with colleagues (20.5%). Specific to the pandemic, the three most common stressors were fear of getting infected (39.7%), reduced income (28.0%), and stringent infection control measures adding to workload (18.5%). Conclusion: Burnout is common in gastroenterologists in Southeast Asia;however;better safeguards for mental health are urgently needed.

7.
Journal of Intelligent & Fuzzy Systems ; 41(1):1151-1171, 2021.
Artículo en Inglés | Web of Science | ID: covidwho-1374228

RESUMEN

In this article, we investigate the multi-criteria decision-making complications under Pythagorean fuzzy soft information. The Pythagorean fuzzy soft set (PFSS) is a proper extension of the Pythagorean fuzzy set (PFS) which discusses the parametrization of the attributes of alternatives. It is also a generalization of the intuitionistic fuzzy soft set (IFSS). The PFSS is used to precisely evaluate the deficiencies, anxiety, and hesitation in decision-making (DM). The most essential determination of the current study is to advance some operational laws along with aggregation operators (AOs) within the Pythagorean fuzzy soft environs such as Pythagorean fuzzy soft interaction weighted average (PFSIWA) and Pythagorean fuzzy soft interaction weighted geometric (PFSIWG) operators with their desirable features. Furthermore, a DM technique has been established based on the developed operators to solve multi-criteria decision-making (MCDM) problems. Moreover, an application of the projected method is presented for the selection of an effective hand sanitizer during the COVID-19 pandemic. A comparative analysis with the merits, effectivity, tractability, along with some available research deduces the effectiveness of this approach.

8.
Sci Adv ; 7(31)2021 Jul.
Artículo en Inglés | MEDLINE | ID: covidwho-1334521

RESUMEN

Interpretation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serosurveillance studies is limited by poorly defined performance of antibody assays over time in individuals with different clinical presentations. We measured antibody responses in plasma samples from 128 individuals over 160 days using 14 assays. We found a consistent and strong effect of disease severity on antibody magnitude, driven by fever, cough, hospitalization, and oxygen requirement. Responses to spike protein versus nucleocapsid had consistently higher correlation with neutralization. Assays varied substantially in sensitivity during early convalescence and time to seroreversion. Variability was dramatic for individuals with mild infection, who had consistently lower antibody titers, with sensitivities at 6 months ranging from 33 to 98% for commercial assays. Thus, the ability to detect previous infection by SARS-CoV-2 is highly dependent on infection severity, timing, and the assay used. These findings have important implications for the design and interpretation of SARS-CoV-2 serosurveillance studies.

9.
Nat Biotechnol ; 39(8): 928-935, 2021 08.
Artículo en Inglés | MEDLINE | ID: covidwho-1152862

RESUMEN

Current serology tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies mainly take the form of enzyme-linked immunosorbent assays, chemiluminescent microparticle immunoassays or lateral flow assays, which are either laborious, expensive or lacking sufficient sensitivity and scalability. Here we present the development and validation of a rapid, low-cost, solution-based assay to detect antibodies in serum, plasma, whole blood and to a lesser extent saliva, using rationally designed split luciferase antibody biosensors. This new assay, which generates quantitative results in 30 min, substantially reduces the complexity and improves the scalability of coronavirus disease 2019 (COVID-19) antibody tests. This assay is well-suited for point-of-care, broad population testing, and applications in low-resource settings, for monitoring host humoral responses to vaccination or viral infection.


Asunto(s)
Anticuerpos Antivirales/sangre , Técnicas Biosensibles/métodos , Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , Sistemas de Atención de Punto , SARS-CoV-2/inmunología , COVID-19/virología , Humanos , Luminiscencia
10.
MAbs ; 13(1): 1893426, 2021.
Artículo en Inglés | MEDLINE | ID: covidwho-1118886

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 , COVID-19/inmunología , Epítopos/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , SARS-CoV-2/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 , COVID-19/genética , Epítopos/genética , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Tratamiento Farmacológico de COVID-19
11.
medrxiv; 2021.
Preprint en Inglés | medRxiv | ID: ppzbmed-10.1101.2021.03.03.21251639

RESUMEN

Serosurveillance studies are critical for estimating SARS-CoV-2 transmission and immunity, but interpretation of results is currently limited by poorly defined variability in the performance of antibody assays to detect seroreactivity over time in individuals with different clinical presentations. We measured longitudinal antibody responses to SARS-CoV-2 in plasma samples from a diverse cohort of 128 individuals over 160 days using 14 binding and neutralization assays. For all assays, we found a consistent and strong effect of disease severity on antibody magnitude, with fever, cough, hospitalization, and oxygen requirement explaining much of this variation. We found that binding assays measuring responses to spike protein had consistently higher correlation with neutralization than those measuring responses to nucleocapsid, regardless of assay format and sample timing. However, assays varied substantially with respect to sensitivity during early convalescence and in time to seroreversion. Variations in sensitivity and durability were particularly dramatic for individuals with mild infection, who had consistently lower antibody titers and represent the majority of the infected population, with sensitivities often differing substantially from reported test characteristics (e.g., amongst commercial assays, sensitivity at 6 months ranged from 33% for ARCHITECT IgG to 98% for VITROS Total Ig). Thus, the ability to detect previous infection by SARS-CoV-2 is highly dependent on the severity of the initial infection, timing relative to infection, and the assay used. These findings have important implications for the design and interpretation of SARS-CoV-2 serosurveillance studies.


Asunto(s)
Fiebre , Tos
12.
Proc Natl Acad Sci U S A ; 117(45): 28046-28055, 2020 11 10.
Artículo en Inglés | MEDLINE | ID: covidwho-889324

RESUMEN

An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2-pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.


Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/química , Diseño de Fármacos , Ingeniería de Proteínas/métodos , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/genética , Antivirales/metabolismo , Sitios de Unión , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Mutación , Biblioteca de Péptidos , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Glicoproteína de la Espiga del Coronavirus/química
13.
Nat Chem Biol ; 17(1): 113-121, 2021 01.
Artículo en Inglés | MEDLINE | ID: covidwho-882912

RESUMEN

Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC50) of 4.0 nM (180 ng ml-1). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy.


Asunto(s)
Enzima Convertidora de Angiotensina 2/química , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Anticuerpos de Cadena Única/química , Glicoproteína de la Espiga del Coronavirus/química , Enzima Convertidora de Angiotensina 2/antagonistas & inhibidores , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/inmunología , Animales , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Sitios de Unión de Anticuerpos/genética , Sitios de Unión de Anticuerpos/inmunología , Chlorocebus aethiops , Microscopía por Crioelectrón , Células HEK293 , Humanos , Modelos Moleculares , Biblioteca de Péptidos , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , SARS-CoV-2 , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Células Vero
14.
Journal of Xi'an Jiaotong University (Medical Sciences) ; 41(5):777-782, 2020.
Artículo en Chino | EMBASE | ID: covidwho-846095

RESUMEN

Objective: To discuss the key points of reconstruction from the surgical intensive care unit (SICU) to the negative pressure isolation ward which can treat patients with novel coronavirus pneumonia (NCP, COVID-19). Methods: The key points of the reconstruction are as follows: establishment of "three zones and two passages", transformation of the ventilation system, process management of personnel access room, and introduction of the visual management concept. Results: The reconstructed SICU can maintain the original space area. As for space layout, there are separate clean zone, semi-polluted zone and polluted zone as well as a separate passage for patients and medical personnel respectively, without the two groups of people contacting each other. To achieve positive air pressure in the clean zone, negative air pressure in the semi-polluted zone is -5 Pa to -10 Pa, and negative air pressure in the patient zone is -10 Pa to -20 Pa. And we ensure that the pressure difference (negative pressure) in adjacent rooms with the same pollution level is not less than 5 Pa. Under normal circumstances, the pressure can be switched to positive one for admission of general ICU patients. Results: Through the reconstruction, the SICU can meet the requirements of treatment of COVID-19 patients. The main points of reconstruction are space layout and the air conditioning and ventilation system.

15.
Cell Rep Med ; 1(7): 100123, 2020 10 20.
Artículo en Inglés | MEDLINE | ID: covidwho-793949

RESUMEN

Comprehensive understanding of the serological response to SARS-CoV-2 infection is important for both pathophysiologic insight and diagnostic development. Here, we generate a pan-human coronavirus programmable phage display assay to perform proteome-wide profiling of coronavirus antigens enriched by 98 COVID-19 patient sera. Next, we use ReScan, a method to efficiently sequester phage expressing the most immunogenic peptides and print them onto paper-based microarrays using acoustic liquid handling, which isolates and identifies nine candidate antigens, eight of which are derived from the two proteins used for SARS-CoV-2 serologic assays: spike and nucleocapsid proteins. After deployment in a high-throughput assay amenable to clinical lab settings, these antigens show improved specificity over a whole protein panel. This proof-of-concept study demonstrates that ReScan will have broad applicability for other emerging infectious diseases or autoimmune diseases that lack a valid biomarker, enabling a seamless pipeline from antigen discovery to diagnostic using one recombinant protein source.


Asunto(s)
Antígenos Virales/inmunología , Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/aislamiento & purificación , Anticuerpos Antivirales/sangre , COVID-19/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Biblioteca de Péptidos , Análisis por Matrices de Proteínas , Proteoma/inmunología , Reproducibilidad de los Resultados , SARS-CoV-2/inmunología , Sensibilidad y Especificidad , Proteínas Virales/inmunología
16.
mSphere ; 5(5)2020 09 16.
Artículo en Inglés | MEDLINE | ID: covidwho-772263

RESUMEN

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread around the world, there is an urgent need for new assay formats to characterize the humoral response to infection. Here, we present an efficient, competitive serological assay that can simultaneously determine an individual's seroreactivity against the SARS-CoV-2 Spike protein and determine the proportion of anti-Spike antibodies that block interaction with the human angiotensin-converting enzyme 2 (ACE2) required for viral entry. In this approach based on the use of enzyme-linked immunosorbent assays (ELISA), we present natively folded viral Spike protein receptor-binding domain (RBD)-containing antigens via avidin-biotin interactions. Sera are then competed with soluble ACE2-Fc, or with a higher-affinity variant thereof, to determine the proportion of ACE2 blocking anti-RBD antibodies. Assessment of sera from 144 SARS-CoV-2 patients ultimately revealed that a remarkably consistent and high proportion of antibodies in the anti-RBD pool targeted the epitope responsible for ACE2 engagement (83% ± 11%; 50% to 107% signal inhibition in our largest cohort), further underscoring the importance of tailoring vaccines to promote the development of such antibodies.IMPORTANCE With the emergence and continued spread of the SARS-CoV-2 virus, and of the associated disease, coronavirus disease 2019 (COVID-19), there is an urgent need for improved understanding of how the body mounts an immune response to the virus. Here, we developed a competitive SARS-CoV-2 serological assay that can simultaneously determine whether an individual has developed antibodies against the SARS-CoV-2 Spike protein receptor-binding domain (RBD) and measure the proportion of these antibodies that block interaction with the human angiotensin-converting enzyme 2 (ACE2) required for viral entry. Using this assay and 144 SARS-CoV-2 patient serum samples, we found that a majority of anti-RBD antibodies compete for ACE2 binding. These results not only highlight the need to design vaccines to generate such blocking antibodies but also demonstrate the utility of this assay to rapidly screen patient sera for potentially neutralizing antibodies.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Peptidil-Dipeptidasa A/inmunología , Pruebas Serológicas/métodos , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2 , Antígenos Virales/inmunología , Sitios de Unión/inmunología , COVID-19 , Infecciones por Coronavirus/prevención & control , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Pandemias/prevención & control , Neumonía Viral/prevención & control , Unión Proteica , Dominios Proteicos/inmunología , SARS-CoV-2
17.
medrxiv; 2020.
Preprint en Inglés | medRxiv | ID: ppzbmed-10.1101.2020.08.17.20176925

RESUMEN

Current serology tests for SARS-CoV-2 antibodies mainly take the form of enzyme-linked immunosorbent assays or lateral flow assays, with the former being laborious and the latter being expensive and often lacking sufficient sensitivity and scalability. Here we present the development and validation of a rapid, low-cost solution-based assay to detect antibodies in serum, plasma, whole blood, and saliva, using rationally designed split luciferase antibody biosensors (spLUC). This new assay, which generates quantitative results in as short as 5 minutes, substantially reduces the complexity and improves the scalability of COVID-19 antibody tests for point-of-care and broad population testing.


Asunto(s)
COVID-19
18.
biorxiv; 2020.
Preprint en Inglés | bioRxiv | ID: ppzbmed-10.1101.2020.08.08.242511

RESUMEN

Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domain binders with high affinity toward neutralizing epitopes without the need for high-resolution structural information. We constructed a VH-phage library and targeted a known neutralizing site, the angiotensin-converting enzyme 2 (ACE2) binding interface of the trimeric SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified 85 unique VH binders to two non-overlapping epitopes within the ACE2 binding site on Spike-RBD. This enabled us to systematically link these VH domains into multivalent and bi-paratopic formats. These multivalent and bi-paratopic VH constructs showed a marked increase in affinity to Spike (up to 600-fold) and neutralization potency (up to 1400-fold) on pseudotyped SARS-CoV-2 virus when compared to the standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with half-minimal inhibitory concentration (IC50) of 4.0 nM (180 ng/mL). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain bound an RBD at the ACE2 binding site, explaining its increased neutralization potency and confirming our original design strategy. Our results demonstrate that targeted selection and engineering campaigns using a VH-phage library can enable rapid assembly of highly avid and potent molecules towards therapeutically important protein interfaces.


Asunto(s)
COVID-19
19.
biorxiv; 2020.
Preprint en Inglés | bioRxiv | ID: ppzbmed-10.1101.2020.07.31.231746

RESUMEN

An essential mechanism for SARS-CoV-1 and -2 infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human Fc domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2 pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50) in the tens of ng/ml range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-utilizing coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be pre-designed for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated or generated from convalescent patients.


Asunto(s)
COVID-19
20.
medrxiv; 2020.
Preprint en Inglés | medRxiv | ID: ppzbmed-10.1101.2020.05.27.20114652

RESUMEN

As SARS-CoV-2 continues to spread around the world, there is an urgent need for new assay formats to characterize the humoral response to infection. Convalescent serum is being used for treatment and for isolation of patient-derived antibodies. However, currently there is not a simple means to estimate serum bulk neutralizing capability. Here we present an efficient competitive serological assay that can simultaneously determine an individual's seropositivity against the SARS-CoV-2 Spike protein and estimate the neutralizing capacity of anti-Spike antibodies to block interaction with the human angiotensin converting enzyme 2 (ACE2) required for viral entry. In this ELISA-based assay, we present natively-folded viral Spike protein receptor binding domain (RBD)-containing antigens via avidin-biotin interactions. Sera are then supplemented with soluble ACE2-Fc to compete for RBD-binding serum antibodies, and antibody binding quantified. Comparison of signal from untreated serum and ACE2-Fc-treated serum reveals the presence of antibodies that compete with ACE2 for RBD binding, as evidenced by loss of signal with ACE2-Fc treatment. In our test cohort of nine convalescent SARS-CoV-2 patients, we found all patients had developed anti-RBD antibodies targeting the epitope responsible for ACE2 engagement. This assay provides a simple and high-throughput method to screen patient sera for potentially neutralizing anti-Spike antibodies to enable identification of candidate sera for therapeutic use.


Asunto(s)
Síndrome Respiratorio Agudo Grave
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