RESUMO
Aim: Our aim was to investigate the differences between healthy people and COVID-19 patients in terms of some immunological biomolecules, especially including those related to the inflammation process. Materials & methods: A total of 180 participants (90 healthy controls and 90 COVID-19 patients) were included. The expression levels of eight different inflammation-related biomolecules were measured by the ELISA technique. Results: The mean levels of ACE2, ANG1-7, GAL3, GAL9, SCUBE1, SCUBE2 and SCUBE3 were elevated in COVID-19 patients when compared with healthy controls, while the mean level of GAL2 was lower in COVID-19 patients than controls. Conclusion: To understand the cytokine storm mechanism and related parameters, more detailed studies should be performed investigating more related biomolecules and related signaling pathways.
Assuntos
COVID-19 , Humanos , Enzima de Conversão de Angiotensina 2 , SARS-CoV-2 , Prognóstico , Galectinas , Inflamação , Proteínas de Ligação ao Cálcio , Proteínas Adaptadoras de Transdução de SinalRESUMO
Angiotensin-converting enzyme II (ACE2) is a monocarboxypeptidase expressed throughout multiple tissues and its catalysis of bioactive peptides regulates the renin-angiotensin system mediating blood pressure homeostasis. ACE2 is implicated in a variety of diseases, including obesity, diabetes, and cardiovascular diseases, and is the obligate entry receptor for SARS-CoV-2 infection. Disease-associated genetic variants of ACE2 are increasingly being identified but are poorly characterized. To aid this problem, we introduce a fluorometric cell-based assay for evaluating surface-expressed ACE2 catalytic activity that preserves the native glycosylation of the host environment and is amenable to high-throughput analysis of ACE2 variants in multi-well plates. We demonstrate sensitivity to detecting catalysis of the key ACE2 substrates, Angiotensin II, Apelin-13, and des-Arg9-bradykinin, and impact of a catalytically-deficient ACE2 variant. Normalizing catalytic measures to surface ACE2 expression accounts for variability in ACE2 variant transfection, surface delivery or stability. This assay provides a convenient and powerful approach for investigating the catalytic characteristics of ACE2 variants involved in cardiovascular peptide cascades and homeostasis of multiple organs.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Humanos , Enzima de Conversão de Angiotensina 2/genética , SARS-CoV-2/genética , Angiotensina II , CatáliseRESUMO
The current epidemic of corona virus disease (COVID-19) has resulted in an immense health burden that became the third leading cause of death and potentially contributed to a decline in life expectancy in the United States. The severe acute respiratory syndrome-related coronavirus-2 binds to the surface-bound peptidase angiotensin-converting enzyme 2 (ACE2, EC 3.4.17.23) leading to tissue infection and viral replication. ACE2 is an important enzymatic component of the renin-angiotensin system (RAS) expressed in the lung and other organs. The peptidase regulates the levels of the peptide hormones Ang II and Ang-(1-7), which have distinct and opposing actions to one another, as well as other cardiovascular peptides. A potential consequence of severe acute respiratory syndrome-related coronavirus-2 infection is reduced ACE2 activity by internalization of the viral-ACE2 complex and subsequent activation of the RAS (higher ratio of Ang II:Ang-[1-7]) that may exacerbate the acute inflammatory events in COVID-19 patients and possibly contribute to the effects of long COVID-19. Moreover, COVID-19 patients present with an array of autoantibodies to various components of the RAS including the peptide Ang II, the enzyme ACE2, and the AT1 AT2 and Mas receptors. Greater disease severity is also evident in male COVID-19 patients, which may reflect underlying sex differences in the regulation of the 2 distinct functional arms of the RAS. The current review provides a critical evaluation of the evidence for an activated RAS in COVID-19 subjects and whether this system contributes to the greater severity of severe acute respiratory syndrome-related coronavirus-2 infection in males as compared with females.
Assuntos
COVID-19 , Sistema Renina-Angiotensina , Humanos , Masculino , Feminino , Sistema Renina-Angiotensina/fisiologia , Enzima de Conversão de Angiotensina 2/metabolismo , Peptidil Dipeptidase A/fisiologia , SARS-CoV-2 , Caracteres Sexuais , Síndrome de COVID-19 Pós-Aguda , Inibidores da Enzima Conversora de Angiotensina/farmacologiaRESUMO
SARS-CoV-2, the causative agent of the debilitating COVID-19, is mainly transmitted by first infecting nose and lung epithelial cells. The mouth is also believed to be a viral portal site since certain types of oral epithelial cells were shown to express ACE2 receptor. However, it is unclear whether oral epithelial cells are directly infected by SARS-CoV-2. In this study, we addressed whether epithelial cells of the oral gingiva were susceptible to infection. Interestingly, we found that KRT5+ and KRT18+ gingival epithelial cells do not express ACE2 but highly express TMPRSS2 and Furin as well as CD147, which was proposed to be an alternative receptor for SARS-CoV-2. However, using SARS-CoV-2 pseudoviruses containing the spike protein, we observed that gingival epithelial cells were not susceptible to infection due to the lack of ACE2 expression and the inability of CD147 to mediate viral entry. These results strongly suggest that epithelial cells from the gingiva are not susceptible to SARS-CoV-2 and CD147 is not a receptor for the SARS-CoV-2 virus. The susceptibility of oral cells from other oral structures under healthy and pathological conditions still needs to be confirmed to better understand the role of the oral cavity in COVID-19 infection and transmission.
Assuntos
Basigina , Receptores de Coronavírus , SARS-CoV-2 , Humanos , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19 , Suscetibilidade a Doenças , Células Epiteliais/metabolismo , Gengiva , SARS-CoV-2/metabolismo , Basigina/metabolismo , Receptores de Coronavírus/metabolismoRESUMO
The continued emergence of new SARS-CoV-2 variants has accentuated the growing need for fast and reliable methods for the design of potentially neutralizing antibodies (Abs) to counter immune evasion by the virus. Here, we report on the de novo computational design of high-affinity Ab variable regions (Fv) through the recombination of VDJ genes targeting the most solvent-exposed hACE2-binding residues of the SARS-CoV-2 spike receptor binding domain (RBD) protein using the software tool OptMAVEn-2.0. Subsequently, we carried out computational affinity maturation of the designed variable regions through amino acid substitutions for improved binding with the target epitope. Immunogenicity of designs was restricted by preferring designs that match sequences from a 9-mer library of "human Abs" based on a human string content score. We generated 106 different antibody designs and reported in detail on the top five that trade-off the greatest computational binding affinity for the RBD with human string content scores. We further describe computational evaluation of the top five designs produced by OptMAVEn-2.0 using a Rosetta-based approach. We used Rosetta SnugDock for local docking of the designs to evaluate their potential to bind the spike RBD and performed "forward folding" with DeepAb to assess their potential to fold into the designed structures. Ultimately, our results identified one designed Ab variable region, P1.D1, as a particularly promising candidate for experimental testing. This effort puts forth a computational workflow for the de novo design and evaluation of Abs that can quickly be adapted to target spike epitopes of emerging SARS-CoV-2 variants or other antigenic targets.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes , Epitopos/química , Região Variável de Imunoglobulina , Glicoproteína da Espícula de Coronavírus/metabolismo , Anticorpos Antivirais/metabolismoRESUMO
After more than two years, COVID-19 still represents a global health burden of unprecedented extent and assessing the degree of immunity of individuals against SARS-CoV-2 remains a challenge. Virus neutralization assays represent the gold standard for assessing antibody-mediated protection against SARS-CoV-2 in sera from recovered and/or vaccinated individuals. Neutralizing antibodies block the interaction of viral spike protein with human angiotensin-converting enzyme 2 (ACE2) receptor in vitro and prevent viral entry into host cells. Classical viral neutralization assays using full replication-competent viruses are restricted to specific biosafety level 3-certified laboratories, limiting their utility for routine and large-scale applications. We developed therefore a cell-fusion-based assay building on the interaction between viral spike and ACE2 receptor expressed on two different cell lines, substantially reducing biosafety risks associated with classical viral neutralization assays. This chapter describes this simple, sensitive, safe and cost-effective approach for rapid and high-throughput evaluation of SARS-CoV-2 neutralizing antibodies relying on high-affinity NanoLuc® luciferase complementation technology (HiBiT). When applied to a variety of standards and patient samples, this method yields highly reproducible results in 96-well, as well as in 384-well format. The use of novel NanoLuc® substrates with increased signal stability like Nano-Glo® Endurazine™ furthermore allows for high flexibility in assay set-up and full automatization of all reading processes. Lastly, the assay is suitable to evaluate the neutralizing capacity of sera against the existing spike variants, and potentially variants that will emerge in the future.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Anticorpos Neutralizantes , Anticorpos Antivirais , Fusão Celular , Humanos , Luciferases , Testes de Neutralização/métodos , Peptidil Dipeptidase A/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Virus-like particles (VLPs) resemble authentic virus while not containing any genomic information. Here, we present a fast and powerful method for the production of SARS-CoV-2 VLP in insect cells and the application of these VLPs to evaluate the inhibition capacity of monoclonal antibodies and sera of vaccinated donors. Our method avoids the baculovirus-based approaches commonly used in insect cells by employing direct plasmid transfection to co-express SARS-CoV-2 envelope, membrane, and spike protein that self-assemble into VLPs. After optimization of the expression plasmids and vector ratios, VLPs with an ~145 nm diameter and the typical "Corona" aura were obtained, as confirmed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Fusion of the membrane protein to GFP allowed direct quantification of binding inhibition to angiotensin II-converting enzyme 2 (ACE2) on cells by therapeutic antibody candidates or sera from vaccinated individuals. Neither VLP purification nor fluorescent labeling by secondary antibodies are required to perform these flow cytometric assays.
Assuntos
Baculoviridae , COVID-19 , Humanos , Animais , Baculoviridae/genética , SARS-CoV-2/genética , Enzima de Conversão de Angiotensina 2 , Glicoproteína da Espícula de Coronavírus/genética , Angiotensina II , Insetos , Anticorpos MonoclonaisRESUMO
Background and Objectives: Consequences due to infection with SARS-CoV-2 virus can have a direct impact on skeletal muscle, due to the fact that both cardiac and skeletal muscle tissue show robust ACE2(angiotensin-converting enzyme 2) expression, suggesting a potential susceptibility to SARS-CoV-2 infection in both types of tissues. From the articles analyzed we concluded that the musculoskeletal damage is firstly produced by the inflammatory effects, cytokine storm and muscle catabolism. However, myopathy, polyneuropathy and therapies such as corticoids were also considered important factors in muscle fatigue and functional incapacity. Pulmonary rehabilitation programs and early mobilization had a highly contribution during the acute phase and post-illness recovery process and helped patients to reduce dyspnea, increase the capacity of physical effort, overcome psychological disorders and improved the quality of their life. Materials and Methods: We have included in this review 33 articles that contain data on muscle damage following SARS-CoV-2 infection. We used the following keywords to search for articles: SARS-CoV-2, COVID-19, muscle weakness, muscle disease, muscle fatigue, neurological disorders. As a search strategy we used PubMed, Cochrane Database of Systematic Reviews; Database of Abstracts of Reviews of Effects and Health Technology Assessment Database to collect the information. We also have chosen the most recent articles published in the last 5 years. Conclusions: Muscular damage, as well as the decrease in the quality of life, are often a consequence of severe SARS-CoV-2 infection through: systemic inflammation, corticotherapy, prolonged bed rest and other unknown factors. Pulmonary rehabilitation programs and early mobilization had a highly contribution during the acute phase and post-illness recovery process and helped patients to reduce dyspnea, increase the capacity of physical effort, overcome psychological disorders and improve the quality of their life.
Assuntos
COVID-19 , Doenças Musculares , Enzima de Conversão de Angiotensina 2 , Dispneia , Humanos , Fadiga Muscular , Músculo Esquelético/metabolismo , Doenças Musculares/complicações , Qualidade de Vida , SARS-CoV-2 , Revisões Sistemáticas como AssuntoRESUMO
The hypermutated receptor binding domain (RBD) of the Omicron (B.1.1.529) lineage exhibits a different binding interface with human angiotensin-converting enzyme 2 (ACE2) relative to that of the wild-type Wuhan Hu-1, yet how the altered interaction will affect viral evolution is largely unknown. Here, we used molecular dynamics simulation to characterize the binding features of the Omicron BA.1/hACE2 complex and used free energy perturbation calculations to assess the ongoing and putative variations. The complex reveals a substantial rearrangement of the interfacial hydrogen-bond network: R493 of RBD forms a dynamic electrostatic interaction with both E35 and D38 of hACE2, which prohibits the hydrogen bonds of R498-D38 and Y449-D38. Whereas most circulating mutations minimally affect RBD binding to hACE2, the charge-altering mutation R493Q attenuates the affinity by abolishing the electrostatic interaction. However, the potential variants H505Y or N417K/R493Q could restore and gain even greater binding affinities than BA.1 as a result of their optimized interaction network and epistasis effects.
Assuntos
Enzima de Conversão de Angiotensina 2 , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Epistasia Genética , Humanos , Hidrogênio , Mutação , Peptidil Dipeptidase A/química , Ligação Proteica , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/químicaRESUMO
Neutralizing antibody (NAb) titer is a key biomarker of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but point-of-care methods for assessing NAb titer are not widely available. Here, we present a lateral flow assay that captures SARS-CoV-2 receptor-binding domain (RBD) that has been neutralized from binding angiotensin-converting enzyme 2 (ACE2). Quantification of neutralized RBD in this assay correlates with NAb titer from vaccinated and convalescent patients. This methodology demonstrated superior performance in assessing NAb titer compared with either measurement of total anti-spike immunoglobulin G titer or quantification of the absolute reduction in binding between ACE2 and RBD. Our testing platform has the potential for mass deployment to aid in determining at population scale the degree of protective immunity individuals may have following SARS-CoV-2 vaccination or infection and can enable simple at-home assessment of NAb titer.
Assuntos
Anticorpos Neutralizantes , COVID-19 , Humanos , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Vacinas contra COVID-19 , Sistemas Automatizados de Assistência Junto ao Leito , Anticorpos Antivirais , COVID-19/diagnósticoRESUMO
BACKGROUND: SARS-CoV-2 is a positive-sense single-stranded RNA virus. It is enveloped by four structural proteins. The entry of the virus into the host cells is mediated by spike protein binding to the angiotensin converting enzyme 2 (ACE2) and proteolytic cleavage by transmembrane protease serine 2 (TMPRSS2). In this study, we analyzed the expression of the ACE2 receptor and TMPRSS2 in cases under investigation for SARS-CoV-2 infection. METHODS: The study was carried out using the viral transport medium of consecutive nasopharyngeal swabs from 300 people under examination for SARS-CoV-2 infection. All samples underwent the SARS-CoV-2 transcriptase-mediated amplification assay (Procleix® SARS-CoV-2) to detect the virus. Immunocytochemistry was used in each sample to detect the presence of the SARS-CoV-2 nucleoprotein, the ACE2 receptor, and TMPRSS2. RESULTS: An immunocytochemical study with monoclonal antibody against SARS-CoV-2 viral nucleoprotein showed positivity in squamous cells. ACE2 were not detected in the squamous cells obtained from the nasopharyngeal samples. CONCLUSIONS: SARS-CoV-2 predominantly localizes to squamous cells in cytology samples of patients with positive transcriptase-mediated amplification SARS-CoV-2 assay results. The immunocytochemical negativity for ACE2 evidenced in the present study could be related to the cellular heterogeneity present in the nasopharyngeal smear samples and could be related to variations at the genomic level. Our results suggest that SARS-CoV-2 might be present in the nasopharyngeal region because viral cell junctions are weaker. This facilitates viral concentration, infective capacity and migration to specific organs, where SARS-CoV-2 infects target cells by binding to their receptors and then entering.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , COVID-19/diagnóstico , Humanos , Nasofaringe/metabolismo , Proteólise , SARS-CoV-2 , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
The Omicron variant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has now spread throughout the world. We used computational tools to assess the spike infectivity, transmission, and pathogenicity of Omicron (BA.1) and sub-variants (BA.1.1, BA.2, and BA.3) in this study. BA.1 has 39 mutations, BA.1.1 has 40 mutations, BA.2 has 31 mutations, and BA.3 has 34 mutations, with 21 shared mutations between all. We observed 11 common mutations in Omicron's receptor-binding domain (RBD) and sub-variants. In pathogenicity analysis, the Y505H, N786K, T95I, N211I, N856K, and V213R mutations in omicron and sub-variants are predicted to be deleterious. Due to the major effect of the mutations characterizing in the RBD, we found that Omicron and sub-variants had a higher positive electrostatic surface potential. This could increase interaction between RBD and negative electrostatic surface potential human angiotensin-converting enzyme 2 (hACE2). Omicron and sub-variants had a higher affinity for hACE2 and the potential for increased transmission when compared to the wild-type (WT). Negative electrostatic potential of N-terminal domain (NTD) of the spike protein value indicates that the Omicron variant binds receptors less efficiently than the WT. Given that at least one receptor is highly expressed in lung and bronchial cells, the electrostatic potential of NTD negative value could be one of the factors contributing to why the Omicron variant is thought to be less harmful to the lower respiratory tract. Among Omicron sub-lineages, BA.2 and BA.3 have a higher transmission potential than BA.1 and BA.1.1. We predicted that mutated residues in BA.1.1 (K478), BA.2 (R400, R490, and R495), and BA.3 (R397 and H499) formation of new salt bridges and hydrogen bonds. Omicron and sub-variant mutations at Receptor-binding Motif (RBM) residues such as Q493R, N501Y, Q498, T478K, and Y505H all contribute significantly to binding affinity with human ACE2. Interactions with Omicron variant mutations at residues 493, 496, 498, and 501 seem to restore ACE2 binding effectiveness lost due to other mutations like K417N.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Enzima de Conversão de Angiotensina 2/genética , Humanos , Peptidil Dipeptidase A/metabolismo , Receptores Virais/metabolismo , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , VirulênciaRESUMO
The severe acute respiratory syndrome (SARS) coronavirus 2 (CoV-2) variant Omicron spread more rapid than the other variants of SARS-CoV-2 virus. Mutations on the Spike (S) protein receptor-binding domain (RBD) are critical for the antibody resistance and infectivity of the SARS-CoV-2 variants. In this study, we have used accelerated molecular dynamics (aMD) simulations and free energy calculations to present a systematic analysis of the affinity and conformational dynamics along with the interactions that drive the binding between Spike protein RBD and human angiotensin-converting enzyme 2 (ACE2) receptor. We evaluate the impacts of the key mutation that occur in the RBDs Omicron and other variants in the binding with the human ACE2 receptor. The results show that S protein Omicron has stronger binding to the ACE2 than other variants. The evaluation of the decomposition energy per residue shows the mutations N440K, T478K, Q493R and Q498R observed in Spike protein of SARS-CoV-2 provided a stabilization effect for the interaction between the SARS-CoV-2 RBD and ACE2. Overall, the results demonstrate that faster spreading of SARS-CoV-2 Omicron may be correlated with binding affinity of S protein RBD to ACE2 and mutations of uncharged residues to positively charged residues such as Lys and Arg in key positions in the RBD.
Assuntos
COVID-19 , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2/genética , COVID-19/genética , Humanos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Mathematical modelling of infection processes in cells is of fundamental interest. It helps to understand the SARS-CoV-2 dynamics in detail and can be useful to define the vulnerability steps targeted by antiviral treatments. We previously developed a deterministic mathematical model of the SARS-CoV-2 life cycle in a single cell. Despite answering many questions, it certainly cannot accurately account for the stochastic nature of an infection process caused by natural fluctuation in reaction kinetics and the small abundance of participating components in a single cell. In the present work, this deterministic model is transformed into a stochastic one based on a Markov Chain Monte Carlo (MCMC) method. This model is employed to compute statistical characteristics of the SARS-CoV-2 life cycle including the probability for a non-degenerate infection process. Varying parameters of the model enables us to unveil the inhibitory effects of IFN and the effects of the ACE2 binding affinity. The simulation results show that the type I IFN response has a very strong effect on inhibition of the total viral progeny whereas the effect of a 10-fold variation of the binding rate to ACE2 turns out to be negligible for the probability of infection and viral production.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Interferon Tipo I/imunologia , Modelos Teóricos , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/imunologia , Simulação por Computador , Humanos , Cinética , Estágios do Ciclo de Vida , Cadeias de Markov , Ligação Proteica , SARS-CoV-2/crescimento & desenvolvimento , Processos EstocásticosRESUMO
Coronavirus disease 2019, caused by SARS-CoV-2, remains an on-going pandemic, partly due to the emergence of variant viruses that can "break-through" the protection of the current vaccines and neutralizing antibodies (nAbs), highlighting the needs for broadly nAbs and next-generation vaccines. We report an antibody that exhibits breadth and potency in binding the receptor-binding domain (RBD) of the virus spike glycoprotein across SARS coronaviruses. Initially, a lead antibody was computationally discovered and crystallographically validated that binds to a highly conserved surface of the RBD of wild-type SARS-CoV-2. Subsequently, through experimental affinity enhancement and computational affinity maturation, it was further developed to bind the RBD of all concerning SARS-CoV-2 variants, SARS-CoV-1 and pangolin coronavirus with pico-molar binding affinities, consistently exhibited strong neutralization activity against wild-type SARS-CoV-2 and the Alpha and Delta variants. These results identify a vulnerable target site on coronaviruses for development of pan-sarbecovirus nAbs and vaccines.
Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Antígenos Virais/química , Antígenos Virais/genética , Anticorpos Amplamente Neutralizantes/genética , Anticorpos Amplamente Neutralizantes/metabolismo , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Humanos , Fragmentos de Imunoglobulinas/imunologia , Simulação de Acoplamento Molecular , Método de Monte Carlo , Testes de Neutralização , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Domínios Proteicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Serology tests for SARS-CoV-2 have proven to be important tools to fight against the COVID-19 pandemic. These serological tests can be used in low-income and remote areas for patient contact tracing, epidemiologic studies and vaccine efficacy evaluations. In this study, we used a semi-stable mammalian episomal expression system to produce high quantities of the receptor-binding domain-RBD of SARS-CoV-2 in a simple and very economical way. The recombinant antigen was tested in an in-house IgG ELISA for COVID-19 with a panel of human sera. A performance comparison of this serology test with a commercial test based on the full-length spike protein showed 100% of concordance between tests. Thus, this serological test can be an attractive and inexpensive option in scenarios of limited resources to face the COVID-19 pandemic.
Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/economia , Teste Sorológico para COVID-19/economia , Custos e Análise de Custo , Ensaio de Imunoadsorção Enzimática , Engenharia Genética , Humanos , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Contribution of the renin-angiotensinogen system in the risk of COVID-19 and related complications have been assessed by several groups. However, the results are not consistent. We examined levels of ACE1 and ACE2 in the circulation of two groups of COVID-19 patients (ICU-admitted and general ward-admitted patients) compared with healthy controls. We also genotyped two polymorphisms in ACE1 gene (the ACE1-I/D polymorphism rs1799752 and rs4359) to appraise their association with expression levels of ACE1 and ACE2. Expression level of ACE1 was significantly higher in ICU patients compared with non-ICU patients (P value = 0.02). However, its expression was not significantly different between total COVID-19 patients and total controls (P value = 0.34). ACE2 expression was not different ether between two groups of COVID-19 patients (P value = 0.12) or between total COVID-19 patients and total controls (P value = 0.79). While distribution of rs1799752 and rs4359 alleles was similar between study groups, genotype frequencies of rs1799752 were differently distributed among total COVID-19 patients and controls (P value = 0.00001). Moreover, genotypes of the other polymorphism tended to be distinctively distributed among these two groups (P value = 0.06). In the total population of patients and controls, different ACE1 mRNA levels were observed among carriers of different rs1799752 genotypes; of note, ID genotype carriers showed a higher expression of ACE1 compared with II genotype carriers (P = 0.01). ACE1 polymorphisms might affect risk of COVID-19 and expression of ACE transcripts.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Genótipo , Humanos , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Polimorfismo Genético , SARS-CoV-2RESUMO
Highly accurate serological tests are key to assessing the prevalence of SARS-CoV-2 antibodies and the level of immunity in the population. This is important to predict the current and future status of the pandemic. With the recent emergence of new and more infectious SARS-CoV-2 variants, assays allowing for high throughput analysis of antibodies able to neutralize SARS-CoV-2 become even more important. Here, we report the development and validation of a robust, high throughput method, which enables the assessment of antibodies inhibiting the binding between the SARS-CoV-2 spike protein and angiotensin converting enzyme 2 (ACE2). The assay uses recombinantly produced spike-f and ACE2 and is performed in a bead array format, which allows analysis of up to 384 samples in parallel per instrument over seven hours, demanding only one hour of manual handling. The method is compared to a microneutralization assay utilising live SARS-CoV-2 and is shown to deliver highly correlating data. Further, a comparison with a serological method that measures all antibodies recognizing the spike protein shows that this type of assessment provides important insights into the neutralizing efficiency of the antibodies, especially for individuals with low antibody levels. This method can be an important and valuable tool for large-scale assessment of antibody-based neutralization, including neutralization of new spike variants that might emerge.
Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , COVID-19 , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2/imunologia , COVID-19/imunologia , Ensaios de Triagem em Larga Escala , Humanos , Testes de Neutralização , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
SARS-CoV-2 infects humans and causes Coronavirus disease 2019 (COVID-19). The S1 domain of the spike glycoprotein of SARS-CoV-2 binds to human angiotensin-converting enzyme 2 (hACE2) via its receptor-binding domain, while the S2 domain facilitates fusion between the virus and the host cell membrane for entry. The spike glycoprotein of circulating SARS-CoV-2 genomes is a mutation hotspot. Some mutations may affect the binding affinity for hACE2, while others may modulate S-glycoprotein expression, or they could result in a virus that can escape from antibodies generated by infection with the original variant or by vaccination. Since a large number of variants are emerging, it is of vital importance to be able to rapidly assess their characteristics: while changes of binding affinity alone do not always cause direct advantages for the virus, they still can provide important insights on where the evolutionary pressure is directed. Here, we propose a simple and cost-effective computational protocol based on Molecular Dynamics simulations to rapidly screen the ability of mutated spike protein to bind to the hACE2 receptor and selected neutralizing biomolecules. Our results show that it is possible to achieve rapid and reliable predictions of binding affinities. A similar approach can be used to perform preliminary screenings of the potential effects of S-RBD mutations, helping to prioritize the more time-consuming and expensive experimental work.
Assuntos
Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes/imunologia , Simulação por Computador , Simulação de Dinâmica Molecular , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Afinidade de Anticorpos , Humanos , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Spike glycoprotein (Sgp) is liable for binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the host receptors. Since Sgp is the main target for vaccine and drug designing, elucidating its mutation pattern could help in this regard. This study is aimed at investigating the correspondence of specific residues to the SgpSARS-CoV-2 functionality by explorative interpretation of sequence alignments. Centrality analysis of the Sgp dissects the importance of these residues in the interaction network of the RBD-ACE2 (receptor-binding domain) complex and furin cleavage site. Correspondence of RBD to threonine500 and asparagine501 and furin cleavage site to glutamine675, glutamine677, threonine678, and alanine684 was observed; all residues are exactly located at the interaction interfaces. The harmonious location of residues dictates the RBD binding property and the flexibility, hydrophobicity, and accessibility of the furin cleavage site. These species-specific residues can be assumed as real targets of evolution, while other substitutions tend to support them. Moreover, all these residues are parts of experimentally identified epitopes. Therefore, their substitution may affect vaccine efficacy. Higher rate of RBD maintenance than furin cleavage site was predicted. The accumulation of substitutions reinforces the probability of the multi-host circulation of the virus and emphasizes the enduring evolutionary events.