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1.
Sci Rep ; 11(1): 20143, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34635788

RESUMO

Rapid, high-throughput diagnostic tests are essential to decelerate the spread of the novel coronavirus disease 2019 (COVID-19) pandemic. While RT-PCR tests performed in centralized laboratories remain the gold standard, rapid point-of-care antigen tests might provide faster results. However, they are associated with markedly reduced sensitivity. Bedside breath gas analysis of volatile organic compounds detected by ion mobility spectrometry (IMS) may enable a quick and sensitive point-of-care testing alternative. In this proof-of-concept study, we investigated whether gas analysis by IMS can discriminate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from other respiratory viruses in an experimental set-up. Repeated gas analyses of air samples collected from the headspace of virus-infected in vitro cultures were performed for 5 days. A three-step decision tree using the intensities of four spectrometry peaks correlating to unidentified volatile organic compounds allowed the correct classification of SARS-CoV-2, human coronavirus-NL63, and influenza A virus H1N1 without misassignment when the calculation was performed with data 3 days post infection. The forward selection assignment model allowed the identification of SARS-CoV-2 with high sensitivity and specificity, with only one of 231 measurements (0.43%) being misclassified. Thus, volatile organic compound analysis by IMS allows highly accurate differentiation of SARS-CoV-2 from other respiratory viruses in an experimental set-up, supporting further research and evaluation in clinical studies.


Assuntos
Antígenos Virais/isolamento & purificação , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Testes Imediatos , SARS-CoV-2/isolamento & purificação , Animais , COVID-19/imunologia , COVID-19/virologia , Teste Sorológico para COVID-19/instrumentação , Chlorocebus aethiops , Coronavirus Humano NL63/imunologia , Coronavirus Humano NL63/isolamento & purificação , Diagnóstico Diferencial , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Espectrometria de Mobilidade Iônica , Estudo de Prova de Conceito , SARS-CoV-2/imunologia , Células Vero
2.
Nat Commun ; 12(1): 4740, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362897

RESUMO

Unraveling the long-term kinetics of antibodies to SARS-CoV-2 and the individual characteristics influencing it, including the impact of pre-existing antibodies to human coronaviruses causing common cold (HCoVs), is essential to understand protective immunity to COVID-19 and devise effective surveillance strategies. IgM, IgA and IgG levels against six SARS-CoV-2 antigens and the nucleocapsid antigen of the four HCoV (229E, NL63, OC43 and HKU1) were quantified by Luminex, and antibody neutralization capacity was assessed by flow cytometry, in a cohort of health care workers followed up to 7 months (N = 578). Seroprevalence increases over time from 13.5% (month 0) and 15.6% (month 1) to 16.4% (month 6). Levels of antibodies, including those with neutralizing capacity, are stable over time, except IgG to nucleocapsid antigen and IgM levels that wane. After the peak response, anti-spike antibody levels increase from ~150 days post-symptom onset in all individuals (73% for IgG), in the absence of any evidence of re-exposure. IgG and IgA to HCoV are significantly higher in asymptomatic than symptomatic seropositive individuals. Thus, pre-existing cross-reactive HCoVs antibodies could have a protective effect against SARS-CoV-2 infection and COVID-19 disease.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Coronavirus Humano 229E/imunologia , Coronavirus Humano NL63/imunologia , SARS-CoV-2/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Resfriado Comum/imunologia , Resfriado Comum/virologia , Proteção Cruzada/imunologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue
3.
Front Immunol ; 12: 696370, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34386006

RESUMO

The COVID-19 pandemic is caused by SARS-CoV-2, a novel zoonotic coronavirus. Emerging evidence indicates that preexisting humoral immunity against other seasonal human coronaviruses (HCoVs) plays a critical role in the specific antibody response to SARS-CoV-2. However, current work to assess the effects of preexisting and cross-reactive anti-HCoVs antibodies has been limited. To address this issue, we have adapted our previously reported multiplex assay to simultaneously and quantitatively measure anti-HCoV antibodies. The full mPlex-CoV panel covers the spike (S) and nucleocapsid (N) proteins of three highly pathogenic HCoVs (SARS-CoV-1, SARS-CoV-2, MERS) and four human seasonal strains (OC43, HKU1, NL63, 229E). Combining this assay with volumetric absorptive microsampling (VAMS), we measured the anti-HCoV IgG, IgA, and IgM antibodies in fingerstick blood samples. The results demonstrate that the mPlex-CoV assay has high specificity and sensitivity. It can detect strain-specific anti-HCoV antibodies down to 0.1 ng/ml with 4 log assay range and with low intra- and inter-assay coefficients of variation (%CV). We also estimate multiple strain HCoVs IgG, IgA and IgM concentration in VAMS samples in three categories of subjects: pre-COVID-19 (n=21), post-COVID-19 convalescents (n=19), and COVID-19 vaccine recipients (n=14). Using metric multidimensional scaling (MDS) analysis, HCoVs IgG concentrations in fingerstick blood samples were well separated between the pre-COVID-19, post-COVID-19 convalescents, and COVID-19 vaccine recipients. In addition, we demonstrate how multi-dimensional scaling analysis can be used to visualize IgG mediated antibody immunity against multiple human coronaviruses. We conclude that the combination of VAMS and the mPlex-Cov assay is well suited to performing remote study sample collection under pandemic conditions to monitor HCoVs antibody responses in population studies.


Assuntos
Anticorpos Antivirais/sangue , Coronavirus/imunologia , Reações Cruzadas/imunologia , Imunoensaio/métodos , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , COVID-19/imunologia , Coronavirus Humano 229E/imunologia , Coronavirus Humano NL63/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Coronavirus Humano OC43/imunologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Vírus da SARS/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia
4.
Virol J ; 18(1): 166, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389034

RESUMO

The emergence of a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and more recently, the independent evolution of multiple SARS-CoV-2 variants has generated renewed interest in virus evolution and cross-species transmission. While all known human coronaviruses (HCoVs) are speculated to have originated in animals, very little is known about their evolutionary history and factors that enable some CoVs to co-exist with humans as low pathogenic and endemic infections (HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1), while others, such as SARS-CoV, MERS-CoV and SARS-CoV-2 have evolved to cause severe disease. In this review, we highlight the origins of all known HCoVs and map positively selected for mutations within HCoV proteins to discuss the evolutionary trajectory of SARS-CoV-2. Furthermore, we discuss emerging mutations within SARS-CoV-2 and variants of concern (VOC), along with highlighting the demonstrated or speculated impact of these mutations on virus transmission, pathogenicity, and neutralization by natural or vaccine-mediated immunity.


Assuntos
Vacinas contra COVID-19 , COVID-19/virologia , SARS-CoV-2/genética , Animais , COVID-19/transmissão , Coronavirus Humano 229E/genética , Coronavirus Humano 229E/imunologia , Coronavirus Humano 229E/patogenicidade , Coronavirus Humano NL63/genética , Coronavirus Humano NL63/imunologia , Coronavirus Humano NL63/patogenicidade , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/imunologia , Coronavirus Humano OC43/patogenicidade , Humanos , Imunidade , Mutação , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade
5.
Viruses ; 13(8)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34452443

RESUMO

The novel coronavirus SARS-CoV-2 is the seventh identified human coronavirus. Understanding the extent of pre-existing immunity induced by seropositivity to endemic seasonal coronaviruses and the impact of cross-reactivity on COVID-19 disease progression remains a key research question in immunity to SARS-CoV-2 and the immunopathology of COVID-2019 disease. This paper describes a panel of lentiviral pseudotypes bearing the spike (S) proteins for each of the seven human coronaviruses (HCoVs), generated under similar conditions optimized for high titre production allowing a high-throughput investigation of antibody neutralization breadth. Optimal production conditions and most readily available permissive target cell lines were determined for spike-mediated entry by each HCoV pseudotype: SARS-CoV-1, SARS-CoV-2 and HCoV-NL63 best transduced HEK293T/17 cells transfected with ACE2 and TMPRSS2, HCoV-229E and MERS-CoV preferentially entered HUH7 cells, and CHO cells were most permissive for the seasonal betacoronavirus HCoV-HKU1. Entry of ACE2 using pseudotypes was enhanced by ACE2 and TMPRSS2 expression in target cells, whilst TMPRSS2 transfection rendered HEK293T/17 cells permissive for HCoV-HKU1 and HCoV-OC43 entry. Additionally, pseudotype viruses were produced bearing additional coronavirus surface proteins, including the SARS-CoV-2 Envelope (E) and Membrane (M) proteins and HCoV-OC43/HCoV-HKU1 Haemagglutinin-Esterase (HE) proteins. This panel of lentiviral pseudotypes provides a safe, rapidly quantifiable and high-throughput tool for serological comparison of pan-coronavirus neutralizing responses; this can be used to elucidate antibody dynamics against individual coronaviruses and the effects of antibody cross-reactivity on clinical outcome following natural infection or vaccination.


Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , COVID-19/imunologia , Coronavirus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Amplamente Neutralizantes/sangue , Linhagem Celular , Coronavirus Humano 229E/imunologia , Coronavirus Humano 229E/fisiologia , Coronavirus Humano NL63/imunologia , Coronavirus Humano NL63/fisiologia , Coronavirus Humano OC43/imunologia , Coronavirus Humano OC43/fisiologia , Reações Cruzadas , Humanos , Lentivirus/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Testes de Neutralização , Plasmídeos , SARS-CoV-2/fisiologia , Transfecção , Internalização do Vírus
6.
Viruses ; 13(8)2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34452482

RESUMO

The COVID-19 pandemic has highlighted the importance of understanding the immune response to seasonal human coronavirus (HCoV) infections such as HCoV-NL63, how existing neutralising antibodies to HCoV may modulate responses to SARS-CoV-2 infection, and the utility of seasonal HCoV as human challenge models. Therefore, in this study we quantified HCoV-NL63 neutralising antibody titres in a healthy adult population using plasma from 100 blood donors in Australia. A microneutralisation assay was performed with plasma diluted from 1:10 to 1:160 and tested with the HCoV-NL63 Amsterdam-1 strain. Neutralising antibodies were detected in 71% of the plasma samples, with a median geometric mean titre of 14. This titre was similar to those reported in convalescent sera taken from individuals 3-7 months following asymptomatic SARS-CoV-2 infection, and 2-3 years post-infection from symptomatic SARS-CoV-1 patients. HCoV-NL63 neutralising antibody titres decreased with increasing age (R2 = 0.042, p = 0.038), but did not differ by sex. Overall, this study demonstrates that neutralising antibody to HCoV-NL63 is detectable in approximately 71% of the healthy adult population of Australia. Similar titres did not impede the use of another seasonal human coronavirus (HCoV-229E) in a human challenge model, thus, HCoV-NL63 may be useful as a human challenge model for more pathogenic coronaviruses.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Infecções por Coronavirus/epidemiologia , Coronavirus Humano NL63/imunologia , Adulto , Fatores Etários , Idoso , Austrália/epidemiologia , COVID-19/imunologia , Teste Sorológico para COVID-19 , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/imunologia , Estudos Soroepidemiológicos , Adulto Jovem
7.
mBio ; 12(3): e0122921, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34182775

RESUMO

We sought to discover links between antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and patient clinical variables, cytokine profiles, and antibodies to endemic coronaviruses. Serum samples from 30 patients of younger (26 to 39 years) and older (69 to 83 years) age groups and with varying clinical severities ranging from outpatient to mechanically ventilated were collected and used to probe a novel multi-coronavirus protein microarray. This microarray contained variable-length overlapping fragments of SARS-CoV-2 spike (S), envelope (E), membrane (M), nucleocapsid (N), and open reading frame (ORF) proteins created through in vitro transcription and translation (IVTT). The array also contained SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus OC43 (HCoV-OC43), and HCoV-NL63 proteins. IgG antibody responses to specific epitopes within the S1 protein region spanning amino acids (aa) 500 to 650 and within the N protein region spanning aa 201 to 300 were found to be significantly higher in older patients and further significantly elevated in those older patients who were ventilated. Additionally, there was a noticeable overlap between antigenic regions and known mutation locations in selected emerging SARS-CoV-2 variants of current clinical consequence (B.1.1.7, B1.351, P.1, CAL20.C, and B.1.526). Moreover, the older age group displayed more consistent correlations of antibody reactivity with systemic cytokine and chemokine responses than the younger adult group. A subset of patients, however, had little or no response to SARS-CoV-2 antigens and disproportionately severe clinical outcomes. Further characterization of these slow-low-responding individuals with cytokine analysis revealed significantly higher interleukin-10 (IL-10), IL-15, and interferon gamma-induced protein 10 (IP-10) levels and lower epidermal growth factor (EGF) and soluble CD40 ligand (sCD40L) levels than those of seroreactive patients in the cohort. IMPORTANCE As numerous viral variants continue to emerge in the coronavirus disease 2019 (COVID-19) pandemic, determining antibody reactivity to SARS-CoV-2 epitopes becomes essential in discerning changes in the immune response to infection over time. This study enabled us to identify specific areas of antigenicity within the SARS-CoV-2 proteome, allowing us to detect correlations of epitopes with clinical metadata and immunological signals to gain holistic insight into SARS-CoV-2 infection. This work also emphasized the risk of mutation accumulation in viral variants and the potential for evasion of the adaptive immune responses in the event of reinfection. We additionally highlighted the correlation of antigenicity between structural proteins of SARS-CoV-2 and endemic HCoVs, raising the possibility of cross-protection between homologous lineages. Finally, we identified a subset of patients with minimal antibody reactivity to SARS-CoV-2 infection, prompting discussion of the potential consequences of this alternative immune response.


Assuntos
Anticorpos Antivirais/sangue , Coronavirus Humano NL63/imunologia , Coronavirus Humano OC43/imunologia , Citocinas/sangue , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , SARS-CoV-2/imunologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Proteínas do Envelope de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Masculino , Fosfoproteínas/imunologia , Análise Serial de Proteínas , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologia
8.
Medicine (Baltimore) ; 100(25): e26446, 2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34160438

RESUMO

RATIONALE: Viruses are the most common pathogens that can cause infection-related non-recurrent death after transplantation, occurring mostly from the early stages of hematopoietic stem cell transplantation (HSCT) to within 1 year after transplantation. Human coronavirus (HCoV)-NL63 is a coronavirus that could cause mortality among patients with underlying disease complications. Serological tests are of limited diagnostic value in immunocompromised hosts and cases of latent infection reactivation. In contrast, macro-genomic high-throughput (DNA and RNA) sequencing allows for rapid and accurate diagnosis of infecting pathogens for targeted treatment. PATIENT CONCERNS: In this report, we describe a patient who exhibited acute B-lymphocytic leukemia and developed complicated pulmonary HCoV-NL63 infection after a second allogeneic HSCT (allo-HSCT). Six months after the second allo-HSCT, he developed sudden-onset hyperthermia and cough with decreased oxygen saturation. Chest computed tomography (CT) suggested bilateral multiple rounded ground-glass opacities with the pulmonary lobules as units. DIAGNOSES: HCoV-NL63 was detected by metagenomic next-generation sequencing (NGS), and HCoV-NL63 viral pneumonia was diagnosed. INTERVENTIONS: The treatment was mainly based on the use of antiviral therapy, hormone administration, and gamma-globulin. OUTCOMES: After the therapy, the body temperature returned to normal, the chest CT findings had improved on review, and the viral copy number eventually became negative. LESSONS: The latest NGS is an effective method for early infection diagnosis. The HCoV-NL63 virus can cause inflammatory factor storm and alter the neutrophil-to-lymphocyte ratio (NLR). This case suggests that the patient's NLR and cytokine levels could be monitored during the clinical treatment to assess the disease and its treatment outcome in a timely manner.


Assuntos
Infecções por Coronavirus/diagnóstico , Coronavirus Humano NL63/isolamento & purificação , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Leucemia de Células B/terapia , Pneumonia Viral/diagnóstico , Antivirais/administração & dosagem , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Coronavirus Humano NL63/genética , Coronavirus Humano NL63/imunologia , Quimioterapia Combinada/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hospedeiro Imunocomprometido , Leucemia de Células B/imunologia , Pulmão/diagnóstico por imagem , Masculino , Metagenômica , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Tomografia Computadorizada por Raios X , Transplante Homólogo/efeitos adversos , Adulto Jovem , gama-Globulinas/administração & dosagem
9.
J Clin Invest ; 131(10)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-33822770

RESUMO

Recent studies have shown T cell cross-recognition of SARS-CoV-2 and common cold coronavirus spike proteins. However, the effect of SARS-CoV-2 vaccines on T cell responses to common cold coronaviruses (CCCs) remains unknown. In this study, we analyzed CD4+ T cell responses to spike peptides from SARS-CoV-2 and 3 CCCs (HCoV-229E, HCoV-NL63, and HCoV-OC43) before and after study participants received Pfizer-BioNTech (BNT162b2) or Moderna (mRNA-1273) mRNA-based COVID-19 vaccines. Vaccine recipients showed broad T cell responses to the SARS-CoV-2 spike protein, and we identified 23 distinct targeted peptides in 9 participants, including 1 peptide that was targeted in 6 individuals. Only 4 of these 23 targeted peptides would potentially be affected by mutations in the UK (B.1.1.7) and South African (B.1.351) variants, and CD4+ T cells from vaccine recipients recognized the 2 variant spike proteins as effectively as they recognized the spike protein from the ancestral virus. Interestingly, we observed a 3-fold increase in the CD4+ T cell responses to HCoV-NL63 spike peptides after vaccination. Our results suggest that T cell responses elicited or enhanced by SARS-CoV-2 mRNA vaccines may be able to control SARS-CoV-2 variants and lead to cross-protection against some endemic coronaviruses.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Vacinas contra COVID-19/imunologia , Coronavirus Humano 229E , Coronavirus Humano NL63 , Coronavirus Humano OC43 , RNA Mensageiro , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Adulto , Coronavirus Humano 229E/genética , Coronavirus Humano 229E/imunologia , Coronavirus Humano NL63/genética , Coronavirus Humano NL63/imunologia , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/imunologia , Reações Cruzadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/imunologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
10.
Sci Rep ; 10(1): 21447, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33293664

RESUMO

Currently, infections with SARS-Coronavirus-2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, are responsible for substantial morbidity and mortality worldwide. Older adults subjects > 60 years of age account for > 95% of the over one million fatal cases reported to date. It is unclear why in this age group SARS-CoV-2 infection causes more severe disease than in young adults. We hypothesized that differences in SARS-CoV-2 cross-reactive cellular immunity induced after infection with human coronaviruses (HCoVs), like OC43 and NL63, were at the basis of the differential mortality (and morbidity) observed after SARS-CoV-2 infection, because a small proportion of HCoV-specific T cells cross-react with SARS-CoV-2. Our data demonstrate that pre-existing T cell immunity induced by circulating human alpha- and beta-HCoVs is present in young adult individuals, but virtually absent in older adult subjects. Consequently, the frequency of cross-reactive T cells directed to the novel pandemic SARS-CoV-2 was minimal in most older adults. To the best of our knowledge, this is the first time that the presence of cross-reactive T cells to SARS-CoV-2 is compared in young and older adults. Our findings provide at least a partial explanation for the more severe clinical outcome of SARS-CoV-2 infection observed in the elderly. Moreover, this information could help to design efficacious vaccines for this age group, aiming at the induction of cell-mediated immunity.


Assuntos
Anticorpos Antivirais/imunologia , Coronavirus Humano NL63/imunologia , Coronavirus Humano OC43/imunologia , SARS-CoV-2/imunologia , Linfócitos T/imunologia , Adulto , Idoso , COVID-19/imunologia , COVID-19/patologia , Reações Cruzadas/imunologia , Humanos , Imunidade Celular/imunologia , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto Jovem
11.
J Clin Invest ; 130(12): 6631-6638, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32966269

RESUMO

BACKGROUNDT cell responses to the common cold coronaviruses have not been well characterized. Preexisting T cell immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been reported, and a recent study suggested that this immunity was due to cross-recognition of the novel coronavirus by T cells specific for the common cold coronaviruses.METHODSWe used the enzyme-linked immunospot (ELISPOT) assay to characterize the T cell responses against peptide pools derived from the spike protein of 3 common cold coronaviruses (HCoV-229E, HCoV-NL63, and HCoV-OC43) and SARS-CoV-2 in 21 healthy donors (HDs) who were seronegative for SARS-CoV-2 and had no known exposure to the virus. An in vitro expansion culture assay was also used to analyze memory T cell responses.RESULTSWe found responses to the spike protein of the 3 common cold coronaviruses in many of the donors. We then focused on HCoV-NL63 and detected broad T cell responses to the spike protein and identified 22 targeted peptides. Interestingly, only 1 study participant had a significant response to SARS-CoV-2 spike or nucleocapsid protein in the ELISPOT assay. In vitro expansion studies suggested that T cells specific for the HCoV-NL63 spike protein in this individual could also recognize SARS-CoV-2 spike protein peptide pools.CONCLUSIONHDs have circulating T cells specific for the spike proteins of HCoV-NL63, HCoV-229E, and HCoV-OC43. T cell responses to SARS-CoV-2 spike and nucleocapsid proteins were present in only 1 participant and were potentially the result of cross-recognition by T cells specific for the common cold coronaviruses. Further studies are needed to determine whether this cross-recognition influences coronavirus disease 2019 (COVID-19) outcomes.


Assuntos
COVID-19/imunologia , Resfriado Comum/imunologia , Coronavirus Humano 229E/imunologia , Coronavirus Humano NL63/imunologia , Coronavirus Humano OC43/imunologia , Imunidade Celular , SARS-CoV-2/imunologia , Linfócitos T/imunologia , Adulto , Reações Cruzadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
12.
SLAS Technol ; 25(6): 545-552, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32815769

RESUMO

As of July 22, 2020, more than 14.7 million infections of SARS-CoV-2, the virus responsible for Coronavirus Disease 2019 (COVID-19), have been confirmed globally. Serological assays are essential for community screening, assessing infection prevalence, aiding identification of infected patients, and enacting appropriate treatment and quarantine protocols in the battle against this rapidly expanding pandemic. Antibody detection by agglutination-PCR (ADAP) is a pure solution phase immunoassay that generates a PCR amplifiable signal when patient antibodies agglutinate DNA-barcoded antigen probes into a dense immune complex. Here, we present an ultrasensitive and high-throughput automated liquid biopsy assay based on the Hamilton Microlab ADAP STAR automated liquid-handling platform, which was developed and validated for the qualitative detection of total antibodies against spike protein 1 (S1) of SARS-CoV-2 that uses as little as 4 µL of serum. To assess the clinical performance of the ADAP assay, 57 PCR-confirmed COVID-19 patients and 223 control patients were tested. The assay showed a sensitivity of 98% (56/57) and a specificity of 99.55% (222/223). Notably, the SARS-CoV-2-negative control patients included individuals with other common coronaviral infections, such as CoV-NL63 and CoV-HKU, which did not cross-react. In addition to high performance, the hands-free automated workstation enabled high-throughput sample processing to reduce screening workload while helping to minimize analyst contact with biohazardous samples. Therefore, the ADAP STAR liquid-handling workstation can be used as a valuable tool to address the COVID-19 global pandemic.


Assuntos
Alphacoronavirus/imunologia , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Coronavirus Humano NL63/imunologia , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Automação Laboratorial , Quirópteros , Técnicas de Laboratório Clínico , Reações Cruzadas , Ensaios de Triagem em Larga Escala , Humanos , Imunoensaio , Pandemias , Reação em Cadeia da Polimerase , Procedimentos Cirúrgicos Robóticos , Sensibilidade e Especificidade
13.
Nature ; 587(7833): 270-274, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32726801

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the rapidly unfolding coronavirus disease 2019 (COVID-19) pandemic1,2. Clinical manifestations of COVID-19 vary, ranging from asymptomatic infection to respiratory failure. The mechanisms that determine such variable outcomes remain unresolved. Here we investigated CD4+ T cells that are reactive against the spike glycoprotein of SARS-CoV-2 in the peripheral blood of patients with COVID-19 and SARS-CoV-2-unexposed healthy donors. We detected spike-reactive CD4+ T cells not only in 83% of patients with COVID-19 but also in 35% of healthy donors. Spike-reactive CD4+ T cells in healthy donors were primarily active against C-terminal epitopes in the spike protein, which show a higher homology to spike glycoproteins of human endemic coronaviruses, compared with N-terminal epitopes. Spike-protein-reactive T cell lines generated from SARS-CoV-2-naive healthy donors responded similarly to the C-terminal region of the spike proteins of the human endemic coronaviruses 229E and OC43, as well as that of SARS-CoV-2. This results indicate that spike-protein cross-reactive T cells are present, which were probably generated during previous encounters with endemic coronaviruses. The effect of pre-existing SARS-CoV-2 cross-reactive T cells on clinical outcomes remains to be determined in larger cohorts. However, the presence of spike-protein cross-reactive T cells in a considerable fraction of the general population may affect the dynamics of the current pandemic, and has important implications for the design and analysis of upcoming trials investigating COVID-19 vaccines.


Assuntos
Betacoronavirus/imunologia , Linfócitos T CD4-Positivos/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19 , Linhagem Celular , Coronavirus Humano 229E/imunologia , Coronavirus Humano NL63/imunologia , Coronavirus Humano OC43/imunologia , Reações Cruzadas , Epitopos de Linfócito T/imunologia , Feminino , Voluntários Saudáveis , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Pandemias , SARS-CoV-2
15.
Nat Struct Mol Biol ; 23(10): 899-905, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27617430

RESUMO

The threat of a major coronavirus pandemic urges the development of strategies to combat these pathogens. Human coronavirus NL63 (HCoV-NL63) is an α-coronavirus that can cause severe lower-respiratory-tract infections requiring hospitalization. We report here the 3.4-Å-resolution cryo-EM reconstruction of the HCoV-NL63 coronavirus spike glycoprotein trimer, which mediates entry into host cells and is the main target of neutralizing antibodies during infection. The map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass spectrometry, provides a structural framework to understand the accessibility to antibodies. The structure reveals the complete architecture of the fusion machinery including the triggering loop and the C-terminal domains, which contribute to anchoring the trimer to the viral membrane. Our data further suggest that HCoV-NL63 and other coronaviruses use molecular trickery, based on epitope masking with glycans and activating conformational changes, to evade the immune system of infected hosts.


Assuntos
Infecções por Coronavirus/virologia , Coronavirus Humano NL63/química , Epitopos/química , Polissacarídeos/análise , Glicoproteína da Espícula de Coronavírus/química , Animais , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Infecções por Coronavirus/imunologia , Coronavirus Humano NL63/imunologia , Microscopia Crioeletrônica , Drosophila , Epitopos/imunologia , Humanos , Modelos Moleculares , Polissacarídeos/imunologia , Conformação Proteica , Multimerização Proteica , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/ultraestrutura
16.
Protein Cell ; 5(12): 912-27, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25311841

RESUMO

Autophagy plays important roles in modulating viral replication and antiviral immune response. Coronavirus infection is associated with the autophagic process, however, little is known about the mechanisms of autophagy induction and its contribution to coronavirus regulation of host innate responses. Here, we show that the membrane-associated papain-like protease PLP2 (PLP2-TM) of coronaviruses acts as a novel autophagy-inducing protein. Intriguingly, PLP2-TM induces incomplete autophagy process by increasing the accumulation of autophagosomes but blocking the fusion of autophagosomes with lysosomes. Furthermore, PLP2-TM interacts with the key autophagy regulators, LC3 and Beclin1, and promotes Beclin1 interaction with STING, the key regulator for antiviral IFN signaling. Finally, knockdown of Beclin1 partially reverses PLP2-TM's inhibitory effect on innate immunity which resulting in decreased coronavirus replication. These results suggested that coronavirus papain-like protease induces incomplete autophagy by interacting with Beclin1, which in turn modulates coronavirus replication and antiviral innate immunity.


Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Coronavirus Humano NL63/imunologia , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Proteínas de Membrana/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Papaína/imunologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Autofagia , Proteína Beclina-1 , Coronavirus Humano NL63/genética , Proteases Semelhantes à Papaína de Coronavírus , Células HEK293 , Células HeLa , Humanos , Evasão da Resposta Imune , Imunidade Inata , Interferon gama/genética , Interferon gama/imunologia , Lisossomos/metabolismo , Lisossomos/virologia , Células MCF-7 , Fusão de Membrana , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Papaína/genética , Fagossomos/metabolismo , Fagossomos/virologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Transdução de Sinais , Replicação Viral
17.
Cytokine Growth Factor Rev ; 25(6): 669-79, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25212897

RESUMO

STING has emerged in recent years as an important signalling adaptor in the activation of type I interferon responses during infection with DNA viruses and bacteria. An increasing body of evidence suggests that STING also modulates responses to RNA viruses, though the mechanisms remain less clear. In this review, we give a brief overview of the ways in which STING facilitates sensing of RNA viruses. These include modulation of RIG-I-dependent responses through STING's interaction with MAVS, and more speculative mechanisms involving the DNA sensor cGAS and sensing of membrane remodelling events. We then provide an in-depth literature review to summarise the known mechanisms by which RNA viruses of the families Flaviviridae and Coronaviridae evade sensing through STING. Our own work has shown that the NS2B/3 protease complex of the flavivirus dengue virus binds and cleaves STING, and that an inability to degrade murine STING may contribute to host restriction in this virus. We contrast this to the mechanism employed by the distantly related hepacivirus hepatitis C virus, in which STING is bound and inactivated by the NS4B protein. Finally, we discuss STING antagonism in the coronaviruses SARS coronavirus and human coronavirus NL63, which disrupt K63-linked polyubiquitination and dimerisation of STING (both of which are required for STING-mediated activation of IRF-3) via their papain-like proteases. We draw parallels with less-well characterised mechanisms of STING antagonism in related viruses, and place our current knowledge in the context of species tropism restrictions that potentially affect the emergence of new human pathogens.


Assuntos
Coronavirus Humano NL63/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Proteínas de Membrana/imunologia , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Transdução de Sinais/imunologia , Animais , Coronavirus Humano NL63/genética , Dengue/genética , Vírus da Dengue/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Vírus da SARS/genética , Síndrome Respiratória Aguda Grave/genética , Transdução de Sinais/genética , Proteínas Virais/genética , Proteínas Virais/imunologia
18.
J Med Virol ; 86(12): 2146-53, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24760654

RESUMO

Although human coronavirus (HCoV)-NL63 was once considered a possible causative agent of Kawasaki disease based on RT-PCR analyses, subsequent studies could not confirm the result. In this study, this possibility was explored using serological tests. To evaluate the role of HCoV infection in patients with Kawasaki disease, immunofluorescence assays and virus neutralizing tests were performed. Paired serum samples were obtained from patients with Kawasaki disease who had not been treated with γ-globulin. HCoV-NL63 and two antigenically different isolates of HCoV-229E (ATCC-VR740 and a new isolate, Sendai-H) were examined as controls. Immunofluorescence assays detected no difference in HCoV-NL63 antibody positivity between the patients with Kawasaki disease and controls, whereas the rate of HCoV-229E antibody positivity was higher in the patients with Kawasaki disease than that in controls. The neutralizing tests revealed no difference in seropositivity between the acute and recovery phases of patients with Kawasaki disease for the two HCoV-229Es. However, the Kawasaki disease specimens obtained from patients in recovery phase displayed significantly higher positivity for Sendai-H, but not for ATCC-VR740, as compared to the controls. The serological test supported no involvement of HCoV-NL63 but suggested the possible involvement of HCoV-229E in the development of Kawasaki disease.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Coronaviridae/complicações , Infecções por Coronaviridae/virologia , Coronavirus Humano 229E/imunologia , Coronavirus Humano NL63/imunologia , Síndrome de Linfonodos Mucocutâneos/etiologia , Síndrome de Linfonodos Mucocutâneos/virologia , Criança , Pré-Escolar , Feminino , Imunofluorescência , Humanos , Lactente , Masculino , Testes de Neutralização
19.
J Clin Virol ; 53(2): 135-9, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22188723

RESUMO

BACKGROUND: It is unknown to what extent the human coronaviruses (HCoVs) OC43, HKU1, 229E and NL63 infect healthy children. Frequencies of infections are only known for hospitalized children. OBJECTIVES: Comparing infection frequencies in children who have mild infections with frequencies in children needing hospital uptake will determine whether infection by one of the four HCoVs leads to more severe disease. In addition, the sequence of seroconversions can reveal whether infection by one HCoV protects from infection by other HCoVs. STUDY DESIGN: Two distinct study groups were monitored: healthy children and children hospitalized due to respiratory infection. HCoV natural infection rates in healthy children were obtained by serology in 25 newborns (followed 0-20months). The frequencies of severe HCoVs infection was determined by real time RT-PCR among 1471 hospitalized infants (<2-years old) with acute respiratory tract disease. RESULTS: The majority of healthy children seroconverted for HCoV-OC43 (n=19) and HCoV-NL63 (n=17), less for HCoV-HKU1 (n=9) and HCoV-229E (n=5). Notably, HCoV-HKU1 seroconversion was absent after HCoV-OC43 infection. Also HCoV-229E infection was rarely observed after HCoV-NL63 infection (1 out of 5). In the hospital 207 (14%) out of 1471 children were HCoV positive. Again we observed most infection by HCoV-OC43 (n=85) and HCoV-NL63 (n=60), followed by HCoV-HKU1 (n=47) and HCoV-229E (n=15). CONCLUSIONS: HCoV-NL63 and HCoV-OC43 infections occur frequently in early childhood, more often than HCoV-HKU1 or HCoV-229E infections. HCoV-OC43 and HCoV-NL63 may elicit immunity that protects from subsequent HCoV-HKU1 and HCoV-229E infection, respectively, which would explain why HCoV-OC43 and HCoV-NL63 are the most frequently infecting HCoVs. There are no indications that infection by one of the HCoVs is more pathogenic than others.


Assuntos
Infecções por Coronavirus/epidemiologia , Coronavirus Humano NL63/isolamento & purificação , Coronavirus Humano OC43/isolamento & purificação , Infecções Respiratórias/epidemiologia , Anticorpos Antivirais/sangue , Infecções por Coronavirus/fisiopatologia , Infecções por Coronavirus/virologia , Coronavirus Humano NL63/imunologia , Coronavirus Humano NL63/patogenicidade , Coronavirus Humano OC43/imunologia , Coronavirus Humano OC43/patogenicidade , Feminino , Hospitalização/estatística & dados numéricos , Humanos , Lactente , Recém-Nascido , Masculino , Infecções Respiratórias/fisiopatologia , Infecções Respiratórias/virologia , Índice de Gravidade de Doença
20.
Clin Vaccine Immunol ; 18(1): 113-8, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21084464

RESUMO

Human coronaviruses (HCoVs) are responsible for respiratory tract infections ranging from common colds to severe acute respiratory syndrome. HCoV-NL63 and HCoV-229E are two of the four HCoVs that circulate worldwide and are close phylogenetic relatives. HCoV infections can lead to hospitalization of children, elderly individuals, and immunocompromised patients. Globally, approximately 5% of all upper and lower respiratory tract infections in hospitalized children are caused by HCoV-229E and HCoV-NL63. The latter virus has recently been associated with the childhood disease croup. Thus, differentiation between the two viruses is relevant for epidemiology studies. The aim of this study was to develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) as a potential tool for identification and differentiation between HCoV-NL63 and HCoV-229E. The nucleocapsid (N) proteins of HCoV-NL63 and HCoV-229E were expressed in an Escherichia coli system and used to immunize mice in order to obtain monoclonal antibodies (MAbs) specific for each virus. Three specific MAbs to HCoV-NL63, one MAb specific to HCoV-229E, and four MAbs that recognized both viruses were obtained. After their characterization, three MAbs were selected in order to develop a differential DAS-ELISA. The described assay could detect up to 3 ng/ml of N protein and 50 50% tissue culture infective doses/ml of virus stock. No cross-reactivity with other human coronaviruses or closely related animal coronaviruses was found. The newly developed DAS-ELISA was species specific, and therefore, it could be considered a potential tool for detection and differentiation of HCoV-NL63 and HCoV-229E infections.


Assuntos
Anticorpos Monoclonais , Especificidade de Anticorpos , Coronavirus Humano 229E/classificação , Infecções por Coronavirus/diagnóstico , Coronavirus Humano NL63/classificação , Proteínas do Nucleocapsídeo/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Coronavirus Humano 229E/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/microbiologia , Coronavirus Humano NL63/imunologia , Proteínas do Nucleocapsídeo de Coronavírus , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/administração & dosagem , Proteínas do Nucleocapsídeo/química , Especificidade da Espécie
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