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1.
BMC Res Notes ; 14(1): 152, 2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879229

RESUMO

OBJECTIVE: Herd immunity is achieved when in a population, immune individuals are in a sufficiently large proportion. Neutralizing antibodies specific to SARS-CoV-2 that are produced following infection or vaccination are critical for controlling the spread of COVID-19. The objective of the present work was to investigate the rate of SARS-CoV-2 natural immunization in Gabonese. RESULTS: One thousand, four hundred and ninety two people were enrolled. The overall prevalence of anti-SARS-CoV-2 antibodies was 36.2%. Moreover, 76.4% of people who developed a humoral response to SARS-CoV-2 produced both anti-SARS-CoV-2 N-protein antibodies and anti-SARS-CoV-2 S-protein antibodies, which correspond to 27.7% of the total population. In infants (0-9 month), children (1-17 years) and adults, the prevalence of anti-SARS-CoV-2 antibodies was relatively the same, between 33 and 37% (any antibody types) and between 25 and 28.6% (neutralizing antibodies). In this African context, one-third (1/3) of the screened population was exposed to SARS-CoV-2 and three-quarter (3/4) of those exposed individuals developed neutralizing antibodies against SARS-CoV-2. This data suggest that herd immunity is not yet to be achieved in Gabon.


Assuntos
Anticorpos Antivirais/imunologia , /imunologia , Imunidade Coletiva , Glicoproteína da Espícula de Coronavírus/imunologia , Adolescente , Adulto , Idoso , Anticorpos Neutralizantes/imunologia , Criança , Pré-Escolar , Feminino , Gabão/epidemiologia , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/imunologia , Adulto Jovem
2.
Front Immunol ; 12: 614436, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33790892

RESUMO

The novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic of the coronavirus disease 2019 (COVID-19), which elicits a wide variety of symptoms, ranging from mild to severe, with the potential to lead to death. Although used as the standard method to screen patients for SARS-CoV-2 infection, real-time PCR has challenges in dealing with asymptomatic patients and those with an undetectable viral load. Serological tests are therefore considered potent diagnostic tools to complement real-time PCR-based diagnosis and are used for surveillance of seroprevalence in populations. However, the dynamics of the antibody response against SARS-CoV-2 currently remain to be investigated. Here, through analysis of plasma samples from 84 patients with COVID-19, we observed that the response of virus-specific antibodies against three important antigens, RBD, N and S, dynamically changed over time and reached a peak 5-8 weeks after the onset of symptoms. The antibody responses were irrespective of sex. Severe cases were found to have higher levels of antibody response, larger numbers of inflammatory cells and C-reactive protein levels. Within the mild/moderate cases, pairwise comparison indicated moderate association between anti-RBD vs. anti-N, anti-RBD vs. anti-S1S2, and anti-N vs. anti-S1S2. Furthermore, the majority of cases could achieve IgM and IgG seroconversion at 2 weeks since the disease onset. Analysis of neutralizing antibodies indicated that these responses were able to last for more than 112 days but decline significantly after the peak. In summary, our findings demonstrate the longitudinally dynamic changes in antibody responses against SARS-CoV-2, which can contribute to the knowledge of humoral immune response after SARS-CoV-2 infection and are informative for future development of vaccine and antibody-based therapies.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , /imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Pequim , China , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/imunologia , Domínios Proteicos/imunologia , Soroconversão , Índice de Gravidade de Doença , Centros de Atenção Terciária
3.
BMC Infect Dis ; 21(1): 325, 2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33827460

RESUMO

BACKGROUND: Rapid and simple serological assays for characterizing antibody responses are important in the current COVID-19 pandemic caused by SARS-CoV-2. Multiplex immunoblot (IB) assays termed COVID-19 IB assays were developed for detecting IgG and IgM antibodies to SARS-CoV-2 virus proteins in COVID-19 patients. METHODS: Recombinant nucleocapsid protein and the S1, S2 and receptor binding domain (RBD) of the spike protein of SARS-CoV-2 were used as target antigens in the COVID-19 IBs. Specificity of the IB assay was established with 231 sera from persons with allergy, unrelated viral infections, autoimmune conditions and suspected tick-borne diseases, and 32 goat antisera to human influenza proteins. IgG and IgM COVID-19 IBs assays were performed on 84 sera obtained at different times after a positive RT-qPCR test from 37 COVID-19 patients with mild symptoms. RESULTS: Criteria for determining overall IgG and IgM antibody positivity using the four SARS-CoV-2 proteins were developed by optimizing specificity and sensitivity in the COVID-19 IgG and IgM IB assays. The estimated sensitivities and specificities of the COVID-19 IgG and IgM IBs for IgG and IgM antibodies individually or for either IgG or IgM antibodies meet the US recommendations for laboratory serological diagnostic tests. The proportion of IgM-positive sera from the COVID-19 patients following an RT-qPCR positive test was maximal at 83% before 10 days and decreased to 0% after 100 days, while the proportions of IgG-positive sera tended to plateau between days 11 and 65 at 78-100% and fall to 44% after 100 days. Detection of either IgG or IgM antibodies was better than IgG or IgM alone for assessing seroconversion in COVID-19. Both IgG and IgM antibodies detected RBD less frequently than S1, S2 and N proteins. CONCLUSIONS: The multiplex COVID-19 IB assays offer many advantages for simultaneously evaluating antibody responses to different SARS-CoV-2 proteins in COVID-19 patients.


Assuntos
Anticorpos Antivirais/sangue , Formação de Anticorpos , /imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Humanos , Immunoblotting , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Pandemias , Fosfoproteínas/imunologia , Sensibilidade e Especificidade , Soroconversão , Testes Sorológicos
4.
BMC Bioinformatics ; 22(1): 182, 2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33832440

RESUMO

BACKGROUND: The rapid spread of the COVID-19 demands immediate response from the scientific communities. Appropriate countermeasures mean thoughtful and educated choice of viral targets (epitopes). There are several articles that discuss such choices in the SARS-CoV-2 proteome, other focus on phylogenetic traits and history of the Coronaviridae genome/proteome. However none consider viral protein low complexity regions (LCRs). Recently we created the first methods that are able to compare such fragments. RESULTS: We show that five low complexity regions (LCRs) in three proteins (nsp3, S and N) encoded by the SARS-CoV-2 genome are highly similar to regions from human proteome. As many as 21 predicted T-cell epitopes and 27 predicted B-cell epitopes overlap with the five SARS-CoV-2 LCRs similar to human proteins. Interestingly, replication proteins encoded in the central part of viral RNA are devoid of LCRs. CONCLUSIONS: Similarity of SARS-CoV-2 LCRs to human proteins may have implications on the ability of the virus to counteract immune defenses. The vaccine targeted LCRs may potentially be ineffective or alternatively lead to autoimmune diseases development. These findings are crucial to the process of selection of new epitopes for drugs or vaccines which should omit such regions.


Assuntos
Proteoma , Homologia de Sequência , /imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Fosfoproteínas/imunologia , Filogenia , Fatores de Risco , Glicoproteína da Espícula de Coronavírus/imunologia , Proteínas não Estruturais Virais/imunologia
5.
Emerg Microbes Infect ; 10(1): 774-781, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33830901

RESUMO

Monitoring the humoral protective immune response and its durability after SARS-CoV-2 infections is essential for risk assessment of reinfections, the improvement of diagnostic methods and the evaluation of vaccine trials. We have analyzed neutralizing antibodies and IgG responses specific to different antigens, including the inactivated whole-virion of SARS-CoV-2, the spike subunit 1 protein and its receptor binding domain, as well as the nucleocapsid protein. We show the dynamic developments of the responses from the early convalescent stages up to 9 months post symptoms onset in follow-up samples from 57 COVID-19 patients with varying clinical severity. By correlating antibody signals to neutralizing titres, valid diagnostic markers for the estimation of neutralizing protection could be identified.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , /virologia , Imunidade Humoral , /imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Estudos de Coortes , Feminino , Seguimentos , Alemanha , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Pandemias , Fosfoproteínas/imunologia , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologia , Fatores de Tempo , Vírion/imunologia , Adulto Jovem
6.
Viruses ; 13(2)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33672213

RESUMO

This study aimed to clarify whether infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is prevalent among the staff of a hospital providing treatment to patients with severe coronavirus disease 2019 (COVID-19) using radioligand assay (RLA). One thousand samples from the staff of a general hospital providing treatment to patients with severe COVID-19 were assayed for SARS-CoV-2 nucleocapsid protein (N) IgG using RLA. Nine patients with COVID-19 who had been treated in inpatient settings and had already recovered were used as control subjects, and 186 blood donor samples obtained more than 10 years ago were used as negative controls. Four of the 1000 samples showed apparently positive results, and approximately 10 or more samples showed slightly high counts. Interestingly, a few among the blood donor samples also showed slightly high values. To validate the results, antibody examinations using ELISA and neutralizing antibody tests were performed on 21 samples, and chemiluminescence immunoassay (CLIA) was performed on 201 samples, both resulting in a very high correlation. One blood donor sample showed slightly positive results in both RLA and CLIA, suggesting a cross-reaction. This study showed that five months after the pandemic began in Japan, the staff of a general hospital with a tertiary emergency medical facility had an extremely low seroprevalence of the antibodies against SARS-CoV-2. Further investigation will be needed to determine whether the slightly high results were due to cross-reactions or a low titer of anti-SARS-CoV-2 antibodies. The quantitative RLA was considered sensitive enough to detect low titers of antibodies.


Assuntos
Anticorpos Antivirais/sangue , Imunoglobulina G/sangue , Recursos Humanos em Hospital , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/sangue , /imunologia , Feminino , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Pandemias , Fosfoproteínas/imunologia , Prevalência , Estudos Soroepidemiológicos , Adulto Jovem
7.
Front Immunol ; 12: 631226, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33679778

RESUMO

Coronavirus disease-2019 (COVID-19) is a novel respiratory disease induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It remains poorly understood how the host immune system responds to the infection during disease progression. We applied microarray analysis of the whole genome transcriptome to peripheral blood mononuclear cells (PBMCs) taken from severe and mild COVID-19 patients as well as healthy controls. Functional enrichment analysis of genes associated with COVID-19 severity indicated that disease progression is featured by overactivation of myeloid cells and deficient T cell function. The upregulation of TLR6 and MMP9, which promote the neutrophils-mediated inflammatory response, and the downregulation of SKAP1 and LAG3, which regulate T cells function, were associated with disease severity. Importantly, the regulation of these four genes was absent in patients with influenza A (H1N1). And compared with stimulation with hemagglutinin (HA) of H1N1 virus, the regulation pattern of these genes was unique in PBMCs response to Spike protein of SARS-CoV-2 ex vivo. Our data also suggested that severe SARS-CoV-2 infection largely silenced the response of type I interferons (IFNs) and altered the proportion of immune cells, providing a potential mechanism for the hypercytokinemia. This study indicates that SARS-CoV-2 infection impairs inflammatory and immune signatures in patients, especially those at severe stage. The potential mechanisms underpinning severe COVID-19 progression include overactive myeloid cells, impaired function of T cells, and inadequate induction of type I IFNs signaling.


Assuntos
/imunologia , Leucócitos Mononucleares/imunologia , Transdução de Sinais/imunologia , Adolescente , Adulto , Idoso , Antígenos CD/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Interferon Tipo I/imunologia , Masculino , Metaloproteinase 9 da Matriz/imunologia , Pessoa de Meia-Idade , Fosfoproteínas/imunologia , Receptor 6 Toll-Like/imunologia
8.
PLoS One ; 16(3): e0247711, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33661990

RESUMO

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


Assuntos
Anticorpos Antivirais/sangue , /diagnóstico , Imunoglobulina G/sangue , Imunoglobulina M/sangue , /isolamento & purificação , Anticorpos Antivirais/imunologia , /epidemiologia , /imunologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Japão/epidemiologia , Medições Luminescentes/métodos , Fosfoproteínas/imunologia , Fosfoproteínas/isolamento & purificação , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/isolamento & purificação
9.
Sci Transl Med ; 13(590)2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33723016

RESUMO

Long-term immunological memory to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for the development of population-level immunity, which is the aim of vaccination approaches. Reports on rapidly decreasing antibody titers have led to questions regarding the efficacy of humoral immunity alone. The relevance of T cell memory after coronavirus disease 2019 (COVID-19) remains unclear. Here, we investigated SARS-CoV-2 antibody and T cell responses in matched samples of COVID-19 convalescent individuals up to 6 months after infection. Longitudinal analysis revealed decreasing and stable spike- and nucleocapsid-specific antibody responses, respectively. In contrast, functional T cell responses remained robust, and even increased, in both frequency and intensity. Single peptide mapping of T cell diversity over time identified open reading frame-independent, dominant T cell epitopes mediating long-term SARS-CoV-2 T cell responses. Identification of these epitopes may be fundamental for COVID-19 vaccine design.


Assuntos
/imunologia , Memória Imunológica , Linfócitos T/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Convalescença , Epitopos de Linfócito T/imunologia , Humanos , Epitopos Imunodominantes/imunologia , Cinética , Mapeamento de Peptídeos , Fosfoproteínas/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia
10.
Virology ; 558: 28-37, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33714753

RESUMO

To help fight COVID-19, new molecular tools specifically targeting critical components of the causative agent of COVID-19, SARS-Coronavirus-2 (SARS-CoV-2), are desperately needed. The SARS-CoV-2 nucleocapsid protein is critical for viral replication, integral to viral particle assembly, and a major diagnostic marker for infection and immune protection. Currently the limited available antibody reagents targeting the nucleocapsid protein are not specific to SARS-CoV-2 nucleocapsid protein, and sequences for these antibodies are not publicly available. In this work we developed and characterized a series of new mouse monoclonal antibodies against the SARS-CoV-2 nucleocapsid protein, with a specific clone, mBG86, targeting only SARS-CoV-2 nucleocapsid protein. The monoclonal antibodies were validated in ELISA, Western blot, and immunofluorescence analyses. The variable regions from six select clones were cloned and sequenced, and preliminary epitope mapping of the sequenced clones was performed. Overall, these new antibody reagents will be of significant value in the fight against COVID-19.


Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , /imunologia , /imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Clonagem Molecular , Escherichia coli , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fosfoproteínas/imunologia , Proteínas Recombinantes/imunologia
11.
Sci Rep ; 11(1): 6614, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758278

RESUMO

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


Assuntos
Antígenos/imunologia , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Anticorpos Antivirais/sangue , /imunologia , Feminino , Humanos , Imunoensaio , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/imunologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto Jovem
12.
J Med Microbiol ; 70(3)2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33734961

RESUMO

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


Assuntos
Anticorpos Antivirais/sangue , /imunologia , Adulto , Idoso , /imunologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Cinética , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/imunologia , RNA Viral/análise , /isolamento & purificação , Glicoproteína da Espícula de Coronavírus/imunologia
13.
EBioMedicine ; 65: 103259, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33662833

RESUMO

BACKGROUND: SARS-CoV-2 serology is used to identify prior infection at individual and at population level. Extended longitudinal studies with multi-timepoint sampling to evaluate dynamic changes in antibody levels are required to identify the time horizon in which these applications of serology are valid, and to explore the longevity of protective humoral immunity. METHODS: Healthcare workers were recruited to a prospective cohort study from the first SARS-CoV-2 epidemic peak in London, undergoing weekly symptom screen, viral PCR and blood sampling over 16-21 weeks. Serological analysis (n =12,990) was performed using semi-quantitative Euroimmun IgG to viral spike S1 domain and Roche total antibody to viral nucleocapsid protein (NP) assays. Comparisons were made to pseudovirus neutralizing antibody measurements. FINDINGS: A total of 157/729 (21.5%) participants developed positive SARS-CoV-2 serology by one or other assay, of whom 31.0% were asymptomatic and there were no deaths. Peak Euroimmun anti-S1 and Roche anti-NP measurements correlated (r = 0.57, p<0.0001) but only anti-S1 measurements correlated with near-contemporary pseudovirus neutralising antibody titres (measured at 16-18 weeks, r = 0.57, p<0.0001). By 21 weeks' follow-up, 31/143 (21.7%) anti-S1 and 6/150 (4.0%) anti-NP measurements reverted to negative. Mathematical modelling revealed faster clearance of anti-S1 compared to anti-NP (median half-life of 2.5 weeks versus 4.0 weeks), earlier transition to lower levels of antibody production (median of 8 versus 13 weeks), and greater reductions in relative antibody production rate after the transition (median of 35% versus 50%). INTERPRETATION: Mild SARS-CoV-2 infection is associated with heterogeneous serological responses in Euroimmun anti-S1 and Roche anti-NP assays. Anti-S1 responses showed faster rates of clearance, more rapid transition from high to low level production rate and greater reduction in production rate after this transition. In mild infection, anti-S1 serology alone may underestimate incident infections. The mechanisms that underpin faster clearance and lower rates of sustained anti-S1 production may impact on the longevity of humoral immunity. FUNDING: Charitable donations via Barts Charity, Wellcome Trust, NIHR.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , /imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Pessoal de Saúde/estatística & dados numéricos , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Fosfoproteínas/imunologia , Domínios Proteicos/imunologia
14.
Emerg Infect Dis ; 27(4): 1155-1158, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33734962

RESUMO

Prospective serosurveillance of severe acute respiratory syndrome coronavirus 2 in 1,069 healthcare workers in London, UK, demonstrated that nucleocapsid antibody titers were stable and sustained for <12 weeks in 312 seropositive participants. This finding was consistent across demographic and clinical variables and contrasts with reports of short-term antibody waning.


Assuntos
Anticorpos Antivirais/sangue , Formação de Anticorpos/imunologia , Pessoal de Saúde/estatística & dados numéricos , Adulto , /epidemiologia , /métodos , Feminino , Humanos , Londres/epidemiologia , Masculino , Fosfoproteínas/imunologia , Soroconversão , Estudos Soroepidemiológicos
15.
Nat Commun ; 12(1): 1152, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33608538

RESUMO

The humoral immune response to SARS-CoV-2 is a benchmark for immunity and detailed analysis is required to understand the manifestation and progression of COVID-19, monitor seroconversion within the general population, and support vaccine development. The majority of currently available commercial serological assays only quantify the SARS-CoV-2 antibody response against individual antigens, limiting our understanding of the immune response. To overcome this, we have developed a multiplex immunoassay (MultiCoV-Ab) including spike and nucleocapsid proteins of SARS-CoV-2 and the endemic human coronaviruses. Compared to three broadly used commercial in vitro diagnostic tests, our MultiCoV-Ab achieves a higher sensitivity and specificity when analyzing a well-characterized sample set of SARS-CoV-2 infected and uninfected individuals. We find a high response against endemic coronaviruses in our sample set, but no consistent cross-reactive IgG response patterns against SARS-CoV-2. Here we show a robust, high-content-enabled, antigen-saving multiplex assay suited to both monitoring vaccination studies and facilitating epidemiologic screenings for humoral immunity towards pandemic and endemic coronaviruses.


Assuntos
Anticorpos Antivirais/imunologia , /imunologia , Reações Cruzadas , Imunidade Humoral , /diagnóstico , Humanos , Imunoensaio , Imunoglobulina G/imunologia , Fosfoproteínas/imunologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/imunologia
16.
J Clin Lab Anal ; 35(4): e23735, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33608968

RESUMO

BACKGROUND: The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated a pandemic with alarming rates of fatality worldwide. This situation has had a major impact on clinical laboratories that have attempted to answer the urgent need for diagnostic tools, since the identification of coronavirus disease 2019 (COVID-19). Development of a reliable serological diagnostic immunoassay, with high levels of sensitivity and specificity to detect SARS-CoV-2 antibodies with improved differential diagnosis from other circulating viruses, is mandatory. METHODS: An enzyme-linked immunosorbent assay (ELISA) using whole inactivated virus cultured in vitro, was developed to detect viral antigens. WB and ELISA investigations were carried out with sera of convalescent patients and negative sera samples. Both analyses were concurrently performed with recombinant MABs to verify the findings. RESULTS: Preliminary data from 10 sera (5 patients with COVID-19, and 5 healthy controls) using this immunoassay are very promising, successfully identifying all of the confirmed SARS-CoV-2-positive individuals. CONCLUSION: This ELISA appears to be a specific and reliable method for detecting COVID-19 antibodies (IgG, IgM, and IgA), and a useful tool for identifying individuals which have developed immunity to the virus.


Assuntos
Antígenos Virais , /diagnóstico , Cultura de Vírus/métodos , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/química , Antígenos Virais/imunologia , Antígenos Virais/isolamento & purificação , Western Blotting , /virologia , Chlorocebus aethiops , /imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Fosfoproteínas/química , Fosfoproteínas/imunologia , Fosfoproteínas/isolamento & purificação , /imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Células Vero
17.
Virology ; 557: 15-22, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33582454

RESUMO

Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58-419 aa) was expressed in E. coli in soluble form, purified and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of ß-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively). Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection.


Assuntos
Anticorpos Antivirais/sangue , /imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/sangue , /imunologia , Sequência de Aminoácidos , /imunologia , Estudos de Casos e Controles , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática/normas , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Soros Imunes/química , Imunoglobulina M/sangue , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade
18.
ACS Synth Biol ; 10(2): 379-390, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33534552

RESUMO

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


Assuntos
/epidemiologia , /genética , Sondas Moleculares/genética , Pandemias , /imunologia , Anticorpos Antivirais/genética , Especificidade de Anticorpos/genética , Doenças Transmissíveis Emergentes/virologia , Escherichia coli/genética , Imunofluorescência , Genes Sintéticos , Genes Virais , Células HEK293 , Humanos , Sondas Moleculares/imunologia , Pandemias/prevenção & controle , Biblioteca de Peptídeos , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos de Domínio Único/genética , Biologia Sintética
19.
Commun Biol ; 4(1): 129, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514825

RESUMO

Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.


Assuntos
Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , /química , Glicoproteína da Espícula de Coronavírus/química , Adulto , /imunologia , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , /virologia , Convalescença , /metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Soros Imunes/química , Imunidade Humoral , Lentivirus/genética , Lentivirus/imunologia , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Fosfoproteínas/química , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Ligação Proteica , Receptores Virais/química , Receptores Virais/imunologia , Receptores Virais/metabolismo , /patogenicidade , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Análise de Sobrevida
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