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1.
Emerg Microbes Infect ; 9(1): 2105-2113, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32893735

RESUMO

The global pandemic of coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable neutralization assay is very important for the development of vaccines and novel drugs. In this study, a G protein-deficient vesicular stomatitis virus (VSVdG) bearing a truncated spike protein (S with C-terminal 18 amino acid truncation) was compared to that bearing the full-length spike protein of SARS-CoV-2 and showed much higher efficiency. A neutralization assay was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and hACE2-overexpressing BHK21 cells (BHK21-hACE2 cells). The experimental results can be obtained by automatically counting the number of EGFP-positive cells at 12 h after infection, making the assay convenient and high-throughput. The serum neutralizing titer measured by the VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with that measured by the wild type SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 S protein were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Testes de Neutralização/métodos , Pneumonia Viral/diagnóstico , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Pandemias , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia
2.
Emerg Microbes Infect ; 9(1): 2076-2090, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32897177

RESUMO

The current coronavirus disease 2019 (COVID-19) pandemic was the result of the rapid transmission of a highly pathogenic coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which there is no efficacious vaccine or therapeutic. Toward the development of a vaccine, here we expressed and evaluated as potential candidates four versions of the spike (S) protein using an insect cell expression system: receptor binding domain (RBD), S1 subunit, the wild-type S ectodomain (S-WT), and the prefusion trimer-stabilized form (S-2P). We showed that RBD appears as a monomer in solution, whereas S1, S-WT, and S-2P associate as homotrimers with substantial glycosylation. Cryo-electron microscopy analyses suggested that S-2P assumes an identical trimer conformation as the similarly engineered S protein expressed in 293 mammalian cells but with reduced glycosylation. Overall, the four proteins confer excellent antigenicity with convalescent COVID-19 patient sera in enzyme-linked immunosorbent assay (ELISA), yet show distinct reactivities in immunoblotting. RBD, S-WT and S-2P, but not S1, induce high neutralization titres (>3-log) in mice after a three-round immunization regimen. The high immunogenicity of S-2P could be maintained at the lowest dose (1 µg) with the inclusion of an aluminium adjuvant. Higher doses (20 µg) of S-2P can elicit high neutralization titres in non-human primates that exceed 40-times the mean titres measured in convalescent COVID-19 subjects. Our results suggest that the prefusion trimer-stabilized SARS-CoV-2 S-protein from insect cells may offer a potential candidate strategy for the development of a recombinant COVID-19 vaccine.


Assuntos
Antígenos Virais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Imunogenicidade da Vacina/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linhagem Celular , Infecções por Coronavirus/imunologia , Microscopia Crioeletrônica , Ensaio de Imunoadsorção Enzimática , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Peptidil Dipeptidase A/metabolismo , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Células Sf9 , Glicoproteína da Espícula de Coronavírus/genética , Spodoptera , Vacinação , Proteínas do Envelope Viral/imunologia
3.
Emerg Microbes Infect ; 9(1): 2091-2093, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32930052

RESUMO

We studied plasma antibody responses of 35 patients about 1 month after SARS-CoV-2 infection. Titers of antibodies binding to the viral nucleocapsid and spike proteins were significantly higher in patients with severe disease. Likewise, mean antibody neutralization titers against SARS-CoV-2 pseudovirus and live virus were higher in the sicker patients, by ∼5-fold and ∼7-fold, respectively. These findings have important implications for those pursuing plasma therapy, isolation of neutralizing monoclonal antibodies, and determinants of immunity.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Nucleocapsídeo/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Infecções por Coronavirus/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Pandemias , Pneumonia Viral/imunologia , Índice de Gravidade de Doença , Proteínas do Envelope Viral/imunologia
4.
Signal Transduct Target Ther ; 5(1): 180, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32879307

RESUMO

COVID-19 patients exhibit differential disease severity after SARS-CoV-2 infection. It is currently unknown as to the correlation between the magnitude of neutralizing antibody (NAb) responses and the disease severity in COVID-19 patients. In a cohort of 59 recovered patients with disease severity including severe, moderate, mild, and asymptomatic, we observed the positive correlation between serum neutralizing capacity and disease severity, in particular, the highest NAb capacity in sera from the patients with severe disease, while a lack of ability of asymptomatic patients to mount competent NAbs. Furthermore, the compositions of NAb subtypes were also different between recovered patients with severe symptoms and with mild-to-moderate symptoms. These results reveal the tremendous heterogeneity of SARS-CoV-2-specific NAb responses and their correlations to disease severity, highlighting the needs of future vaccination in COVID-19 patients recovered from asymptomatic or mild illness.


Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Adulto , Betacoronavirus/patogenicidade , Estudos de Casos e Controles , Convalescença , Infecções por Coronavirus/sangue , Infecções por Coronavirus/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/virologia , Índice de Gravidade de Doença
5.
JCI Insight ; 5(19)2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-32870820

RESUMO

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector-based assay.


Assuntos
Anticorpos Neutralizantes/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/terapia , Pneumonia Viral/imunologia , Pneumonia Viral/terapia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Antivirais/sangue , Área Sob a Curva , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização Passiva/métodos , Testes de Neutralização , Pandemias , Análise de Regressão , Amostragem , Resultado do Tratamento , Proteínas do Envelope Viral/imunologia
6.
Cell Mol Immunol ; 17(10): 1095-1097, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32895485
7.
Viruses ; 12(9)2020 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-32927639

RESUMO

The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on "pseudotyping" can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Lentivirus/genética , Testes de Neutralização/métodos , Pandemias , Pneumonia Viral/virologia , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Linhagem Celular , Infecções por Coronavirus/epidemiologia , Humanos , Soros Imunes/imunologia , Itália/epidemiologia , Plasmídeos/genética , Pneumonia Viral/epidemiologia , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/biossíntese , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/fisiologia , Transfecção , Vesiculovirus/genética , Carga Viral
8.
Artigo em Inglês | MEDLINE | ID: mdl-32738193

RESUMO

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, define correlates of immune protection, and down-select candidate antivirals. Here, we generate a highly infectious recombinant vesicular stomatitis virus (VSV) bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein and show that this recombinant virus, rVSV-SARS-CoV-2 S, closely resembles SARS-CoV-2 in its entry-related properties. The neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in a high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S, and neutralization of rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific therapeutics and for mechanistic studies of viral entry and its inhibition.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/fisiologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Animais , Antivirais/farmacologia , Betacoronavirus/genética , Betacoronavirus/fisiologia , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/terapia , Avaliação Pré-Clínica de Medicamentos , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Mutação , Testes de Neutralização , Pandemias/prevenção & controle , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/fisiologia , Pneumonia Viral/prevenção & controle , Pneumonia Viral/terapia , Receptores Virais/genética , Receptores Virais/fisiologia , Recombinação Genética , Serina Endopeptidases/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vacinas Virais/genética , Vacinas Virais/imunologia , Internalização do Vírus , Replicação Viral/genética
9.
Tidsskr Nor Laegeforen ; 140(11)2020 08 18.
Artigo em Inglês, Norueguês | MEDLINE | ID: mdl-32815342

RESUMO

BACKGROUND: Robust serological assays for SARS-CoV-2 are essential for determining prior infection and the suitability of donated convalescent plasma for plasma therapy. We compared two in-house and three commercial serological assays in a family cohort with SARS-CoV-2-infected members. CASE PRESENTATION: Three individuals in a family of five developed COVID-19 confirmed by PCR, following a trip abroad. Three to four weeks after the onset of symptoms, three commercial ELISAs and an in-house immunofluorescence test demonstrated antibodies to SARS-CoV-2. An in-house neutralisation test also demonstrated neutralising antibodies. INTERPRETATION: The in-house assays and one commercial assay gave congruent results, which were also consistent with the initial PCR and/or clinical evaluation, indicating prior infection in three of the five family members. The other commercial assays indicated possible infection in a fourth family member, but this result was likely due to cross-reactivity. The neutralising antibodies suggest complete or partial immunity against reinfection.


Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Anticorpos Neutralizantes/imunologia , Betacoronavirus , Ensaio de Imunoadsorção Enzimática , Saúde da Família , Imunofluorescência , Humanos , Testes de Neutralização , Pandemias , Testes Sorológicos
10.
Clin Microbiol Rev ; 33(4)2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32792417

RESUMO

Convalescent plasma (CP) therapy has been used since the early 1900s to treat emerging infectious diseases; its efficacy was later associated with the evidence that polyclonal neutralizing antibodies can reduce the duration of viremia. Recent large outbreaks of viral diseases for which effective antivirals or vaccines are still lacking has renewed the interest in CP as a life-saving treatment. The ongoing COVID-19 pandemic has led to the scaling up of CP therapy to unprecedented levels. Compared with historical usage, pathogen reduction technologies have now added an extra layer of safety to the use of CP, and new manufacturing approaches are being explored. This review summarizes historical settings of application, with a focus on betacoronaviruses, and surveys current approaches for donor selection and CP collection, pooling technologies, pathogen inactivation systems, and banking of CP. We additionally list the ongoing registered clinical trials for CP throughout the world and discuss the trial results published thus far.


Assuntos
Infecções por Coronavirus/terapia , Pneumonia Viral/terapia , Anticorpos Neutralizantes/análise , Bancos de Espécimes Biológicos/normas , Seleção do Doador/métodos , Seleção do Doador/normas , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização Passiva/efeitos adversos , Imunização Passiva/normas , Testes de Neutralização/normas , Pandemias , Síndrome Respiratória Aguda Grave/terapia
11.
J Infect Dis ; 222(8): 1280-1288, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32761124

RESUMO

BACKGROUND: Robust serological assays are essential for long-term control of the COVID-19 pandemic. Many recently released point-of-care (PoCT) serological assays have been distributed with little premarket validation. METHODS: Performance characteristics for 5 PoCT lateral flow devices approved for use in Australia were compared to a commercial enzyme immunoassay (ELISA) and a recently described novel surrogate virus neutralization test (sVNT). RESULTS: Sensitivities for PoCT ranged from 51.8% (95% confidence interval [CI], 43.1%-60.4%) to 67.9% (95% CI, 59.4%-75.6%), and specificities from 95.6% (95% CI, 89.2%-98.8%) to 100.0% (95% CI, 96.1%-100.0%). ELISA sensitivity for IgA or IgG detection was 67.9% (95% CI, 59.4%-75.6%), increasing to 93.8% (95% CI, 85.0%-98.3%) for samples >14 days post symptom onset. sVNT sensitivity was 60.9% (95% CI, 53.2%-68.4%), rising to 91.2% (95% CI, 81.8%-96.7%) for samples >14 days post symptom onset, with specificity 94.4% (95% CI, 89.2%-97.5%). CONCLUSIONS: Performance characteristics for COVID-19 serological assays were generally lower than those reported by manufacturers. Timing of specimen collection relative to onset of illness or infection is crucial in reporting of performance characteristics for COVID-19 serological assays. The optimal algorithm for implementing serological testing for COVID-19 remains to be determined, particularly in low-prevalence settings.


Assuntos
Infecções por Coronavirus/sangue , Pneumonia Viral/sangue , Algoritmos , Anticorpos Antivirais/sangue , Austrália/epidemiologia , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Testes de Neutralização/métodos , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Prevalência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Testes Sorológicos/métodos , Testes Sorológicos/normas
12.
Cell Host Microbe ; 28(3): 475-485.e5, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32735849

RESUMO

Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, which engages with host ACE2 receptor for entry. Using an infectious molecular clone of vesicular stomatitis virus (VSV) expressing eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput-imaging-based neutralization assay at biosafety level 2. We also developed a focus-reduction neutralization test with a clinical isolate of SARS-CoV-2 at biosafety level 3. Comparing the neutralizing activities of various antibodies and ACE2-Fc soluble decoy protein in both assays revealed a high degree of concordance. These assays will help define correlates of protection for antibody-based countermeasures and vaccines against SARS-CoV-2. Additionally, replication-competent VSV-eGFP-SARS-CoV-2 provides a tool for testing inhibitors of SARS-CoV-2 mediated entry under reduced biosafety containment.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/terapia , Peptidil Dipeptidase A/imunologia , Pneumonia Viral/terapia , Animais , Betacoronavirus/genética , Betacoronavirus/fisiologia , Chlorocebus aethiops , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Proteínas de Fluorescência Verde/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Imunização Passiva , Testes de Neutralização , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia , Internalização do Vírus , Replicação Viral
13.
Emerg Microbes Infect ; 9(1): 1965-1973, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32819220

RESUMO

Serology is a crucial part of the public health response to the ongoing SARS-CoV-2 pandemic. Here, we describe the development, validation and clinical evaluation of a protein micro-array as a quantitative multiplex immunoassay that can identify S and N-directed SARS-CoV-2 IgG antibodies with high specificity and sensitivity and distinguish them from all currently circulating human coronaviruses. The method specificity was 100% for SARS-CoV-2 S1 and 96% for N antigen based on extensive syndromic (n=230 cases) and population panel (n=94) testing that also confirmed the high prevalence of seasonal human coronaviruses. To assess its potential role for both SARS-CoV-2 patient diagnostics and population studies, we evaluated a large heterogeneous COVID-19 cohort (n=330) and found an overall sensitivity of 89% (≥ 21 days post onset symptoms (dps)), ranging from 86% to 96% depending on severity of disease. For a subset of these patients longitudinal samples were provided up to 56 dps. Mild cases showed absent or delayed, and lower SARS-CoV-2 antibody responses. Overall, we present the development and extensive clinical validation of a multiplex coronavirus serological assay for syndromic testing, to answer research questions regarding to antibody responses, to support SARS-CoV-2 diagnostics and to evaluate epidemiological developments efficiently and with high-throughput.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Proteínas do Nucleocapsídeo/sangue , Pneumonia Viral/diagnóstico , Glicoproteína da Espícula de Coronavírus/sangue , Idoso , Antígenos Virais/sangue , Antígenos Virais/imunologia , Betacoronavirus/patogenicidade , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/mortalidade , Infecções por Coronavirus/virologia , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Testes de Neutralização , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/mortalidade , Pneumonia Viral/virologia , Análise Serial de Proteínas , Vírus da SARS/imunologia , Vírus da SARS/patogenicidade , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologia
14.
Indian J Med Res ; 152(1 & 2): 82-87, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32859866

RESUMO

Background & objectives: The global pandemic caused by SARS-CoV-2 virus has challenged public health system worldwide due to the unavailability of approved preventive and therapeutic options. Identification of neutralizing antibodies (NAb) and understanding their role is important. However, the data on kinetics of NAb response among COVID-19 patients are unclear. To understand the NAb response in COVID-19 patients, we compared the findings of microneutralization test (MNT) and plaque reduction neutralization test (PRNT) for the SARS-CoV-2. Further, the kinetics of NAb response among COVID-19 patients was assessed. Methods: A total of 343 blood samples (89 positive, 58 negative for SARS-CoV-2 and 17 cross-reactive and 179 serum from healthy individuals) were collected and tested by MNT and PRNT. SARS-CoV-2 virus was prepared by propagating the virus in Vero CCL-81 cells. The intra-class correlation was calculated to assess the correlation between MNT and PRNT. The neutralizing endpoint as the reduction in the number of plaque count by 90 per cent (PRNT90) was also calculated. Results: The analysis of MNT and PRNT quantitative results indicated that the intra-class correlation was 0.520. Of the 89 confirmed COVID-19 patients, 64 (71.9%) showed NAb response. Interpretation & conclusions: The results of MNT and PRNT were specific with no cross-reactivity. In the early stages of infection, the NAb response was observed with variable antibody kinetics. The neutralization assays can be used for titration of NAb in recovered/vaccinated or infected COVID-19 patients.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Infecções por Coronavirus/sangue , Testes de Neutralização , Pandemias , Pneumonia Viral/sangue , Adolescente , Adulto , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Criança , Chlorocebus aethiops/imunologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Células Vero/imunologia , Adulto Jovem
15.
Sci Transl Med ; 12(559)2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32817357

RESUMO

It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their differing antibody response profiles. Here, we performed a pilot study of four serological assays to assess the amounts of anti-SARS-CoV-2 antibodies in serum samples obtained from 491 healthy individuals before the SARS-CoV-2 pandemic, 51 individuals hospitalized with COVID-19, 209 suspected cases of COVID-19 with mild symptoms, and 200 healthy blood donors. We used two ELISA assays that recognized the full-length nucleoprotein (N) or trimeric spike (S) protein ectodomain of SARS-CoV-2. In addition, we developed the S-Flow assay that recognized the S protein expressed at the cell surface using flow cytometry, and the luciferase immunoprecipitation system (LIPS) assay that recognized diverse SARS-CoV-2 antigens including the S1 domain and the carboxyl-terminal domain of N by immunoprecipitation. We obtained similar results with the four serological assays. Differences in sensitivity were attributed to the technique and the antigen used. High anti-SARS-CoV-2 antibody titers were associated with neutralization activity, which was assessed using infectious SARS-CoV-2 or lentiviral-S pseudotype virus. In hospitalized patients with COVID-19, seroconversion and virus neutralization occurred between 5 and 14 days after symptom onset, confirming previous studies. Seropositivity was detected in 32% of mildly symptomatic individuals within 15 days of symptom onset and in 3% of healthy blood donors. The four antibody assays that we used enabled a broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in different subpopulations within one region.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Citometria de Fluxo/métodos , França/epidemiologia , Voluntários Saudáveis , Humanos , Imunoprecipitação/métodos , Luciferases , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/imunologia , Pesquisa Médica Translacional , Adulto Jovem
16.
Viruses ; 12(7)2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32646015

RESUMO

Standard precautions to minimize the risk of SARS-CoV-2 transmission implies that infected cell cultures and clinical specimens may undergo some sort of inactivation to reduce or abolish infectivity. We evaluated three heat inactivation protocols (56 °C-30 min, 60 °C-60 min and 92 °C-15 min) on SARS-CoV-2 using (i) infected cell culture supernatant, (ii) virus-spiked human sera (iii) and nasopharyngeal samples according to the recommendations of the European norm NF EN 14476-A2. Regardless of the protocol and the type of samples, a 4 Log10 TCID50 reduction was observed. However, samples containing viral loads > 6 Log10 TCID50 were still infectious after 56 °C-30 min and 60 °C-60 min, although infectivity was < 10 TCID50. The protocols 56 °C-30 min and 60 °C-60 min had little influence on the RNA copies detection, whereas 92 °C-15 min drastically reduced the limit of detection, which suggests that this protocol should be avoided for inactivation ahead of molecular diagnostics. Lastly, 56 °C-30 min treatment of serum specimens had a negligible influence on the results of IgG detection using a commercial ELISA test, whereas a drastic decrease in neutralizing titers was observed.


Assuntos
Betacoronavirus , Contenção de Riscos Biológicos/métodos , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Testes Sorológicos/métodos , Inativação de Vírus , Anticorpos Neutralizantes/imunologia , Betacoronavirus/imunologia , Contenção de Riscos Biológicos/normas , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Temperatura Alta , Humanos , Testes de Neutralização , Pandemias/prevenção & controle , Pneumonia Viral/diagnóstico , Pneumonia Viral/prevenção & controle , Testes Sorológicos/normas
17.
J Infect ; 81(3): e31-e32, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32622905

RESUMO

We followed-up a mild COVID-19 patient for 91 days and serially monitored his serum antibodies to four SARS-CoV-2 related antigens (NP, RBD, S1 and ECD) and neutralization activities. Our data revealed a profile of serial antibody responses during the progress and a quick decline of neutralization activities after discharge.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Adulto , Antígenos Virais/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Estudos Longitudinais , Masculino , Testes de Neutralização , Pandemias , RNA Viral , Estudos Retrospectivos
18.
J Clin Virol ; 129: 104539, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32679298

RESUMO

BACKGROUND: Serological tests for anti-SARS-CoV-2 antibodies are becoming of great interest to determine seroprevalence in a given population, define previous exposure and identify highly reactive human donors for the generation of convalescent serum as therapeutic. OBJECTIVES: We evaluated the diagnostic performance of the Abbott ARCHITECT SARS-CoV-2 IgG test, a fully automated indirect immunoassay that detects antibodies directed to a recombinant SARS-CoV-2 Nucleocapsid antigen. STUDY DESIGN: Abbott ARCHITECT SARS-CoV-2 IgG immunoassay was compared to an indirect immunofluorescence assay (IFA) on sera from patients with COVID-19 collected at different days after symptoms onset or infected by other human coronaviruses. Comparison with neutralization test was also performed. RESULTS: After 7, 14 and >14 days after onset ARCHITECT was positive on 8.3 %; 61.9 % and 100 % of the tested samples compared to 58.3 %; 85.7 % and 100 % by IFA. The sensitivity was 72 % vs. IFA and 66.7 % vs. a real-time PCR, the specificity was 100 %. On 18 samples with neutralizing activity, 17 were positive by Abbott ARCHITECT SARS-CoV-2 IgG. CONCLUSIONS: In our study, Abbott ARCHITECT SARS-CoV-2 IgG assay showed a satisfactory performance, with a very high specificity. IgG reactivity against SARSCoV-2 N antigen was detectable in all patients by two weeks after symptoms onset. In addition, concordance between this serological response and viral neutralization suggests that a strong humoral response may be predictive of a neutralization activity, regardless of the target antigens. This finding supports the use of this automated serological assay in diagnostic algorithm and public health intervention, especially for high loads of testing.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Técnica Indireta de Fluorescência para Anticorpo/métodos , Testes de Neutralização/métodos , Pneumonia Viral/diagnóstico , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/sangue , Criança , Pré-Escolar , Feminino , Humanos , Imunoglobulina G/sangue , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Pandemias , Sensibilidade e Especificidade , Adulto Jovem
20.
Medicina (B Aires) ; 80 Suppl 3: 1-6, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658841

RESUMO

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab')2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.


Assuntos
Anticorpos Antivirais , Infecções por Coronavirus/terapia , Soros Imunes/imunologia , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/isolamento & purificação , Pandemias , Pneumonia Viral , Glicoproteína da Espícula de Coronavírus , Animais , Anticorpos Antivirais/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/isolamento & purificação , Argentina , Betacoronavirus , Cavalos , Humanos , Imunização Passiva , Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/química , Testes de Neutralização
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