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1.
Sci Rep ; 11(1): 5538, 2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33692390

RESUMO

Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Recent studies showed that serum from convalescent patients can display variable neutralization capacities. Still, it remains unclear whether there are specific signatures that can be used to predict neutralization. Here, we performed a detailed analysis of sera from a cohort of 101 recovered healthcare workers and we addressed their SARS-CoV-2 antibody response by ELISA against SARS-CoV-2 Spike receptor binding domain and nucleoprotein. Both ELISA methods detected sustained levels of serum IgG against both antigens. Yet, the majority of individuals from our cohort generated antibodies with low neutralization capacity and only 6% showed high neutralizing titers against both authentic SARS-CoV-2 virus and the Spike pseudotyped virus. Interestingly, higher neutralizing sera correlate with detection of -IgG, IgM and IgA antibodies against both antigens, while individuals with positive IgG alone showed poor neutralization response. These results suggest that having a broader repertoire of antibodies may contribute to more potent SARS-CoV-2 neutralization. Altogether, our work provides a cross sectional snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides preliminary evidence that possessing multiple antibody isotypes can play an important role in predicting SARS-CoV-2 neutralization.


Assuntos
Anticorpos Neutralizantes/imunologia , /imunologia , Adulto , Anticorpos Antivirais/imunologia , Estudos de Coortes , Estudos Transversais , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/imunologia , Feminino , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Masculino , Testes de Neutralização/métodos , Pandemias , Soro/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia
2.
PLoS One ; 16(3): e0248348, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33690649

RESUMO

Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.


Assuntos
/metabolismo , Lentivirus/metabolismo , Testes de Neutralização/métodos , /genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293 , Humanos , /patogenicidade , Glicoproteína da Espícula de Coronavírus/genética
3.
Cell Rep ; 34(12): 108890, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33713594

RESUMO

Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines show protective efficacy, which is most likely mediated by neutralizing antibodies recognizing the viral entry protein, spike. Because new SARS-CoV-2 variants are emerging rapidly, as exemplified by the B.1.1.7, B.1.351, and P.1 lineages, it is critical to understand whether antibody responses induced by infection with the original SARS-CoV-2 virus or current vaccines remain effective. In this study, we evaluate neutralization of a series of mutated spike pseudotypes based on divergence from SARS-CoV and then compare neutralization of the B.1.1.7 spike pseudotype and individual mutations. Spike-specific monoclonal antibody neutralization is reduced dramatically; in contrast, polyclonal antibodies from individuals infected in early 2020 remain active against most mutated spike pseudotypes, but potency is reduced in a minority of samples. This work highlights that changes in SARS-CoV-2 spike can alter neutralization sensitivity and underlines the need for effective real-time monitoring of emerging mutations and their effect on vaccine efficacy.


Assuntos
Anticorpos Neutralizantes/imunologia , /genética , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Formação de Anticorpos , /metabolismo , Células HEK293 , Humanos , Testes de Neutralização/métodos , Mutação Puntual , Receptores Virais/genética , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia
4.
mSphere ; 6(1)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627511

RESUMO

The majority of infections with SARS-CoV-2 are asymptomatic or mild without the necessity of hospitalization. It is of importance to reveal if these patients develop an antibody response against SARS-CoV-2 and to define which antibodies confer virus neutralization. We conducted a comprehensive serological survey of 49 patients with a mild course of disease and quantified neutralizing antibody responses against a clinical SARS-CoV-2 isolate employing human cells as targets. Four patients (8%), even though symptomatic, did not develop antibodies against SARS-CoV-2, and two other patients (4%) were positive in only one of the six serological assays employed. For the remaining 88%, antibody response against the S protein correlated with serum neutralization whereas antibodies against the nucleocapsid were poor predictors of virus neutralization. None of the sera enhanced infection of human cells with SARS-CoV-2 at any dilution, arguing against antibody-dependent enhancement of infection in our system. Regarding neutralization, only six patients (12%) could be classified as high neutralizers. Furthermore, sera from several individuals with fairly high antibody levels had only poor neutralizing activity. In addition, employing a novel serological Western blot system to characterize antibody responses against seasonal coronaviruses, we found that antibodies against the seasonal coronavirus 229E might contribute to SARS-CoV-2 neutralization. Altogether, we show that there is a wide breadth of antibody responses against SARS-CoV-2 in patients that differentially correlate with virus neutralization. This highlights the difficulty to define reliable surrogate markers for immunity against SARS-CoV-2.IMPORTANCE There is strong interest in the nature of the neutralizing antibody response against SARS-CoV-2 in infected individuals. For vaccine development, it is especially important which antibodies confer protection against SARS-CoV-2, if there is a phenomenon called antibody-dependent enhancement (ADE) of infection, and if there is cross-protection by antibodies directed against seasonal coronaviruses. We addressed these questions and found in accordance with other studies that neutralization is mediated mainly by antibodies directed against the spike protein of SARS-CoV-2 in general and the receptor binding site in particular. In our test system, utilizing human cells for infection experiments, we did not detect ADE. However, using a novel diagnostic test we found that antibodies against the coronavirus 229E might be involved in cross-protection to SARS-CoV-2.


Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Infecções por Coronavirus/imunologia , /imunologia , Adulto , Anticorpos Neutralizantes/imunologia , Anticorpos Facilitadores/imunologia , Sítios de Ligação/imunologia , Feminino , Hospitalização , Humanos , Masculino , Testes de Neutralização/métodos , Nucleocapsídeo/imunologia , Estações do Ano , Testes Sorológicos/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Inquéritos e Questionários , Vacinas/imunologia
5.
Cell Host Microbe ; 29(3): 477-488.e4, 2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33535027

RESUMO

Neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of COVID-19 vaccines and have received emergency use authorization as therapeutics. However, viral escape mutants could compromise efficacy. To define immune-selected mutations in the S protein, we exposed a VSV-eGFP-SARS-CoV-2-S chimeric virus, in which the VSV glycoprotein is replaced with the S protein, to 19 neutralizing monoclonal antibodies (mAbs) against the receptor-binding domain (RBD) and generated 50 different escape mutants. Each mAb had a unique resistance profile, although many shared residues within an epitope of the RBD. Some variants (e.g., S477N) were resistant to neutralization by multiple mAbs, whereas others (e.g., E484K) escaped neutralization by convalescent sera. Additionally, sequential selection identified mutants that escape neutralization by antibody cocktails. Comparing these antibody-mediated mutations with sequence variation in circulating SARS-CoV-2 revealed substitutions that may attenuate neutralizing immune responses in some humans and thus warrant further investigation.


Assuntos
Anticorpos Monoclonais/sangue , Anticorpos Antivirais/sangue , Mutação , Testes de Neutralização/métodos , Glicoproteína da Espícula de Coronavírus/genética , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , /imunologia , Chlorocebus aethiops , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Ligação Proteica , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Células Vero
6.
PLoS One ; 16(2): e0246864, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33577615

RESUMO

BACKGROUND: The presence of neutralizing antibodies (NAbs) is an indicator of protective immunity for most viral infections. A newly developed surrogate viral neutralization assay (sVNT) offers the ability to detect total receptor binding domain-targeting NAbs in an isotype-independent manner, increasing the test sensitivity. Thus, specimens with low IgM/ IgG antibody levels showed strong neutralization activity in sVNT. METHODS: This study aimed to measure the %inhibition of NAbs measured by sVNT in PCR-confirmed COVID-19 patients. The sensitivity of sVNT for the diagnosis of SARS-CoV-2 infection and its kinetics were determined. RESULTS: Ninety-seven patients with PCR-confirmed SARS-CoV-2 infection were included in this study. Majority of the patients were 21-40 years old (67%) and 63% had mild symptoms. The sensitivity of sVNT for the diagnosis of SARS-CoV-2 infection was 99% (95% confidence interval (CI) 94.4-100%) and the specificity was 100% (95% CI 98.3-100%). The negative predictive value of sVNT from the samples collected before and after 7 days of symptom onset was 99.5% (95% CI 97.4-100%) and 100% (95% CI 93.8-100%), respectively. The level of inhibition at days 8-14 were significantly higher than days 0-7 (p<0.001). The median %inhibition values by severity of COVID-19 symptoms were 79.9% (interquartile range (IQR) 49.7-91.8%); 89.0% (IQR 71.2-92.4%); and 86.6% (IQR 69.5-92.8%), for mild, moderate and severe/critical symptoms respectively. The median level of sVNT %inhibition of severe was significantly higher than the mild group (p = 0.05). CONCLUSION: The sVNT is a practical and robust serological test for SARS-CoV-2 infection and does not require specialized biosafety containment. It can be used clinically to aid diagnosis in both early and late infection especially in cases when the real-time RT-PCR results in weakly negative or weakly positive, and to determine the protective immune response from SARS-CoV-2 infection in patients.


Assuntos
Anticorpos Neutralizantes/isolamento & purificação , /diagnóstico , Testes de Neutralização/métodos , /fisiologia , Adulto , Anticorpos Neutralizantes/análise , Anticorpos Antivirais/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Testes Sorológicos , Glicoproteína da Espícula de Coronavírus/química , Tailândia , Adulto Jovem
7.
Diagn Microbiol Infect Dis ; 99(4): 115294, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33387896

RESUMO

There remains an urgent need for assays to quantify humoral protective immunity to SARS-CoV-2 to understand the immune responses of COVID-19 patients, evaluate efficacy of vaccine candidates in clinical trials, and conduct large-scale epidemiological studies. The plaque-reduction neutralization test (PRNT) is the reference-standard for quantifying antibodies capable of neutralizing SARS-CoV-2. However, the PRNT is logistically demanding, time-consuming, and requires containment level-3 facilities to safely work with live virus. In contrast, a surrogate virus neutralization test (sVNT) manufactured by Genscript is a quick and simple assay that detects antibodies that inhibit the RBD-ACE2 interaction, crucial for virus entry into host cells. In this study, we evaluate the sensitivity, specificity, and cross-reactivity of the sVNT compared with the PRNT using both 50% and 90% SARS-CoV-2 neutralization as a reference-standard. We found that the sVNT provides a high-throughput screening tool prior to confirmatory PRNT testing for the evaluation of SARS-CoV-2 neutralizing antibodies.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Ensaio de Placa Viral/métodos , /metabolismo , Ensaios de Triagem em Larga Escala/métodos , Humanos , Testes de Neutralização/métodos
8.
BMC Vet Res ; 17(1): 51, 2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33494765

RESUMO

BACKGROUND: Infectious bronchitis virus (IBV), a coronavirus, is one of the most important poultry pathogens worldwide due to its multiple serotypes and poor cross-protection. Vaccination plays a vital role in controlling the disease. The efficacy of vaccination in chicken flocks can be evaluated by detecting neutralizing antibodies with the neutralization test. However there are no simple and rapid methods for detecting the neutralizing antibodies. RESULTS: In this study, a peptide enzyme-linked immunosorbent assay (pELISA) as a possible alternative to the neutralization test for evaluating the immune response to IBV vaccine was developed. The pELISA could indirect evaluate neutralizing antibody titers against different types of IBV in all tested sera. The titers measured with the pELISA had a coefficient of 0.83 for neutralizing antibody titers. CONCLUSIONS: The pELISA could detect antibodies against different types of IBV in all tested sera. The pELISA has the potential to evaluate samples for IBV-specific neutralizing antibodies and surveillance the infection of IBV.


Assuntos
Infecções por Coronavirus/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Vírus da Bronquite Infecciosa/imunologia , Testes de Neutralização , Doenças das Aves Domésticas/prevenção & controle , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Galinhas/imunologia , Galinhas/virologia , Infecções por Coronavirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Testes de Neutralização/métodos , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
9.
Virol J ; 18(1): 16, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33435994

RESUMO

BACKGROUND: SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is now classified in the genus Coronavirus with closely related SARS-CoV. SARS-CoV-2 is highly pathogenic in humans and is classified as a biosafety level (BSL)-3 pathogen, which makes manipulating it relatively difficult due to its infectious nature. METHODS: To circumvent the need for BSL-3 laboratories, an alternative assay was developed that avoids live virus and instead uses a recombinant VSV expressing luciferase and possesses the full length or truncated spike proteins of SARS-CoV-2. Furthermore, to measure SARS-CoV-2 neutralizing antibodies under BSL2 conditions, a chemiluminescence reduction neutralization test (CRNT) for SARS-CoV-2 was developed. The neutralization values of the serum samples collected from hospitalized patients with COVID-19 or SARS-CoV-2 PCR-negative donors against the pseudotyped virus infection evaluated by the CRNT were compared with antibody titers determined from an enzyme-linked immunosorbent assay (ELISA) or an immunofluorescence assay (IFA). RESULTS: The CRNT, which used whole blood collected from hospitalized patients with COVID-19, was also examined. As a result, the inhibition of pseudotyped virus infection was specifically observed in both serum and whole blood and was also correlated with the results of the IFA. CONCLUSIONS: In conclusion, the CRNT for COVID-19 is a convenient assay system that can be performed in a BSL-2 laboratory with high specificity and sensitivity for evaluating the occurrence of neutralizing antibodies against SARS-CoV-2.


Assuntos
Anticorpos Neutralizantes/sangue , /sangue , Testes de Neutralização/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Vírus da Estomatite Vesicular Indiana/genética , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linhagem Celular , Convalescença , Humanos , Concentração Inibidora 50 , Luminescência , Glicoproteína da Espícula de Coronavírus/genética
10.
Virol J ; 18(1): 1, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397387

RESUMO

BACKGROUND: Virus neutralization by antibodies is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal murine leukemia virus genome encoding firefly luciferase. This assay design is intended for use in laboratories with biocontainment level 2 and therefore circumvents the need for the biocontainment level 3 that would be required for replication-competent SARS-CoV-2 virus. To validate the pseudovirion assay, we set up comparisons with other available antibody tests including those from Abbott, Euroimmun and Siemens, using archived, known samples. RESULTS: 11 out of 12 SARS-CoV-2-infected patient serum samples showed neutralizing activity against SARS-CoV-2-spike pseudotyped MLV viruses, with neutralizing titers-50 (NT50) that ranged from 1:25 to 1:1,417. Five historical samples from patients hospitalized for severe influenza infection in 2016 tested negative in the neutralization assay (NT50 < 25). Three serum samples with high neutralizing activity against SARS-CoV-2/MLV pseudoviruses showed no detectable neutralizing activity (NT50 < 25) against SARS-CoV-1/MLV pseudovirions. We also compared the semiquantitative Siemens SARS-CoV-2 IgG test, which measures binding of IgG to recombinantly expressed receptor binding domain of SARS-CoV-2 spike glycoprotein with the neutralization titers obtained in the pseudovirion assay and the results show high concordance between the two tests (R2 = 0.9344). CONCLUSIONS: SARS-CoV-2 spike/MLV pseudovirions provide a practical means of assessing neutralizing activity of antibodies in serum or plasma from infected patients under laboratory conditions consistent with biocontainment level 2. This assay offers promise also in evaluating immunogenicity of spike glycoprotein-based candidate vaccines in the near future.


Assuntos
/imunologia , Leucemia/imunologia , Testes de Neutralização/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Vírion/imunologia , /imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Células HEK293 , Humanos , Imunoglobulina G/sangue , Camundongos
11.
Viruses ; 13(1)2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33440618

RESUMO

Toscana phlebovirus (TOSV) and Sicilian phlebovirus (SFSV) are endemic in the Mediterranean area where they are transmitted to humans by infected sandflies. Vertebrates of several species have been postulated to act as reservoirs of these viruses, but convincing evidence is still awaited. Among them, bats have been suggested, however documented evidence is lacking. Here we tested a total of 329 bats belonging to eight species collected from twelve localities in southern Spain for the presence of neutralizing antibodies specific to TOSV and SFSV. Positive sera were detected in Schreiber's long-fingered bat (Miniopterus schreibersii), mouse-eared Myotis (Myotis myotis), European free-tailed bat (Tadarida teniotis), and common serotine (Eptesicus serotinus) with the latter showing the highest prevalence rates for SFSV (22.6%) and TOSV (10%). There was no difference between females and males. Results suggest that bats are not likely to play a major role in the natural cycle of these two sandfly-borne phleboviruses. However, large breeding colonies of bats can be used as sentinels for surveillance of the presence of such viruses in a given locality. In addition, capture-recapture studies should be initiated in order to understand better the dynamics of TOSV and SFSV in bat populations.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Quirópteros/imunologia , Quirópteros/virologia , Testes de Neutralização , Febre por Flebótomos/epidemiologia , Febre por Flebótomos/transmissão , Vírus da Febre do Flebótomo Napolitano/imunologia , Animais , Geografia , Testes de Neutralização/métodos , Febre por Flebótomos/imunologia , Vírus da Febre do Flebótomo Napolitano/isolamento & purificação , Estudos Soroepidemiológicos , Espanha/epidemiologia
12.
Sci Rep ; 11(1): 2062, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33479465

RESUMO

In order to support vaccine development, and to aid convalescent plasma therapy, it would be important to understand the kinetics, timing and persistence of SARS-CoV-2 neutralizing antibodies (NAbs), and their association with clinical disease severity. Therefore, we used a surrogate viral neutralization test to evaluate their levels in patients with varying severity of illness, in those with prolonged shedding and those with mild/asymptomatic illness at various time points. Patients with severe or moderate COVID-19 illness had earlier appearance of NAbs at higher levels compared to those with mild or asymptomatic illness. Furthermore, those who had prolonged shedding of the virus, had NAbs appearing faster and at higher levels than those who cleared the virus earlier. During the first week of illness the NAb levels of those with mild illness was significantly less (p = 0.01), compared to those with moderate and severe illness. At the end of 4 weeks (28 days), although 89% had NAbs, 38/76 (50%) in those with > 90 days had a negative result for the presence of NAbs. The Ab levels significantly declined during convalescence (> 90 days since onset of illness), compared to 4 to 8 weeks since onset of illness. Our data show that high levels of NAbs during early illness associated with clinical disease severity and that these antibodies declined in 50% of individuals after 3 months since onset of illness.


Assuntos
Anticorpos Neutralizantes/imunologia , /imunologia , Imunidade Adaptativa/imunologia , Adulto , Anticorpos Neutralizantes/análise , Anticorpos Antivirais/imunologia , /terapia , Convalescença , Feminino , Humanos , Imunização Passiva/métodos , Masculino , Pessoa de Meia-Idade , Testes de Neutralização/métodos , Índice de Gravidade de Doença , Sri Lanka/epidemiologia
13.
J Virol Methods ; 287: 113995, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33068703

RESUMO

Towards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus (SARS-CoV), emerged in Wuhan, Hubei province of China, and has been responsible for coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals licensed against SARS-CoV-2. The high economical and health impacts of SARS-CoV-2 has placed global pressure on the scientific community to identify effective prophylactic and therapeutic treatments for SARS-CoV-2 infection and associated COVID-19 disease. While some compounds have been already reported to reduce SARS-CoV-2 infection and a handful of monoclonal antibodies (mAbs) have been described that neutralize SARS-CoV-2, there is an urgent need for the development and standardization of assays which can be used in high through-put screening (HTS) settings to identify new antivirals and/or neutralizing mAbs against SARS-CoV-2. Here, we described a rapid, accurate, and highly reproducible plaque reduction microneutralization (PRMNT) assay that can be quickly adapted for the identification and characterization of both neutralizing mAbs and antivirals against SARS-CoV-2. Importantly, our MNA is compatible with HTS settings to interrogate large and/or complex libraries of mAbs and/or antivirals to identify those with neutralizing and/or antiviral activity, respectively, against SARS-CoV-2.


Assuntos
Anticorpos Neutralizantes/imunologia , Antivirais/farmacologia , Testes de Neutralização/métodos , /imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Ensaios de Triagem em Larga Escala , Humanos , Células Vero , Ensaio de Placa Viral , Replicação Viral/efeitos dos fármacos
14.
Rev. esp. quimioter ; 33(6): 392-398, dic. 2020. tab
Artigo em Espanhol | IBECS | ID: ibc-195990

RESUMO

Los coronavirus (CoVs) son un amplio grupo de virus en el que se encuentra el SARS-CoV-2 (familia Coronaviridae, subfamilia Coronavirinae, género Betacoronavirus y subgénero Sarbecovirus). Sus principales proteínas estructurales son la de membrana (M), la de envoltura (E), la nucleocápside (N) y la espicular (S). La respuesta inmune frente a SARS-CoV-2 implica la vertiente celular y humoral, con anticuerpos neutralizantes potencialmente defensivos fundamentalmente dirigidos frente al antígeno S. Aunque los datos de seroprevalencia se asumen muy frecuentemente como marcadores de protección, no necesariamentelo son. En España se estima que al menos cuatro quintas partes de la población deberían estar inmuno-protegidas para asegurar la inmunidad de grupo. Dada la alta tasa de letalidad por COVID-19, la adquisición de esta protección únicamente mediante el contagio natural no es asumible y se debe abogar por otras medidas como puede ser la inmunización masiva. En la actualidad existen varios prototipos de vacunas (que incluyen virus vivos, vectores virológicos, péptidos y proteínas y ácidos nucleicos) en diversas etapas de evaluación clínica. Se prevé que en breve alguna de estas nuevas vacunas se encuentre ya disponible en el mercado. En este texto se revisan aspectos relacionados con estos asuntos


The coronavirus are a wide group of viruses among that the SARS-CoV-2 is included (family Coronaviridae, subfamily Coronavirinae, genus Betacoronavirus and subgenus Sarbecovirus). Its main structural proteins are the membrane (M), the envelope (E), the nucleocapsid (N) and spike (S). The immune response to SARS-CoV-2 involves the cellular and the humoral sides, with neutralizing antibodies fundamentally directed against the S antigen. Although the seroprevalence data are frequently assumed as protection markers, no necessarily they are. In Spain, it is estimated that, to assure the herd immunity, at least four-fifths of the population should be immunoprotected. Due the high fatality rate of COVID-19, the acquisition of the protection only by the natural infection it not assumable and other measures as the mass immunization are required. Currently, there are several vaccine prototypes (including life virus, viral vectors, peptides and proteins and nucleic acid) in different phase of clinical evaluation. Foreseeably, some of these news vaccines would be soon commercially available. In this text, aspects related to these issues are reviewed


Assuntos
Humanos , Infecções por Coronavirus/prevenção & controle , Pneumonia Viral/prevenção & controle , Betacoronavirus/imunologia , Vacinação , Vacinas Virais/imunologia , Pandemias , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Ensaios Clínicos como Assunto/classificação , Reações Cruzadas/imunologia , Imunidade Coletiva/imunologia , Imunização , Testes de Neutralização/métodos , Proteínas Estruturais Virais
15.
BMC Infect Dis ; 20(1): 790, 2020 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-33096994

RESUMO

BACKGROUND: Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus that causes acute febrile illness, meningitis, and meningoencephalitis, mainly among adults. JCV is widely distributed in North America and the number of JCV cases in the U.S. has increased in recent years. Therefore, the central nervous system disease caused by JCV can be considered a potentially re-emerging viral disease. However, the seroprevalence of JCV is unknown in Japan. The purpose of this study is to evaluate the seroprevalence of JCV in the Japanese population. METHODS: We used an IgG enzyme-linked immunosorbent assay (IgG-ELISA) with JCV-infected cell-lysates and/or a neutralizing (NT) antibody assay. The cut-off value of IgG-ELISA was determined using IgG-ELISA to analyze serum specimens from 37 healthy Japanese donors. IgG-ELISA was validated by assessing its sensitivity and specificity, using 38 human serum samples previously tested for the presence or absence of antibodies against JCV and snowshoe hare virus (SSHV), in an in-house NT antibody assay conducted by the Public Health Agency of Canada. The seroepidemiological study was performed using IgG-ELISA and NT antibody assay to analyze 246 human serum samples from the serum bank of the National Institute of Infectious Diseases (NIID) in Japan. RESULTS: The cut-off value of IgG-ELISA was determined at 0.20, based on the mean (- 0.075) and standard deviation (0.092) values using Japanese donors' sera. The sensitivity and the specificity of IgG-ELISA determined using 25 JCV-positive and 4 JCV-negative serum samples were 96 and 100%, respectively. Analysis of the 246 Japanese serum samples revealed that no specimen showed a higher value than the cut-off value of IgG-ELISA, and no sample tested positive by the NT antibody assay. CONCLUSIONS: Our results showed that JCV is not circulating significantly in Japan. To the best of our knowledge, this is the first report to demonstrate the seroprevalence of JCV in the general population in Japan.


Assuntos
Anticorpos Antivirais/imunologia , Vírus da Encefalite da Califórnia/imunologia , Encefalite da Califórnia/epidemiologia , Ensaio de Imunoadsorção Enzimática/métodos , Testes de Neutralização/métodos , Adolescente , Adulto , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Criança , Pré-Escolar , Culicidae/virologia , Encefalite da Califórnia/virologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Lactente , Recém-Nascido , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Adulto Jovem
16.
Emerg Microbes Infect ; 9(1): 2394-2403, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33043818

RESUMO

To understand SARS-CoV-2 immunity after natural infection or vaccination, functional assays such as virus neutralising assays are needed. So far, assays to detect SARS-CoV-2 neutralising antibodies rely on cell-culture based infection assays either using wild type SARS-CoV-2 or pseudotyped viruses. Such assays are labour-intensive, require appropriate biosafety facilities and are difficult to standardize. Recently, a new surrogate virus neutralisation test (sVNT) was described that uses the principle of an ELISA to measure the neutralisation capacity of anti-SARS-CoV-2 antibodies directed against the receptor binding domain. Here, we performed an independent evaluation of the robustness, specificity and sensitivity on an extensive panel of sera from 269 PCR-confirmed COVID-19 cases and 259 unmatched samples collected before 2020 and compared it to cell-based neutralisation assays. We found a high specificity of 99.2 (95%CI: 96.9-99.9) and overall sensitivity of 80.3 (95%CI: 74.9-84.8) for the sVNT. Clinical sensitivity increased between early (<14 days post symptom onset or post diagnosis, dpos/dpd) and late sera (>14 dpos/dpd) from 75.0 (64.7-83.2) to 83.1 (76.5-88.1). Also, higher severity was associated with an increase in clinical sensitivity. Upon comparison with cell-based neutralisation assays we determined an analytical sensitivity of 74.3 (56.4-86.9) and 98.2 (89.4-99.9) for titres ≥10 to <40 and ≥40 to <160, respectively. Only samples with a titre ≥160 were always positive in the sVNT. In conclusion, the sVNT can be used as an additional assay to determine the immune status of COVID-19 infected of vaccinated individuals but its value needs to be assessed for each specific context.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/virologia , Ensaio de Imunoadsorção Enzimática/métodos , Testes de Neutralização/métodos , Pneumonia Viral/virologia , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/genética , Infecções por Coronavirus/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/sangue
17.
Nat Commun ; 11(1): 5214, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060595

RESUMO

A high-throughput platform would greatly facilitate coronavirus disease 2019 (COVID-19) serological testing and antiviral screening. Here we present a high-throughput nanoluciferase severe respiratory syndrome coronavirus 2 (SARS-CoV-2-Nluc) that is genetically stable and replicates similarly to the wild-type virus in cell culture. SARS-CoV-2-Nluc can be used to measure neutralizing antibody activity in patient sera within 5 hours, and it produces results in concordance with a plaque reduction neutralization test (PRNT). Additionally, using SARS-CoV-2-Nluc infection of A549 cells expressing human ACE2 receptor (A549-hACE2), we show that the assay can be used for antiviral screening. Using the optimized SARS-CoV-2-Nluc assay, we evaluate a panel of antivirals and other anti-infective drugs, and we identify nelfinavir, rupintrivir, and cobicistat as the most selective inhibitors of SARS-CoV-2-Nluc (EC50 0.77 to 2.74 µM). In contrast, most of the clinically approved antivirals, including tenofovir alafenamide, emtricitabine, sofosbuvir, ledipasvir, and velpatasvir were inactive at concentrations up to 10 µM. Collectively, this high-throughput platform represents a reliable tool for rapid neutralization testing and antiviral screening for SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Ensaios de Triagem em Larga Escala/métodos , Testes de Neutralização/métodos , Pneumonia Viral/diagnóstico , Células A549 , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/genética , Betacoronavirus/imunologia , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Humanos , Luciferases/genética , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
18.
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
19.
J Virol Methods ; 286: 113979, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32979406

RESUMO

Feline coronaviruses (FCoV) are members of the alphacoronavirus genus that are further characterized by serotype (types I and II) based on the antigenicity of the spike (S) protein and by pathotype based on the associated clinical conditions. Feline enteric coronaviruses (FECV) are associated with the vast majority of infections and are typically asymptomatic. Within individual animals, FECV can mutate and cause a severe and usually fatal disease called feline infectious peritonitis (FIP), the leading infectious cause of death in domestic cat populations. There are no approved antiviral drugs or recommended vaccines to treat or prevent FCoV infection. The plaque reduction neutralization test (PRNT) traditionally employed to assess immune responses and to screen therapeutic and vaccine candidates is time-consuming, low-throughput, and typically requires 2-3 days for the formation and manual counting of cytolytic plaques. Host cells are capable of carrying heavy viral burden in the absence of visible cytolytic effects, thereby reducing the sensitivity of the assay. In addition, operator-to-operator variation can generate uncertainty in the results and digital records are not automatically created. To address these challenges we developed a novel high-throughput viral microneutralization assay, with quantification of virus-infected cells performed in a plate-based image cytometer. Host cell seeding density, microplate surface coating, virus concentration and incubation time, wash buffer and fluorescent labeling were optimized. Subsequently, this FCoV viral neutralization assay was used to explore immune correlates of protection using plasma from naturally FECV-infected cats. We demonstrate that the high-throughput viral neutralization assay using the Celigo Image Cytometer provides a robust and efficient method for the rapid screening of therapeutic antibodies, antiviral compounds, and vaccines. This method can be applied to various viral infectious diseases to accelerate vaccine and antiviral drug discovery and development.


Assuntos
Infecções por Coronavirus/veterinária , Coronavirus Felino/isolamento & purificação , Ensaios de Triagem em Larga Escala/veterinária , Citometria por Imagem/métodos , Testes de Neutralização/métodos , Animais , Doenças do Gato/diagnóstico , Doenças do Gato/virologia , Gatos , Linhagem Celular , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Peritonite Infecciosa Felina/diagnóstico , Peritonite Infecciosa Felina/virologia , Ensaios de Triagem em Larga Escala/métodos , Citometria por Imagem/veterinária , Testes de Neutralização/veterinária , Carga Viral
20.
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
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