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1.
Front Immunol ; 13: 835333, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35359936

RESUMO

Human coronavirus OC43 (HCoV-OC43) is the coronavirus most associated with "common colds", infections of the upper respiratory tract. Previously, we reported that direct interactions of nucleocapsid (N) protein and C-terminal domain of Spike protein (Spike CD) are essential for replication of SARS-CoV-2 and MERS-CoV. Thus, we developed a novel ELISA-based strategy targeting these specific interactions to detect SARS-CoV-2 and MERS-CoV. Here, we investigated whether the same principles apply to HCoV-OC43. We discovered that the S protein of HCoV-OC43 interacts with N protein and that cell penetrating Spike CD peptide inhibits virus protein expression and replication of HCoV-OC43. The interaction between HCoV-OC43 S and N proteins were recapitulated with a recombinant HCoV-OC43 Spike CD fusion protein and a recombinant HCoV-OC43 N fusion protein in vitro. By producing an anti-HCoV-OC43 N protein-specific monoclonal antibody, we established a virus detection system based on the interaction between recombinant Spike CD and N protein of HCoV-OC43. We suggest that the interaction between Spike CD and N protein is conserved in coronaviruses and therefore could be a target for therapeutics against both novel coronavirus and its variants.


Assuntos
COVID-19 , Coronavirus Humano OC43 , Coronavírus da Síndrome Respiratória do Oriente Médio , Proteínas do Nucleocapsídeo de Coronavírus , Humanos , Proteínas do Nucleocapsídeo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus
2.
Cell Rep Med ; 3(2): 100520, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35233545

RESUMO

Effective vaccines are essential for the control of the coronavirus disease 2019 (COVID-19) pandemic. Currently developed vaccines inducing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S)-antigen-specific neutralizing antibodies (NAbs) are effective, but the appearance of NAb-resistant S variant viruses is of great concern. A vaccine inducing S-independent or NAb-independent SARS-CoV-2 control may contribute to containment of these variants. Here, we investigate the efficacy of an intranasal vaccine expressing viral non-S antigens against intranasal SARS-CoV-2 challenge in cynomolgus macaques. Seven vaccinated macaques exhibit significantly reduced viral load in nasopharyngeal swabs on day 2 post-challenge compared with nine unvaccinated controls. The viral control in the absence of SARS-CoV-2-specific NAbs is significantly correlated with vaccine-induced, viral-antigen-specific CD8+ T cell responses. Our results indicate that CD8+ T cell induction by intranasal vaccination can result in NAb-independent control of SARS-CoV-2 infection, highlighting a potential of vaccine-induced CD8+ T cell responses to contribute to COVID-19 containment.


Assuntos
Administração Intranasal/métodos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas contra COVID-19/administração & dosagem , COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Vacinação/métodos , Animais , COVID-19/epidemiologia , COVID-19/virologia , Vacinas contra COVID-19/imunologia , Chlorocebus aethiops , Proteínas do Envelope de Coronavírus/imunologia , Proteínas M de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Modelos Animais de Doenças , Feminino , Macaca fascicularis , Masculino , Pandemias/prevenção & controle , Fosfoproteínas/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Resultado do Tratamento , Células Vero , Carga Viral
3.
Int J Mol Sci ; 23(6)2022 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-35328398

RESUMO

The B and T lymphocytes of the adaptive immune system are important for the control of most viral infections, including COVID-19. Identification of epitopes recognized by these cells is fundamental for understanding how the immune system detects and removes pathogens, and for antiviral vaccine design. Intriguingly, several cross-reactive T lymphocyte epitopes from SARS-CoV-2 with other betacoronaviruses responsible for the common cold have been identified. In addition, antibodies that cross-recognize the spike protein, but not the nucleoprotein (N protein), from different betacoronavirus have also been reported. Using a consensus of eight bioinformatic methods for predicting B-cell epitopes and the collection of experimentally detected epitopes for SARS-CoV and SARS-CoV-2, we identified four surface-exposed, conserved, and hypothetical antigenic regions that are exclusive of the N protein. These regions were analyzed using ELISA assays with two cohorts: SARS-CoV-2 infected patients and pre-COVID-19 samples. Here we describe four epitopes from SARS-CoV-2 N protein that are recognized by the humoral response from multiple individuals infected with COVID-19, and are conserved in other human coronaviruses. Three of these linear surface-exposed sequences and their peptide homologs in SARS-CoV-2 and HCoV-OC43 were also recognized by antibodies from pre-COVID-19 serum samples, indicating cross-reactivity of antibodies against coronavirus N proteins. Different conserved human coronaviruses (HCoVs) cross-reactive B epitopes against SARS-CoV-2 N protein are detected in a significant fraction of individuals not exposed to this pandemic virus. These results have potential clinical implications.


Assuntos
Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Coronavirus Humano OC43/imunologia , Reações Cruzadas/imunologia , Mapeamento de Epitopos/métodos , Epitopos de Linfócito B/imunologia , SARS-CoV-2/imunologia , Adulto , Sequência de Aminoácidos , COVID-19/imunologia , COVID-19/virologia , Estudos de Coortes , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/genética , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/fisiologia , Reações Cruzadas/genética , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos de Linfócito B/metabolismo , Células HEK293 , Pessoal de Saúde/estatística & dados numéricos , Humanos , Domínios Proteicos , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Homologia de Sequência de Aminoácidos , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
5.
Front Immunol ; 13: 816220, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35145522

RESUMO

SARS-CoV-2 variants of concern (VOCs) can trigger severe endemic waves and vaccine breakthrough infections (VBI). We analyzed the cellular and humoral immune response in 8 patients infected with the alpha variant, resulting in moderate to fatal COVID-19 disease manifestation, after double mRNA-based anti-SARS-CoV-2 vaccination. In contrast to the uninfected vaccinated control cohort, the diseased individuals had no detectable high-avidity spike (S)-reactive CD4+ and CD8+ T cells against the alpha variant and wild type (WT) at disease onset, whereas a robust CD4+ T-cell response against the N- and M-proteins was generated. Furthermore, a delayed alpha S-reactive high-avidity CD4+ T-cell response was mounted during disease progression. Compared to the vaccinated control donors, these patients also had lower neutralizing antibody titers against the alpha variant at disease onset. The delayed development of alpha S-specific cellular and humoral immunity upon VBI indicates reduced immunogenicity against the S-protein of the alpha VOC, while there was a higher and earlier N- and M-reactive T-cell response. Our findings do not undermine the current vaccination strategies but underline a potential need for the inclusion of VBI patients in alternative vaccination strategies and additional antigenic targets in next-generation SARS-CoV-2 vaccines.


Assuntos
/imunologia , Anticorpos Neutralizantes/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Afinidade de Anticorpos/imunologia , COVID-19/mortalidade , Proteínas M de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação
6.
Sci Rep ; 12(1): 2853, 2022 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-35181721

RESUMO

Corona Virus Disease 2019 (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This pandemic has brought the world to a standstill and threatened human lives. Many methods are known to date to detect this virus. Due to their relative sensitivity, polymerase chain reaction (PCR)-based assays are the most frequently applied and considered the gold standard. However, due to the rapid mutation rate of the viral genome and the emergence of new variants, existing protocols need to be updated and improved. Designing a fast and accurate PCR-based assay is of great importance for the early detection of this virus and more efficient control of the spread of this disease. This study describes a fast, reliable, easy-to-use, and high-throughput multiplex SARS-CoV-2 RT-PCR detection method. The assay was designed to detect two viral genes (N and RdRP) and a human gene (RP) simultaneously. The performance and the sensitivity of the assay were tested in 28 SARS-CoV-2 positive samples and compared with commercial kits, which showed 100% positive percent agreement with a limit of detection (LOD) value of 1.40 and 0.81 copies/µL or 35.13 and 20.31 copies/reaction for RdRP and N genes, respectively. The current assay is found accurate, reliable, simple, sensitive, and specific. It can be used as an optimized SARS-CoV-2 diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Proteínas do Nucleocapsídeo de Coronavírus/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Ribonuclease P/genética , SARS-CoV-2/isolamento & purificação , Humanos
7.
Front Immunol ; 13: 811952, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35126396

RESUMO

Numerous studies have suggested that the titers of antibodies against SARS-CoV-2 are associated with the COVID-19 severity, however, the types of antibodies associated with the disease maximum severity and the timing at which the associations are best observed, especially within one week after symptom onset, remain controversial. We attempted to elucidate the antibody responses against SARS-CoV-2 that are associated with the maximum severity of COVID-19 in the early phase of the disease, and to investigate whether antibody testing might contribute to prediction of the disease maximum severity in COVID-19 patients. We classified the patients into four groups according to the disease maximum severity (severity group 1 (did not require oxygen supplementation), severity group 2a (required oxygen supplementation at low flow rates), severity group 2b (required oxygen supplementation at relatively high flow rates), and severity group 3 (required mechanical ventilatory support)), and serially measured the titers of IgM, IgG, and IgA against the nucleocapsid protein, spike protein, and receptor-binding domain of SARS-CoV-2 until day 12 after symptom onset. The titers of all the measured antibody responses were higher in severity group 2b and 3, especially severity group 2b, as early as at one week after symptom onset. Addition of data obtained from antibody testing improved the ability of analysis models constructed using a machine learning technique to distinguish severity group 2b and 3 from severity group 1 and 2a. These models constructed with non-vaccinated COVID-19 patients could not be applied to the cases of breakthrough infections. These results suggest that antibody testing might help physicians identify non-vaccinated COVID-19 patients who are likely to require admission to an intensive care unit.


Assuntos
Anticorpos Antivirais/sangue , Vacinas contra COVID-19/sangue , COVID-19/sangue , SARS-CoV-2/imunologia , Índice de Gravidade de Doença , Formação de Anticorpos/imunologia , COVID-19/imunologia , COVID-19/patologia , Vacinas contra COVID-19/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Aprendizado de Máquina , Domínios Proteicos/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Fatores de Tempo , Vacinação
8.
Microb Cell Fact ; 21(1): 21, 2022 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-35123472

RESUMO

We have developed a method for the inexpensive, high-level expression of antigenic protein fragments of SARS-CoV-2 proteins in Escherichia coli. Our approach uses the thermophilic family 9 carbohydrate-binding module (CBM9) as an N-terminal carrier protein and affinity tag. The CBM9 module was joined to SARS-CoV-2 protein fragments via a flexible proline-threonine linker, which proved to be resistant to E. coli proteases. Two CBM9-spike protein fragment fusion proteins and one CBM9-nucleocapsid fragment fusion protein largely resisted protease degradation, while most of the CBM9 fusion proteins were degraded at some site in the SARS-CoV-2 protein fragment. All of the fusion proteins were highly expressed in E. coli and the CBM9-ID-H1 fusion protein was shown to yield 122 mg/L of purified product. Three purified CBM9-SARS-CoV-2 fusion proteins were tested and found to bind antibodies directed to the appropriate SARS-CoV-2 antigenic regions. The largest intact CBM9 fusion protein, CBM9-ID-H1, incorporates spike protein amino acids 540-588, which is a conserved region overlapping and C-terminal to the receptor binding domain that is widely recognized by human convalescent sera and contains a putative protective epitope.


Assuntos
Proteínas do Nucleocapsídeo de Coronavírus/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Antivirais/imunologia , Reações Antígeno-Anticorpo , COVID-19/patologia , COVID-19/virologia , Cromatografia Líquida de Alta Pressão , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Humanos , Espectrometria de Massas , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/metabolismo
9.
Front Immunol ; 13: 817905, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35185909

RESUMO

The duration of humoral and cellular immune memory following SARS-CoV-2 infection in populations in least developed countries remains understudied but is key to overcome the current SARS-CoV-2 pandemic. Sixty-four Cambodian individuals with laboratory-confirmed infection with asymptomatic or mild/moderate clinical presentation were evaluated for Spike (S)-binding and neutralizing antibodies and antibody effector functions during acute phase of infection and at 6-9 months follow-up. Antigen-specific B cells, CD4+ and CD8+ T cells were characterized, and T cells were interrogated for functionality at late convalescence. Anti-S antibody titers decreased over time, but effector functions mediated by S-specific antibodies remained stable. S- and nucleocapsid (N)-specific B cells could be detected in late convalescence in the activated memory B cell compartment and are mostly IgG+. CD4+ and CD8+ T cell immune memory was maintained to S and membrane (M) protein. Asymptomatic infection resulted in decreased antibody-dependent cellular cytotoxicity (ADCC) and frequency of SARS-CoV-2-specific CD4+ T cells at late convalescence. Whereas anti-S antibodies correlated with S-specific B cells, there was no correlation between T cell response and humoral immune memory. Hence, all aspects of a protective immune response are maintained up to nine months after SARS-CoV-2 infection and in the absence of re-infection.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/imunologia , SARS-CoV-2/imunologia , Linfócitos B/imunologia , COVID-19/patologia , Camboja , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Humanos , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Fosfoproteínas/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia
10.
Front Immunol ; 13: 776861, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35185878

RESUMO

Cardiovascular dysfunction and disease are common and frequently fatal complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Indeed, from early on during the SARS-CoV-2 virus pandemic it was recognized that cardiac complications may occur, even in patients with no underlying cardiac disorders, as part of the acute infection, and that these were associated with more severe disease and increased morbidity and mortality. The most common cardiac complication is acute cardiac injury, defined by significant elevation of cardiac troponins. The potential mechanisms of cardiovascular complications include direct viral myocardial injury, systemic inflammation induced by the virus, sepsis, arrhythmia, myocardial oxygen supply-demand mismatch, electrolyte abnormalities, and hypercoagulability. This review is focused on the prevalence, risk factors and clinical course of COVID-19-related myocardial injury, as well as on current data with regard to disease pathogenesis, specifically the interaction of platelets with the vascular endothelium. The latter section includes consideration of the role of SARS-CoV-2 proteins in triggering development of a generalized endotheliitis that, in turn, drives intense activation of platelets. Most prominently, SARS-CoV-2-induced endotheliitis involves interaction of the viral spike protein with endothelial angiotensin-converting enzyme 2 (ACE2) together with alternative mechanisms that involve the nucleocapsid and viroporin. In addition, the mechanisms by which activated platelets intensify endothelial activation and dysfunction, seemingly driven by release of the platelet-derived calcium-binding proteins, SA100A8 and SA100A9, are described. These events create a SARS-CoV-2-driven cycle of intravascular inflammation and coagulation, which contributes significantly to a poor clinical outcome in patients with severe disease.


Assuntos
Plaquetas/metabolismo , COVID-19/patologia , Doenças Cardiovasculares/patologia , Endotélio Vascular/metabolismo , Ativação Plaquetária/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/mortalidade , Doenças Cardiovasculares/virologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Células Endoteliais/metabolismo , Humanos , Miocárdio/patologia , Fosfoproteínas/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
11.
Nat Immunol ; 23(2): 275-286, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35102342

RESUMO

The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in coronavirus disease 2019 (COVID-19). The present study was designed to conduct a systematic investigation of the interaction of human humoral fluid-phase pattern recognition molecules (PRMs) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of 12 PRMs tested, the long pentraxin 3 (PTX3) and mannose-binding lectin (MBL) bound the viral nucleocapsid and spike proteins, respectively. MBL bound trimeric spike protein, including that of variants of concern (VoC), in a glycan-dependent manner and inhibited SARS-CoV-2 in three in vitro models. Moreover, after binding to spike protein, MBL activated the lectin pathway of complement activation. Based on retention of glycosylation sites and modeling, MBL was predicted to recognize the Omicron VoC. Genetic polymorphisms at the MBL2 locus were associated with disease severity. These results suggest that selected humoral fluid-phase PRMs can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.


Assuntos
COVID-19/imunologia , Imunidade Humoral , Receptores de Reconhecimento de Padrão/imunologia , SARS-CoV-2/imunologia , Animais , Proteína C-Reativa/imunologia , Proteína C-Reativa/metabolismo , COVID-19/metabolismo , COVID-19/virologia , Estudos de Casos e Controles , Chlorocebus aethiops , Ativação do Complemento , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Feminino , Glicosilação , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Masculino , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/imunologia , Lectina de Ligação a Manose/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Polimorfismo Genético , Ligação Proteica , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Componente Amiloide P Sérico/imunologia , Componente Amiloide P Sérico/metabolismo , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero
12.
J Virol Methods ; 302: 114486, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35108595

RESUMO

BACKGROUND: Recently, the Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 infection has spread rapidly around the world, becoming a new global pandemic disease. Nucleic acid detection is the primary method for clinical diagnosis of SARS-CoV-2 infection, with the addition of antibody and antigen detection. Nucleocapsid protein (NP) is a kind of conservative structural protein with abundant expression during SARS-CoV-2 infection, which makes it an ideal target for immunoassay. METHODS: The coding sequence for SARS-CoV-2-NP was obtained by chemical synthesis, and then inserted into pET28a(+). The soluble recombinant NP (rNP) with an estimated molecular weight of 49.4 kDa was expressed in E. coli cells after IPTG induction. Six-week-old BALB/c mice were immunized with rNP, and then their spleen cells were fused with SP2/0 cells, to develop hybridoma cell lines that stably secreted monoclonal antibodies (mAbs) against NP. The mAbs were preliminarily evaluated by enzyme-linked immunosorbent assay (ELISA), and then used to develop a magnetic particle-based chemiluminescence enzyme immunoassay (CLEIA) for measurement of SARS-CoV-2-NP. RESULTS: mAb 15B1 and mAb 18G10 were selected as capture and detection antibody respectively to develop CLEIA, due to the highest sensitivity for rNP detection. The proposed CLEIA presented a good linearity for rNP detection at a working range from 0.1 to 160 µg/L, with a precision coefficient of variance below 10 %. CONCLUSION: The newly developed mAbs and CLEIA can serve as potential diagnostic tools for clinical measurement of SARS-CoV-2-NP.


Assuntos
COVID-19 , Proteínas do Nucleocapsídeo de Coronavírus , SARS-CoV-2 , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/metabolismo , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/análise , Proteínas do Nucleocapsídeo de Coronavírus/genética , Escherichia coli/genética , Humanos , Imunoensaio/métodos , Luminescência , Camundongos , Fosfoproteínas/análise , Fosfoproteínas/genética , Sensibilidade e Especificidade
13.
Nat Commun ; 13(1): 601, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35105893

RESUMO

Monitoring SARS-CoV-2 spread and evolution through genome sequencing is essential in handling the COVID-19 pandemic. Here, we sequenced 892 SARS-CoV-2 genomes collected from patients in Saudi Arabia from March to August 2020. We show that two consecutive mutations (R203K/G204R) in the nucleocapsid (N) protein are associated with higher viral loads in COVID-19 patients. Our comparative biochemical analysis reveals that the mutant N protein displays enhanced viral RNA binding and differential interaction with key host proteins. We found increased interaction of GSK3A kinase simultaneously with hyper-phosphorylation of the adjacent serine site (S206) in the mutant N protein. Furthermore, the host cell transcriptome analysis suggests that the mutant N protein produces dysregulated interferon response genes. Here, we provide crucial information in linking the R203K/G204R mutations in the N protein to modulations of host-virus interactions and underline the potential of the nucleocapsid protein as a drug target during infection.


Assuntos
COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , Genoma Viral , Mutação de Sentido Incorreto , SARS-CoV-2/genética , COVID-19/enzimologia , COVID-19/genética , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Fosforilação , Filogenia , Ligação Proteica , SARS-CoV-2/classificação , SARS-CoV-2/fisiologia , Arábia Saudita , Carga Viral , Replicação Viral
14.
PLoS One ; 17(2): e0262591, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35113919

RESUMO

SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/química , COVID-19/sangue , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/química , Imunoglobulina G/sangue , Corpos de Inclusão/química , Redobramento de Proteína , SARS-CoV-2/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Pressão Hidrostática , Imunoglobulina G/imunologia , Fosfoproteínas/química , Fosfoproteínas/imunologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Solubilidade
15.
Microbiol Spectr ; 10(1): e0256321, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35196819

RESUMO

We have previously used composite reference standards and latent class analysis (LCA) to evaluate the performance of laboratory assays in the presence of tarnished gold standards. Here, we apply these techniques to repeated, cross-sectional study of Canadian blood donors, whose sera underwent parallel testing with four separate SARS-CoV-2 antibody assays. We designed a repeated cross-sectional design with random cross-sectional sampling of all available retention samples (n = 1500/month) for a 12 -month period from April 2020 until March 2021. Each sample was evaluated for SARS-CoV-2 IgG antibodies using four assays an Abbott Architect assay targeting the nucleocapsid antigen (Abbott-NP, Abbott, Chicago IL) and three in-house IgG ELISAs recognizing distinct recombinant viral antigens: full-length spike glycoprotein (Spike), spike glycoprotein receptor binding domain (RBD) and nucleocapsid (NP). We used two analytic approaches to estimate SAR-CoV-2 seroprevalence: a composite reference standard and LCA. Using LCA to estimate true seropositivity status based on the results of the four antibody tests, we estimated that seroprevalence increased from 0.8% (95% CI: 0.5-1.4%) in April 2020 to 6.3% (95% CI: 5.1-7.6%) in March 2021. Our study provides further support for the use of LCA in upcoming public health crises, epidemics, and pandemics when a gold standard assay may not be available or identifiable. IMPORTANCE Here, we describe an approach to estimating seroprevalence in a low prevalence setting when multiple assays are available and yet no known gold standard exists. Because serological studies identify cases through both diagnostic testing and surveillance, and otherwise silent, unrecognized infections, serological data can be used to estimate the true infection fatality ratio of a disease. However, seroprevalence studies rely on assays with imperfect sensitivity and specificity. Seroreversion (loss of antibody response) also occurs over time, and with the advent of vaccination, distinction of antibody response resulting from vaccination as opposed to antibody response due to infection has posed an additional challenge. Our approach indicates that seroprevalence on Canadian blood donors by the end of March 2021was less than 10%. Our study supports the use of latent class analysis in upcoming public health crises, epidemics, and pandemics when a gold standard assay may not be available or identifiable.


Assuntos
Anticorpos Antivirais/sangue , Doadores de Sangue/estatística & dados numéricos , COVID-19/sangue , SARS-CoV-2/imunologia , Adulto , Idoso , COVID-19/epidemiologia , COVID-19/virologia , Canadá/epidemiologia , Proteínas do Nucleocapsídeo de Coronavírus/análise , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/genética , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/análise , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto Jovem
16.
Int J Mol Sci ; 23(4)2022 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-35216056

RESUMO

The development of prophylactic agents against the SARS-CoV-2 virus is a public health priority in the search for new surrogate markers of active virus replication. Early detection markers are needed to follow disease progression and foresee patient negativization. Subgenomic RNA transcripts (with a focus on sgN) were evaluated in oro/nasopharyngeal swabs from COVID-19-affected patients with an analysis of 315 positive samples using qPCR technology. Cut-off Cq values for sgN (Cq < 33.15) and sgE (Cq < 34.06) showed correlations to high viral loads. The specific loss of sgN in home-isolated and hospitalized COVID-19-positive patients indicated negativization of patient condition, 3-7 days from the first swab, respectively. A new detection kit for sgN, gene E, gene ORF1ab, and gene RNAse P was developed recently. In addition, in vitro studies have shown that 2'-O-methyl antisense RNA (related to the sgN sequence) can impair SARS-CoV-2 N protein synthesis, viral replication, and syncytia formation in human cells (i.e., HEK-293T cells overexpressing ACE2) upon infection with VOC Alpha (B.1.1.7)-SARS-CoV-2 variant, defining the use that this procedure might have for future therapeutic actions against SARS-CoV-2.


Assuntos
COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , SARS-CoV-2/fisiologia , Replicação Viral/fisiologia , Proteínas do Nucleocapsídeo de Coronavírus/análise , Células Gigantes/efeitos dos fármacos , Células Gigantes/virologia , Células HEK293 , Humanos , Limite de Detecção , Nasofaringe/virologia , Fosfoproteínas/análise , Fosfoproteínas/genética , RNA Antissenso/farmacologia , RNA Viral , Ribonuclease P/genética , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Sensibilidade e Especificidade , Isolamento Social , Carga Viral , Proteínas Viroporinas/genética , Replicação Viral/efeitos dos fármacos
17.
Stem Cell Reports ; 17(3): 522-537, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35180394

RESUMO

Patients with coronavirus disease 2019 (COVID-19) commonly have manifestations of heart disease. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome encodes 27 proteins. Currently, SARS-CoV-2 gene-induced abnormalities of human heart muscle cells remain elusive. Here, we comprehensively characterized the detrimental effects of a SARS-CoV-2 gene, Orf9c, on human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) by preforming multi-omic analyses. Transcriptomic analyses of hPSC-CMs infected by SARS-CoV-2 with Orf9c overexpression (Orf9cOE) identified concordantly up-regulated genes enriched into stress-related apoptosis and inflammation signaling pathways, and down-regulated CM functional genes. Proteomic analysis revealed enhanced expressions of apoptotic factors, whereas reduced protein factors for ATP synthesis by Orf9cOE. Orf9cOE significantly reduced cellular ATP level, induced apoptosis, and caused electrical dysfunctions of hPSC-CMs. Finally, drugs approved by the U.S. Food and Drug Administration, namely, ivermectin and meclizine, restored ATP levels and ameliorated CM death and functional abnormalities of Orf9cOE hPSC-CMs. Overall, we defined the molecular mechanisms underlying the detrimental impacts of Orf9c on hPSC-CMs and explored potentially therapeutic approaches to ameliorate Orf9c-induced cardiac injury and abnormalities.


Assuntos
COVID-19/patologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , Estudo de Associação Genômica Ampla/métodos , SARS-CoV-2/genética , Potenciais de Ação/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , COVID-19/virologia , Regulação para Baixo , Humanos , Ivermectina/farmacologia , Meclizina/farmacologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fosfoproteínas/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Mapas de Interação de Proteínas/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , SARS-CoV-2/isolamento & purificação , Transdução de Sinais/genética , Transcriptoma/efeitos dos fármacos , Regulação para Cima
18.
ACS Appl Mater Interfaces ; 14(8): 10844-10855, 2022 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-35172574

RESUMO

The widespread and long-lasting effect of the COVID-19 pandemic has called attention to the significance of technological advances in the rapid diagnosis of SARS-CoV-2 virus. This study reports the use of a highly stable buffer-based zinc oxide/reduced graphene oxide (bbZnO/rGO) nanocomposite coated on carbon screen-printed electrodes for electrochemical immuno-biosensing of SARS-CoV-2 nuelocapsid (N-) protein antigens in spiked and clinical samples. The incorporation of a salt-based (ionic) matrix for uniform dispersion of the nanomixture eliminates multistep nanomaterial synthesis on the surface of the electrode and enables a stable single-step sensor nanocoating. The immuno-biosensor provides a limit of detection of 21 fg/mL over a linear range of 1-10 000 pg/mL and exhibits a sensitivity of 32.07 ohms·mL/pg·mm2 for detection of N-protein in spiked samples. The N-protein biosensor is successful in discriminating positive and negative clinical samples within 15 min, demonstrating its proof of concept used as a COVID-19 rapid antigen test.


Assuntos
Antígenos Virais/análise , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/análise , Grafite/química , Nanocompostos/química , Óxido de Zinco/química , Anticorpos Imobilizados/imunologia , Antígenos Virais/imunologia , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Humanos , Imunoensaio/instrumentação , Imunoensaio/métodos , Limite de Detecção , Fosfoproteínas/análise , Fosfoproteínas/imunologia , Estudo de Prova de Conceito , SARS-CoV-2/química
19.
Nat Commun ; 13(1): 915, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35177626

RESUMO

Quantitative or qualitative differences in immunity may drive clinical severity in COVID-19. Although longitudinal studies to record the course of immunological changes are ample, they do not necessarily predict clinical progression at the time of hospital admission. Here we show, by a machine learning approach using serum pro-inflammatory, anti-inflammatory and anti-viral cytokine and anti-SARS-CoV-2 antibody measurements as input data, that COVID-19 patients cluster into three distinct immune phenotype groups. These immune-types, determined by unsupervised hierarchical clustering that is agnostic to severity, predict clinical course. The identified immune-types do not associate with disease duration at hospital admittance, but rather reflect variations in the nature and kinetics of individual patient's immune response. Thus, our work provides an immune-type based scheme to stratify COVID-19 patients at hospital admittance into high and low risk clinical categories with distinct cytokine and antibody profiles that may guide personalized therapy.


Assuntos
Anticorpos Antivirais/sangue , COVID-19/patologia , Citocinas/sangue , SARS-CoV-2/imunologia , Índice de Gravidade de Doença , Idoso , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Progressão da Doença , Feminino , Hospitalização , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Imunofenotipagem/métodos , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/imunologia
20.
Sci Rep ; 12(1): 1005, 2022 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-35046461

RESUMO

The pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused a public health emergency, and research on the development of various types of vaccines is rapidly progressing at an unprecedented development speed internationally. Some vaccines have already been approved for emergency use and are being supplied to people around the world, but there are still many ongoing efforts to create new vaccines. Virus-like particles (VLPs) enable the construction of promising platforms in the field of vaccine development. Here, we demonstrate that non-infectious SARS-CoV-2 VLPs can be successfully assembled by co-expressing three important viral proteins membrane (M), envelop (E) and nucleocapsid (N) in plants. Plant-derived VLPs were purified by sedimentation through a sucrose cushion. The shape and size of plant-derived VLPs are similar to native SARS-CoV-2 VLPs without spike. Although the assembled VLPs do not have S protein spikes, they could be developed as formulations that can improve the immunogenicity of vaccines including S antigens, and further could be used as platforms that can carry S antigens of concern for various mutations.


Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/imunologia , Proteínas M de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas Viroporinas/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Proteínas M de Coronavírus/genética , Proteínas M de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Humanos , Tabaco/imunologia , Tabaco/metabolismo , Tabaco/virologia , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/metabolismo , Proteínas Viroporinas/genética , Proteínas Viroporinas/metabolismo
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