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1.
Front Immunol ; 11: 580867, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33133098

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is primarily diagnosed through viral RNA positivity in nasopharyngeal swabs, and it is associated with the early detection of specific immunoglobulins to SARS-CoV-2 proteins. We describe two moderate coronavirus disease 2019 (COVID-19) patients with WHO score 4/5 at the time of hospitalization, pneumonia, and oxygen saturation <94% and with a strong discrepancy between viral RNA and antibodies to SARS-CoV-2. One patient was positive for viral RNA but completely negative for binding and neutralizing antibodies, whereas the second patient was negative for viral RNA but with high levels of both neutralizing and binding antibodies. This observation is relevant to better understand the pathogenesis of this novel infection.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , RNA Viral/análise , Idoso , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Técnicas de Laboratório Clínico , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Feminino , Humanos , Masculino , Mucosa Nasal/virologia , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real , Testes Sorológicos
2.
Euro Surveill ; 25(45)2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33183404

RESUMO

We analysed factors associated with neutralising antibody levels in 330 convalescent plasma donors. Women and younger donors were more likely not to have measurable neutralising antibodies, while higher antibody levels were observed in men, in older donors and in those who had been hospitalised. These data will be of value in the timely recruitment of convalescent plasma donors most likely to have high levels of neutralising antibodies for ongoing studies investigating its effectiveness.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Doadores de Sangue , Infecções por Coronavirus/sangue , Infecções por Coronavirus/terapia , Hospitalização , Pneumonia Viral/sangue , Pneumonia Viral/terapia , Fatores Etários , Idoso , Anticorpos Neutralizantes/uso terapêutico , Doadores de Sangue/estatística & dados numéricos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/imunologia , Inglaterra , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização Passiva , Masculino , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/imunologia , Fatores Sexuais
3.
Emerg Microbes Infect ; 9(1): 2368-2378, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33151135

RESUMO

Managing recovered COVID-19 patients with recurrent-positive SARS-CoV-2 RNA test results is challenging. We performed a population-based observational study to characterize the viral RNA level and serum antibody responses in recurrent-positive patients and evaluate their viral transmission risk. Of 479 recovered COVID-19 patients, 93 (19%) recurrent-positive patients were identified, characterized by younger age, with a median discharge-to-recurrent-positive length of 8 days. After readmission, recurrent-positive patients exhibited mild (28%) or absent (72%) symptoms, with no disease progression. The viral RNA level in recurrent-positive patients ranged from 1.8 to 5.7 log10 copies/mL (median: 3.2), which was significantly lower than the corresponding values at disease onset. There are generally no significant differences in antibody levels between recurrent-positive and non-recurrent-positive patients, or in recurrent-positive patients over time (before, during, or after recurrent-positive detection). Virus isolation of nine representative specimens returned negative results. Whole genome sequencing of six specimens yielded only genomic fragments. 96 close contacts and 1,200 candidate contacts of 23 recurrent-positive patients showed no clinical symptoms; their viral RNA (1,296/1,296) and antibody (20/20) tests were negative. After full recovery (no longer/never recurrent-positive), 60% (98/162) patients had neutralizing antibody titers of ≥1:32. Our findings suggested that an intermittent, non-stable excretion of low-level viral RNA may result in recurrent-positive occurrence, rather than re-infection. Recurrent-positive patients pose a low transmission risk, a relatively relaxed management of recovered COVID-19 patients is recommended.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , RNA Viral/análise , Adulto , Betacoronavirus/genética , Betacoronavirus/imunologia , Infecções por Coronavirus/terapia , Infecções por Coronavirus/transmissão , Feminino , Genoma Viral/genética , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/terapia , Pneumonia Viral/transmissão , Recidiva , Sequenciamento Completo do Genoma , Adulto Jovem
4.
Immunity ; 53(5): 925-933.e4, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33129373

RESUMO

We conducted a serological study to define correlates of immunity against SARS-CoV-2. Compared to those with mild coronavirus disease 2019 (COVID-19) cases, individuals with severe disease exhibited elevated virus-neutralizing titers and antibodies against the nucleocapsid (N) and the receptor binding domain (RBD) of the spike protein. Age and sex played lesser roles. All cases, including asymptomatic individuals, seroconverted by 2 weeks after PCR confirmation. Spike RBD and S2 and neutralizing antibodies remained detectable through 5-7 months after onset, whereas α-N titers diminished. Testing 5,882 members of the local community revealed only 1 sample with seroreactivity to both RBD and S2 that lacked neutralizing antibodies. This fidelity could not be achieved with either RBD or S2 alone. Thus, inclusion of multiple independent assays improved the accuracy of antibody tests in low-seroprevalence communities and revealed differences in antibody kinetics depending on the antigen. We conclude that neutralizing antibodies are stably produced for at least 5-7 months after SARS-CoV-2 infection.


Assuntos
Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Imunidade Humoral , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Arizona/epidemiologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/sangue , Infecções por Coronavirus/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/diagnóstico , Prevalência , Domínios e Motivos de Interação entre Proteínas , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto Jovem
5.
Sci Rep ; 10(1): 19076, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154514

RESUMO

Pseuodotyped particles have significant importance and use in virology as tools for studying the biology of highly pathogenic viruses in a lower biosafety environment. The biological, chemical, and serological studies of the recently emerged SARS-CoV-2 will be greatly aided by the development and optimization of a suitable pseudotyping system. Here, we pseudotyped the SARS-CoV-2 Spike glycoprotein (SPG) on a traditional retroviral (MMLV) as well as a third generation lentiviral (pLV) vector and tested the transduction efficiency in several mammalian cell lines expressing SARS-CoV-2 receptor hACE2. While MMLV pseudotyped the vesicular stomatitis virus G glycoprotein (VSV-G) efficiently, it could not pseudotype the full-length SPG. In contrast, pLV pseudotyped both glycoproteins efficiently; however, much higher titers of pLV-G particles were produced. Among all the tested mammalian cells, 293Ts expressing hACE2 were most efficiently transduced using the pLV-S system. The pLV-S particles were efficiently neutralized by diluted serum (>:640) from recently recovered COVID-19 patients who showed high SARS-CoV-2 specific IgM and IgG levels. In summary, pLV-S pseudotyped virus provides a valid screening tool for the presence of anti SARS-CoV-2 specific neutralizing antibodies in convalescent patient serum.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Lentivirus/genética , Testes Sorológicos/métodos , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular , Vetores Genéticos/genética , Humanos , Transdução Genética
6.
Vaccine ; 38(46): 7205-7212, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33010978

RESUMO

The development of an effective vaccine against SARS-CoV-2 is urgently needed. We generated SARS-CoV-2 RBD-Fc fusion protein and evaluated its potency to elicit neutralizing antibody response in mice. RBD-Fc elicited a higher neutralizing antibodies titer than RBD as evaluated by a pseudovirus neutralization assay and a live virus based microneutralization assay. Furthermore, RBD-Fc immunized sera better inhibited cell-cell fusion, as evaluated by a quantitative cell-cell fusion assay. The cell-cell fusion assay results correlated well with the virus neutralization potency and could be used for high-throughput screening of large panels of anti-SARS-CoV-2 antibodies and vaccines without the requirement of live virus infection in BSL3 containment. Moreover, the anti-RBD sera did not enhance the pseudotyped SARS-CoV-2 infection of K562 cells. These results demonstrate that Fc fusion can significantly improve the humoral immune response to recombinant RBD immunogen, and suggest that RBD-Fc could serve as a useful component of effective vaccines against SARS-CoV-2.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/prevenção & controle , Fragmentos Fc das Imunoglobulinas/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Fusão Celular , Linhagem Celular , Infecções por Coronavirus/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imunidade Humoral/imunologia , Fragmentos Fc das Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Peptidil Dipeptidase A/genética , Domínios Proteicos/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Vacinas de Subunidades/imunologia
7.
PLoS One ; 15(10): e0241104, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33085715

RESUMO

BACKGROUND: To accurately interpret COVID-19 seroprevalence surveys, knowledge of serum-IgG responses to SARS-CoV-2 with a better understanding of patients who do not seroconvert, is imperative. This study aimed to describe serum-IgG responses to SARS-CoV-2 in a cohort of patients with both severe and mild COVID-19, including extended studies of patients who remained seronegative more than 90 days post symptom onset. METHODS: SARS-CoV-2-specific IgG antibody levels were quantified using two clinically validated and widely used commercial serological assays (Architect, Abbott Laboratories and iFlash 1800, YHLO), detecting antibodies against the spike and nucleocapsid proteins. RESULTS: Forty-seven patients (mean age 49 years, 38% female) were included. All (15/15) patients with severe symptoms and 29/32 (90.6%) patients with mild symptoms of COVID-19 developed SARS-CoV-2-specific IgG antibodies in serum. Time to seroconversion was significantly shorter (median 11 vs. 22 days, P = 0.04) in patients with severe compared to mild symptoms. Of the three patients without detectable IgG-responses after >90 days, all had detectable virus-neutralizing antibodies and in two, spike-protein receptor binding domain-specific IgG was detected with an in-house assay. Antibody titers were preserved during follow-up and all patients who seroconverted, irrespective of the severity of symptoms, still had detectable IgG levels >75 days post symptom onset. CONCLUSIONS: Patients with severe COVID-19 both seroconvert earlier and develop higher concentrations of SARS-CoV-2-specific IgG than patients with mild symptoms. Of those patients who not develop detectable IgG antibodies, all have detectable virus-neutralizing antibodies, suggesting immunity. Our results showing that not all COVID-19 patients develop detectable IgG using two validated commercial clinical methods, even over time, are vital for the interpretation of COVID-19 seroprevalence surveys.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Imunoglobulina G/sangue , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Índice de Gravidade de Doença , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/sangue , Betacoronavirus/genética , Infecções por Coronavirus/virologia , Ensaio de Imunoadsorção Enzimática , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real , Soroconversão , Estudos Soroepidemiológicos , Suécia/epidemiologia
8.
mBio ; 11(5)2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067385

RESUMO

In the absence of effective vaccines and with limited therapeutic options, convalescent plasma is being collected across the globe for potential transfusion to coronavirus disease 2019 (COVID-19) patients. The therapy has been deemed safe, and several clinical trials assessing its efficacy are ongoing. While it remains to be formally proven, the presence of neutralizing antibodies is thought to play a positive role in the efficacy of this treatment. Indeed, neutralizing titers of ≥1:160 have been recommended in some convalescent plasma trials for inclusion. Here, we performed repeated analyses at 1-month intervals on 31 convalescent individuals to evaluate how the humoral responses against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein, including neutralization, evolve over time. We observed that the levels of receptor-binding-domain (RBD)-specific IgG and IgA slightly decreased between 6 and 10 weeks after the onset of symptoms but that RBD-specific IgM levels decreased much more abruptly. Similarly, we observed a significant decrease in the capacity of convalescent plasma to neutralize pseudoparticles bearing wild-type SARS-CoV-2 S or its D614G variant. If neutralization activity proves to be an important factor in the clinical efficacy of convalescent plasma transfer, our results suggest that plasma from convalescent donors should be recovered rapidly after resolution of symptoms.IMPORTANCE While waiting for an efficient vaccine to protect against SARS-CoV-2 infection, alternative approaches to treat or prevent acute COVID-19 are urgently needed. Transfusion of convalescent plasma to treat COVID-19 patients is currently being explored; neutralizing activity in convalescent plasma is thought to play a central role in the efficacy of this treatment. Here, we observed that plasma neutralization activity decreased a few weeks after the onset of the symptoms. If neutralizing activity is required for the efficacy of convalescent plasma transfer, our results suggest that convalescent plasma should be recovered rapidly after the donor recovers from active infection.


Assuntos
Betacoronavirus/imunologia , Convalescença , Infecções por Coronavirus/imunologia , Imunidade Humoral , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Infecções por Coronavirus/sangue , Reações Cruzadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/sangue , Glicoproteína da Espícula de Coronavírus/química , Adulto Jovem
9.
Euro Surveill ; 25(42)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33094717

RESUMO

SARS-CoV-2 IgG screening of 1,000 antenatal serum samples in the Oxford area, United Kingdom, between 14 April and 15 June 2020, yielded a 5.3% seroprevalence, mirroring contemporaneous regional data. Among the 53 positive samples, 39 showed in vitro neutralisation activity, correlating with IgG titre (Pearson's correlation p<0.0001). While SARS-CoV-2 seroprevalence in pregnancy cohorts could potentially inform population surveillance, clinical correlates of infection and immunity in pregnancy, and antenatal epidemiology evolution over time need further study.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/epidemiologia , Imunoglobulina G/sangue , Pandemias , Pneumonia Viral/epidemiologia , Vigilância da População , Complicações Infecciosas na Gravidez/sangue , Primeiro Trimestre da Gravidez/sangue , Adolescente , Adulto , Estudos de Coortes , Infecções por Coronavirus/sangue , Inglaterra/epidemiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Pessoa de Meia-Idade , Pneumonia Viral/sangue , Gravidez , Diagnóstico Pré-Natal , Prevalência , Estudos Soroepidemiológicos , Método Simples-Cego , Adulto Jovem
10.
Euro Surveill ; 25(42)2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33094713

RESUMO

BackgroundThe progression and geographical distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the United Kingdom (UK) and elsewhere is unknown because typically only symptomatic individuals are diagnosed. We performed a serological study of blood donors in Scotland in the spring of 2020 to detect neutralising antibodies to SARS-CoV-2 as a marker of past infection and epidemic progression.AimOur objective was to determine if sera from blood bank donors can be used to track the emergence and progression of the SARS-CoV-2 epidemic.MethodsA pseudotyped SARS-CoV-2 virus microneutralisation assay was used to detect neutralising antibodies to SARS-CoV-2. The study comprised samples from 3,500 blood donors collected in Scotland between 17 March and 18 May 2020. Controls were collected from 100 donors in Scotland during 2019.ResultsAll samples collected on 17 March 2020 (n = 500) were negative in the pseudotyped SARS-CoV-2 virus microneutralisation assay. Neutralising antibodies were detected in six of 500 donors from 23 to 26 March. The number of samples containing neutralising antibodies did not significantly rise after 5-6 April until the end of the study on 18 May. We found that infections were concentrated in certain postcodes, indicating that outbreaks of infection were extremely localised. In contrast, other areas remained comparatively untouched by the epidemic.ConclusionAlthough blood donors are not representative of the overall population, we demonstrated that serosurveys of blood banks can serve as a useful tool for tracking the emergence and progression of an epidemic such as the SARS-CoV-2 outbreak.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Doadores de Sangue , Infecções por Coronavirus/epidemiologia , Pandemias , Pneumonia Viral/epidemiologia , Vigilância da População , Adulto , Análise por Conglomerados , Infecções por Coronavirus/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Geografia Médica , Humanos , Concentração Inibidora 50 , Masculino , Modelos Imunológicos , Testes de Neutralização , Pneumonia Viral/sangue , Prevalência , Escócia/epidemiologia , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , População Urbana
11.
Cell ; 183(4): 996-1012.e19, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33010815

RESUMO

Limited knowledge is available on the relationship between antigen-specific immune responses and COVID-19 disease severity. We completed a combined examination of all three branches of adaptive immunity at the level of SARS-CoV-2-specific CD4+ and CD8+ T cell and neutralizing antibody responses in acute and convalescent subjects. SARS-CoV-2-specific CD4+ and CD8+ T cells were each associated with milder disease. Coordinated SARS-CoV-2-specific adaptive immune responses were associated with milder disease, suggesting roles for both CD4+ and CD8+ T cells in protective immunity in COVID-19. Notably, coordination of SARS-CoV-2 antigen-specific responses was disrupted in individuals ≥ 65 years old. Scarcity of naive T cells was also associated with aging and poor disease outcomes. A parsimonious explanation is that coordinated CD4+ T cell, CD8+ T cell, and antibody responses are protective, but uncoordinated responses frequently fail to control disease, with a connection between aging and impaired adaptive immune responses to SARS-CoV-2.


Assuntos
Imunidade Adaptativa , Antígenos Virais/imunologia , Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Doença Aguda , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Betacoronavirus/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Citocinas/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Índice de Gravidade de Doença , Adulto Jovem
12.
BMJ ; 371: m3939, 2020 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-33093056

RESUMO

OBJECTIVE: To investigate the effectiveness of using convalescent plasma to treat moderate coronavirus disease 2019 (covid-19) in adults in India. DESIGN: Open label, parallel arm, phase II, multicentre, randomised controlled trial. SETTING: 39 public and private hospitals across India. PARTICIPANTS: 464 adults (≥18 years) admitted to hospital (screened 22 April to 14 July 2020) with confirmed moderate covid-19 (partial pressure of oxygen in arterial blood/fraction of inspired oxygen (PaO2/FiO2) ratio between 200 mm Hg and 300 mm Hg or a respiratory rate of more than 24/min with oxygen saturation 93% or less on room air): 235 were assigned to convalescent plasma with best standard of care (intervention arm) and 229 to best standard of care only (control arm). INTERVENTIONS: Participants in the intervention arm received two doses of 200 mL convalescent plasma, transfused 24 hours apart. The presence and levels of neutralising antibodies were not measured a priori; stored samples were assayed at the end of the study. MAIN OUTCOME MEASURE: Composite of progression to severe disease (PaO2/FiO2 <100 mm Hg) or all cause mortality at 28 days post-enrolment. RESULTS: Progression to severe disease or all cause mortality at 28 days after enrolment occurred in 44 (19%) participants in the intervention arm and 41 (18%) in the control arm (risk difference 0.008 (95% confidence interval -0.062 to 0.078); risk ratio 1.04, 95% confidence interval 0.71 to 1.54). CONCLUSION: Convalescent plasma was not associated with a reduction in progression to severe covid-19 or all cause mortality. This trial has high generalisability and approximates convalescent plasma use in real life settings with limited laboratory capacity. A priori measurement of neutralising antibody titres in donors and participants might further clarify the role of convalescent plasma in the management of covid-19. TRIAL REGISTRATION: Clinical Trial Registry of India CTRI/2020/04/024775.


Assuntos
Infecções por Coronavirus/terapia , Pneumonia Viral/terapia , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus , Infecções por Coronavirus/mortalidade , Progressão da Doença , Feminino , Humanos , Imunização Passiva , Índia , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/mortalidade , Falha de Tratamento
13.
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
14.
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
15.
PLoS One ; 15(10): e0241471, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33112930

RESUMO

Anecdotal evidence showed a negative correlation between Bacille Calmette-Guérin (BCG) vaccination and incidence of COVID-19. Incidence of the disease in children is much lower than in adults. It is hypothesized that BCG and other childhood vaccinations may provide some protection against SARS-CoV-2 infection through trained or adaptive immune responses. Here, we tested whether BCG, Pneumococcal, Rotavirus, Diphtheria, Tetanus, Pertussis, Hepatitis B, Haemophilus influenzae, Hepatitis B, Meningococcal, Measles, Mumps, and Rubella vaccines provide cross-reactive neutralizing antibodies against SARS-CoV-2 in BALB/c mice. Results indicated that none of these vaccines provided antibodies capable of neutralizing SARS-CoV-2 up to seven weeks post vaccination. We conclude that if such vaccines have any role in COVID-19 immunity, this role is not antibody-mediated.


Assuntos
Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Vacinas/imunologia , Adolescente , Adulto , Idoso , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Criança , Pré-Escolar , Infecções por Coronavirus/imunologia , Reações Cruzadas , Feminino , Humanos , Soros Imunes/imunologia , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Testes de Neutralização , Pneumonia Viral/imunologia , Vacinação , Vacinas de Produtos Inativados/imunologia , Vacinas Virais/imunologia , 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.
Emerg Microbes Infect ; 9(1): 2322-2332, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33028154

RESUMO

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of Coronavirus Disease 2019 (COVID-19) and responsible for the current pandemic. Recent SARS-CoV-2 susceptibility studies in cats show that the virus can replicate in these companion animals and transmit to other cats. Here, we present an in-depth study of SARS-CoV-2 infection, disease and transmission in domestic cats. Cats were challenged with SARS-CoV-2 via intranasal and oral routes. One day post challenge (DPC), two sentinel cats were introduced. Animals were monitored for clinical signs, clinicopathological abnormalities and viral shedding. Postmortem examinations were performed at 4, 7 and 21 DPC. Viral RNA was not detected in blood but transiently in nasal, oropharyngeal and rectal swabs and bronchoalveolar lavage fluid as well as various tissues. Tracheobronchoadenitis of submucosal glands with the presence of viral RNA and antigen was observed in airways of the infected cats. Serology showed that both, principals and sentinels, developed antibodies to SARS-CoV-2. All animals were clinically asymptomatic during the course of the study and capable of transmitting SARS-CoV-2 to sentinels. The results of this study are critical for understanding the clinical course of SARS-CoV-2 in a naturally susceptible host species, and for risk assessment.


Assuntos
Betacoronavirus/isolamento & purificação , Doenças do Gato/transmissão , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/veterinária , Suscetibilidade a Doenças , Pandemias/veterinária , Pneumonia Viral/transmissão , Pneumonia Viral/veterinária , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Líquido da Lavagem Broncoalveolar/química , Doenças do Gato/patologia , Doenças do Gato/virologia , Gatos , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Masculino , Pneumonia Viral/patologia , RNA Viral/análise , RNA Viral/isolamento & purificação , Células Vero , Replicação Viral
18.
Proc Natl Acad Sci U S A ; 117(42): 26382-26388, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-32994343

RESUMO

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached nearly every country in the world with extraordinary person-to-person transmission. The most likely original source of the virus was spillover from an animal reservoir and subsequent adaptation to humans sometime during the winter of 2019 in Wuhan Province, China. Because of its genetic similarity to SARS-CoV-1, it is probable that this novel virus has a similar host range and receptor specificity. Due to concern for human-pet transmission, we investigated the susceptibility of domestic cats and dogs to infection and potential for infected cats to transmit to naive cats. We report that cats are highly susceptible to infection, with a prolonged period of oral and nasal viral shedding that is not accompanied by clinical signs, and are capable of direct contact transmission to other cats. These studies confirm that cats are susceptible to productive SARS-CoV-2 infection, but are unlikely to develop clinical disease. Further, we document that cats developed a robust neutralizing antibody response that prevented reinfection following a second viral challenge. Conversely, we found that dogs do not shed virus following infection but do seroconvert and mount an antiviral neutralizing antibody response. There is currently no evidence that cats or dogs play a significant role in human infection; however, reverse zoonosis is possible if infected owners expose their domestic pets to the virus during acute infection. Resistance to reinfection holds promise that a vaccine strategy may protect cats and, by extension, humans.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Animais , Animais Domésticos , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Antígenos Virais/imunologia , Betacoronavirus/imunologia , Gatos , Infecções por Coronavirus/patologia , Infecções por Coronavirus/transmissão , Modelos Animais de Doenças , Cães , Feminino , Masculino , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/transmissão , Eliminação de Partículas Virais
19.
J Clin Virol ; 131: 104611, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32882666

RESUMO

BACKGROUND: The involvement of SARS-CoV-2 antibodies in mediating immunopathogenetic events in COVID-19 patients has been suggested. By using several experimental approaches, we investigated the potential association between SARS-CoV-2 IgGs recognizing the spike (S) protein receptor-binding domain (RBD), neutralizing antibodies (NtAb) targeting S, and COVID-19 severity. PATIENTS AND METHODS: This unicenter, retrospective, observational study included 51 hospitalized patients (24 at the intensive care unit; ICU). A total of 93 sera from these patients collected at different time points from the onset of symptoms were analyzed. SARS-CoV-2 RBD IgGs were quantitated by ELISA and NtAb50 titers were measured in a GFP reporterbased pseudotyped virus platform. Demographic and clinical data, complete blood counts, as well as serum levels of ferritin, Dimer-D, C reactive protein (CRP), lactose dehydrogenase (LDH), and interleukin-6 (IL-6) were retrieved from clinical charts. RESULTS: The overall correlation between levels of both antibody measurements was good (Rho = 0.82; P = 0 < 0.001). SARS-CoV-2 RBD IgG and NtAb50 levels in sera collected up to day 30 after the onset of symptoms were comparable between ICU and non-ICU patients (P=>0.1). Four ICU patients died; two of these achieved NtAb50 titers ≥1/160 while the other two exhibited a 1/80 titer. Very weak (Rho=>0.0-<0.2) or weak (Rho=>0.2-<0.4) correlations were observed between anti-RBD IgGs, NtAb50, and serum levels pro-inflammatory biomarkers. CONCLUSIONS: The data presented herein do not support an association between SARS-CoV-2 RBD IgG or NtAb50 levels and COVID-19 severity.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Coronavirus/sangue , Hospitalização/estatística & dados numéricos , Inflamação/sangue , Pneumonia Viral/sangue , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Betacoronavirus , Sítios de Ligação de Anticorpos , Biomarcadores/sangue , Infecções por Coronavirus/imunologia , Feminino , Humanos , Inflamação/virologia , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/imunologia , Estudos Retrospectivos , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto Jovem
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