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1.
Proc Natl Acad Sci U S A ; 118(16)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33811184

RESUMO

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection; induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung; and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, whereas PKR activation is evident in iAT2 and iCM. In SARS-CoV-2-infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however, activation of OAS-RNase L and PKR is observed. This is in contrast to Middle East respiratory syndrome (MERS)-CoV, which effectively inhibits IFN signaling and OAS-RNase L and PKR pathways, but is similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.


Assuntos
Células Epiteliais/imunologia , Células Epiteliais/virologia , Imunidade Inata , Pulmão/patologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/virologia , RNA de Cadeia Dupla/metabolismo , /imunologia , Células A549 , Endorribonucleases/metabolismo , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Nariz/virologia , Replicação Viral , eIF-2 Quinase
2.
mBio ; 12(2)2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33653888

RESUMO

There are no approved vaccines against the life-threatening Middle East respiratory syndrome coronavirus (MERS-CoV). Attenuated vaccines have proven their potential to induce strong and long-lasting immune responses. We have previously described that severe acute respiratory syndrome coronavirus (SARS-CoV) envelope (E) protein is a virulence factor. Based on this knowledge, a collection of mutants carrying partial deletions spanning the C-terminal domain of the E protein (rMERS-CoV-E*) has been generated using a reverse genetics system. One of these mutants, MERS-CoV-E*Δ2in, was attenuated and provided full protection in a challenge with virulent MERS-CoV after a single immunization dose. The MERS-CoV-E*Δ2in mutant was stable as it maintained its attenuation after 16 passages in cell cultures and has been selected as a promising vaccine candidate.IMPORTANCE The emergence of the new highly pathogenic human coronavirus SARS-CoV-2 that has already infected more than 80 million persons, killing nearly two million of them, clearly indicates the need to design efficient and safe vaccines protecting from these coronaviruses. Modern vaccines can be derived from virus-host interaction research directed to the identification of signaling pathways essential for virus replication and for virus-induced pathogenesis, in order to learn how to attenuate these viruses and design vaccines. Using a reverse genetics system developed in our laboratory, an infectious cDNA clone of MERS-CoV was engineered. Using this cDNA, we sequentially deleted several predicted and conserved motifs within the envelope (E) protein of MERS-CoV, previously associated with the presence of virulence factors. The in vitro and in vivo evaluation of these deletion mutants highlighted the relevance of predicted linear motifs in viral pathogenesis. Two of them, an Atg8 protein binding motif (Atg8-BM), and a forkhead-associated binding motif (FHA-BM), when deleted, rendered an attenuated virus that was evaluated as a vaccine candidate, leading to full protection against challenge with a lethal dose of MERS-CoV. This approach can be extended to the engineering of vaccines protecting against the new pandemic SARS-CoV-2.


Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , /imunologia , Engenharia Genética/métodos , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Vacinas Atenuadas/uso terapêutico , Vacinas Virais/uso terapêutico
3.
Front Immunol ; 12: 629193, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33732251

RESUMO

Hyper-induction of pro-inflammatory cytokines, also known as a cytokine storm or cytokine release syndrome (CRS), is one of the key aspects of the currently ongoing SARS-CoV-2 pandemic. This process occurs when a large number of innate and adaptive immune cells activate and start producing pro-inflammatory cytokines, establishing an exacerbated feedback loop of inflammation. It is one of the factors contributing to the mortality observed with coronavirus 2019 (COVID-19) for a subgroup of patients. CRS is not unique to the SARS-CoV-2 infection; it was prevalent in most of the major human coronavirus and influenza A subtype outbreaks of the past two decades (H5N1, SARS-CoV, MERS-CoV, and H7N9). With a comprehensive literature search, we collected changing the cytokine levels from patients upon infection with the viral pathogens mentioned above. We analyzed published patient data to highlight the conserved and unique cytokine responses caused by these viruses. Our curation indicates that the cytokine response induced by SARS-CoV-2 is different compared to other CRS-causing respiratory viruses, as SARS-CoV-2 does not always induce specific cytokines like other coronaviruses or influenza do, such as IL-2, IL-10, IL-4, or IL-5. Comparing the collated cytokine responses caused by the analyzed viruses highlights a SARS-CoV-2-specific dysregulation of the type-I interferon (IFN) response and its downstream cytokine signatures. The map of responses gathered in this study could help specialists identify interventions that alleviate CRS in different diseases and evaluate whether they could be used in the COVID-19 cases.


Assuntos
/imunologia , Síndrome da Liberação de Citocina/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Índice de Gravidade de Doença , /sangue , /virologia , Síndrome da Liberação de Citocina/sangue , Síndrome da Liberação de Citocina/virologia , Citocinas/sangue , Humanos , Inflamação/imunologia , Influenza Humana/sangue , Influenza Humana/virologia , Síndrome Respiratória Aguda Grave/sangue , Síndrome Respiratória Aguda Grave/virologia
4.
Arch Immunol Ther Exp (Warsz) ; 69(1): 5, 2021 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-33677719

RESUMO

Coronaviruses share conservative spike protein (S) on their enveloped membrane surface, where S1 subunit recognizes and binds the cellular receptor, and the S2 subunit mediates membrane fusion. This similarity raises the question: does coronaviral infection by one create protection to others? Convalescent SARS-CoV-2 (COVID-19) sera were tested for cross reactivity with peptides from Middle East respiratory syndrome coronavirus (MERS-CoV) which shares 74% homology. Our results showed significant cross-reactivity with a peptide of the heptad repeat 2 (HR2) domain of the MERS-CoV spike protein. Sera samples of 47 validated seropositive convalescent COVID-19 patients and 40 sera samples of control patients, collected in pre-COVID time were used to establish cross-bind reactivity with the MERS-CoV peptide. Significantly stronger binding (p < 0.0001) was observed for IgG antibodies in convalescent COVID-19 patients compared to the control group. In ELISA, MERS-CoV peptide helps to discriminate post-COVID-19 populations and non-infected ones by the presence of antibodies in blood samples. This suggests that polyclonal antibodies established during SARS-CoV-2 infection can recognize and probably decrease severity of MERS-CoV and other coronaviral infections. The high homology of the spike protein domain also suggests that the opposite effect can be true: coronaviral infections produce cross-reactive antibodies effective against SARS-CoV-2. The collected data prove that despite the core HR2 region is hidden in the native viral conformation, its exposure during cell entry makes it highly immunogenic. Since inhibitory peptides to this region were previously described, this opens new possibilities in fighting coronaviral infections and developing vaccines effective even after possible viral mutations.


Assuntos
Anticorpos Antivirais/imunologia , Convalescença , Glicoproteína da Espícula de Coronavírus/imunologia , Reações Cruzadas , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Vírus da SARS/imunologia
5.
Viruses ; 13(2)2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33671602

RESUMO

Middle East respiratory syndrome is a severe respiratory illness caused by an infectious coronavirus. This virus is associated with a high mortality rate, but there is as of yet no effective vaccine or antibody available for human immunity/treatment. Drug design relies on understanding the 3D structures of viral proteins; however, arriving at such understanding is difficult for intrinsically disordered proteins, whose disorder-dependent functions are key to the virus's biology. Disorder is suggested to provide viral proteins with highly flexible structures and diverse functions that are utilized when invading host organisms and adjusting to new habitats. To date, the functional roles of intrinsically disordered proteins in the mechanisms of MERS-CoV pathogenesis, transmission, and treatment remain unclear. In this study, we performed structural analysis to evaluate the abundance of intrinsic disorder in the MERS-CoV proteome and in individual proteins derived from the MERS-CoV genome. Moreover, we detected disordered protein binding regions, namely, molecular recognition features and short linear motifs. Studying disordered proteins/regions in MERS-CoV could contribute to unlocking the complex riddles of viral infection, exploitation strategies, and drug development approaches in the near future by making it possible to target these important (yet challenging) unstructured regions.


Assuntos
Infecções por Coronavirus/virologia , Proteínas Intrinsicamente Desordenadas/química , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Proteínas não Estruturais Virais/química , Bases de Dados de Proteínas , Humanos , Domínios Proteicos
6.
JCI Insight ; 6(5)2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33529170

RESUMO

The development of prophylactic and therapeutic agents for coronavirus disease 2019 (COVID-19) is a current global health priority. Here, we investigated the presence of cross-neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in dromedary camels that were Middle East respiratory syndrome coronavirus (MERS-CoV) seropositive but MERS-CoV free. The tested 229 dromedaries had anti-MERS-CoV camel antibodies with variable cross-reactivity patterns against SARS-CoV-2 proteins, including the S trimer and M, N, and E proteins. Using SARS-CoV-2 competitive immunofluorescence immunoassays and pseudovirus neutralization assays, we found medium-to-high titers of cross-neutralizing antibodies against SARS-CoV-2 in these animals. Through linear B cell epitope mapping using phage immunoprecipitation sequencing and a SARS-CoV-2 peptide/proteome microarray, we identified a large repertoire of Betacoronavirus cross-reactive antibody specificities in these dromedaries and demonstrated that the SARS-CoV-2-specific VHH antibody repertoire is qualitatively diverse. This analysis revealed not only several SARS-CoV-2 epitopes that are highly immunogenic in humans, including a neutralizing epitope, but also epitopes exclusively targeted by camel antibodies. The identified SARS-CoV-2 cross-neutralizing camel antibodies are not proposed as a potential treatment for COVID-19. Rather, their presence in nonimmunized camels supports the development of SARS-CoV-2 hyperimmune camels, which could be a prominent source of therapeutic agents for the prevention and treatment of COVID-19.


Assuntos
Anticorpos Neutralizantes/imunologia , Camelus/imunologia , /imunologia , Anticorpos de Domínio Único/farmacologia , Animais , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Camelus/virologia , Reações Cruzadas , Epitopos , Feminino , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Vírus da SARS/imunologia
7.
Life Sci ; 272: 119245, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33609539

RESUMO

In the past 20 years, infections caused by coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2 have posed a threat to public health since they may cause severe acute respiratory syndrome (SARS) in humans. The Complement System is activated during viral infection, being a central protagonist of innate and acquired immunity. Here, we report some interactions between these three coronaviruses and the Complement System, highlighting the central role of C3 with the severity of these infections. Although it can be protective, its role during coronavirus infections seems to be contradictory. For example, during SARS-CoV-2 infection, Complement System can control the viral infection in asymptomatic or mild cases; however, it can also intensify local and systemic damage in some of severe COVID-19 patients, due to its potent proinflammatory effect. In this last condition, the activation of the Complement System also amplifies the cytokine storm and the pathogenicity of coronavirus infection. Experimental treatment with Complement inhibitors has been an enthusiastic field of intense investigation in search of a promising additional therapy in severe COVID-19 patients.


Assuntos
/imunologia , Proteínas do Sistema Complemento/imunologia , /imunologia , Animais , /tratamento farmacológico , Ativação do Complemento/efeitos dos fármacos , Complemento C3/imunologia , Inativadores do Complemento/farmacologia , Inativadores do Complemento/uso terapêutico , Infecções por Coronavirus/complicações , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Síndrome da Liberação de Citocina/complicações , Síndrome da Liberação de Citocina/tratamento farmacológico , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/patologia , Humanos , Inflamação/complicações , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/complicações , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/patologia
8.
Biomed Res Int ; 2021: 8870425, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33564683

RESUMO

Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and Coronavirus Disease 2019 (COVID-19) infections are the three epidemiological diseases caused by the Coronaviridae family. Perceiving the immune responses in these infections and the escape of viruses could help us design drugs and vaccines for confronting these infections. This review investigates the innate and adaptive immune responses reported in the infections of the three coronaviruses SARS, MERS, and COVID-19. Moreover, the present study can trigger researchers to design and develop new vaccines and drugs based on immune system responses. In conclusion, due to the need for an effective and efficient immune stimulation against coronavirus, a combination of several strategies seems necessary for developing the vaccine.


Assuntos
/imunologia , Infecções por Coronavirus/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Imunidade Adaptativa , Animais , Infecções por Coronavirus/prevenção & controle , Humanos , Imunidade Inata , Síndrome Respiratória Aguda Grave/prevenção & controle
9.
Int J Med Sci ; 18(3): 763-767, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33437211

RESUMO

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is an emerging disease. There has been a rapid increase in cases and deaths since it was identified in Wuhan, China, in early December 2019, with over 4,000,000 cases of COVID-19 including at least 250,000 deaths worldwide as of May 2020. However, limited data about the clinical characteristics of pregnant women with COVID-19 have been reported. Given the maternal physiologic and immune function changes during pregnancy, pregnant women may be at a higher risk of being infected with SARS-CoV-2 and developing more complicated clinical events. Information on severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) may provide insights into the effects of COVID-19's during pregnancy. Even though SARS and MERS have been associated with miscarriage, intrauterine death, fetal growth restriction and high case fatality rates, the clinical course of COVID-19 pneumonia in pregnant women has been reported to be similar to that in non-pregnant women. In addition, pregnant women do not appear to be at a higher risk of catching COVID-19 or suffering from more severe disease than other adults of similar age. Moreover, there is currently no evidence that the virus can be transmitted to the fetus during pregnancy or during childbirth. Babies and young children are also known to only experience mild forms of COVID-19. The aims of this systematic review were to summarize the possible symptoms, treatments, and pregnancy outcomes of women infected with COVID-19 during pregnancy.


Assuntos
/epidemiologia , Transmissão Vertical de Doença Infecciosa , Complicações Infecciosas na Gravidez/epidemiologia , Resultado da Gravidez , /imunologia , Adulto , /terapia , Feminino , Humanos , Recém-Nascido , Exposição Materna , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/terapia , Complicações Infecciosas na Gravidez/virologia , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/epidemiologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/virologia , Índice de Gravidade de Doença
10.
Nat Commun ; 12(1): 6, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397903

RESUMO

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/sangue , /imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , /diagnóstico , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Análise em Microsséries/métodos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Testes de Neutralização , Vírus da SARS/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia
11.
JCI Insight ; 6(1)2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33427208

RESUMO

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), coupled with a lack of therapeutics, has paralyzed the globe. Although significant effort has been invested in identifying antibodies that block infection, the ability of antibodies to target infected cells through Fc interactions may be vital to eliminate the virus. To explore the role of Fc activity in SARS-CoV-2 immunity, the functional potential of a cross-SARS-reactive antibody, CR3022, was assessed. CR3022 was able to broadly drive antibody effector functions, providing critical immune clearance at entry and upon egress. Using selectively engineered Fc variants, no protection was observed after administration of WT IgG1 in mice or hamsters. Conversely, the functionally enhanced Fc variant resulted in increased pathology in both the mouse and hamster models, causing weight loss in mice and enhanced viral replication and weight loss in the more susceptible hamster model, highlighting the pathological functions of Fc-enhancing mutations. These data point to the critical need for strategic Fc engineering for the treatment of SARS-CoV-2 infection.


Assuntos
Anticorpos Neutralizantes/farmacologia , Imunidade Inata/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/uso terapêutico , /fisiopatologia , Cricetinae , Reações Cruzadas , Epitopos , Humanos , Imunidade Inata/imunologia , Imunoglobulina G/genética , Imunoglobulina G/uso terapêutico , Mesocricetus , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Engenharia de Proteínas , Receptores Fc/imunologia , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/imunologia , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologia , Células THP-1 , Carga Viral/efeitos dos fármacos , Perda de Peso/efeitos dos fármacos
12.
Viruses ; 13(1)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33477902

RESUMO

Coronavirus research has gained tremendous attention because of the COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus (nCoV or SARS-CoV-2). In this review, we highlight recent studies that provide atomic-resolution structural details important for the development of monoclonal antibodies (mAbs) that can be used therapeutically and prophylactically and for vaccines against SARS-CoV-2. Structural studies with SARS-CoV-2 neutralizing mAbs have revealed a diverse set of binding modes on the spike's receptor-binding domain and N-terminal domain and highlight alternative targets on the spike. We consider this structural work together with mAb effects in vivo to suggest correlations between structure and clinical applications. We also place mAbs against severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses in the context of the SARS-CoV-2 spike to suggest features that may be desirable to design mAbs or vaccines capable of conferring broad protection.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , /imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Humanos , Imunização Passiva/métodos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/terapia , Glicoproteína da Espícula de Coronavírus/genética , Vacinas Virais/imunologia , Internalização do Vírus/efeitos dos fármacos
13.
Arch Virol ; 166(3): 715-731, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33492524

RESUMO

Coronaviruses (CoV) are a family of viral pathogens that infect both birds and mammals, including humans. Seven human coronaviruses (HCoV) have been recognized so far. HCoV-229E, -OC43, -NL63, and -HKU1 account for one-third of common colds with mild symptoms. The other three members are severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2. These viruses are responsible for SARS, MERS, and CoV disease 2019 (COVID-19), respectively. A variety of diagnostic techniques, including chest X-rays, computer tomography (CT) scans, analysis of viral nucleic acids, proteins, or whole virions, and host antibody detection using serological assays have been developed for the detection of these viruses. In this review, we discuss conventional serological tests, such as enzyme-linked immunosorbent assay (ELISA), western blot (WB), immunofluorescence assay (IFA), lateral flow immunoassay (LFIA), and chemiluminescence immunoassay (CLIA), as well as biosensor-based assays that have been developed for diagnosing HCoV-associated diseases since 2003, with an in-depth focus on COVID-19.


Assuntos
Anticorpos Antivirais/sangue , /diagnóstico , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Síndrome Respiratória Aguda Grave/diagnóstico , Anticorpos Antivirais/imunologia , Técnicas Biossensoriais/métodos , Western Blotting/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Imunofluorescência/métodos , Humanos , Medições Luminescentes/métodos , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/virologia
14.
J Exp Med ; 218(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33464307

RESUMO

Virus-specific T cells play essential roles in protection against multiple virus infections, including SARS-CoV and MERS-CoV. While SARS-CoV-2-specific T cells have been identified in COVID-19 patients, their role in the protection of SARS-CoV-2-infected mice is not established. Here, using mice sensitized for infection with SARS-CoV-2 by transduction with an adenovirus expressing the human receptor (Ad5-hACE2), we identified SARS-CoV-2-specific T cell epitopes recognized by CD4+ and CD8+ T cells in BALB/c and C57BL/6 mice. Virus-specific T cells were polyfunctional and were able to lyse target cells in vivo. Further, type I interferon pathway was proved to be critical for generating optimal antiviral T cell responses after SARS-CoV-2 infection. T cell vaccination alone partially protected SARS-CoV-2-infected mice from severe disease. In addition, the results demonstrated cross-reactive T cell responses between SARS-CoV and SARS-CoV-2, but not MERS-CoV, in mice. Understanding the role of the T cell response will guide immunopathogenesis studies of COVID-19 and vaccine design and validation.


Assuntos
/imunologia , Epitopos de Linfócito T/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Linfócitos T/imunologia , Linfócitos T/virologia , /genética , Animais , Anticorpos Neutralizantes/sangue , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Chlorocebus aethiops , Reações Cruzadas , Mapeamento de Epitopos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Vírus da SARS/imunologia , /patogenicidade , Células Vero
15.
Mol Ther ; 29(3): 1174-1185, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33352107

RESUMO

Self-amplifying RNA (saRNA) is a cutting-edge platform for both nucleic acid vaccines and therapeutics. saRNA is self-adjuvanting, as it activates types I and III interferon (IFN), which enhances the immunogenicity of RNA vaccines but can also lead to inhibition of translation. In this study, we screened a library of saRNA constructs with cis-encoded innate inhibiting proteins (IIPs) and determined the effect on protein expression and immunogenicity. We observed that the PIV-5 V and Middle East respiratory syndrome coronavirus (MERS-CoV) ORF4a proteins enhance protein expression 100- to 500-fold in vitro in IFN-competent HeLa and MRC5 cells. We found that the MERS-CoV ORF4a protein partially abates dose nonlinearity in vivo, and that ruxolitinib, a potent Janus kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor, but not the IIPs, enhances protein expression of saRNA in vivo. Both the PIV-5 V and MERS-CoV ORF4a proteins were found to enhance the percentage of resident cells in human skin explants expressing saRNA and completely rescued dose nonlinearity of saRNA. Finally, we observed that the MERS-CoV ORF4a increased the rabies virus (RABV)-specific immunoglobulin G (IgG) titer and neutralization half-maximal inhibitory concentration (IC50) by ∼10-fold in rabbits, but not in mice or rats. These experiments provide a proof of concept that IIPs can be directly encoded into saRNA vectors and effectively abate the nonlinear dose dependency and enhance immunogenicity.


Assuntos
Imunidade Inata/efeitos dos fármacos , Imunogenicidade da Vacina , Biossíntese de Proteínas/efeitos dos fármacos , Vacinas Sintéticas/farmacologia , Proteínas do Envelope Viral/administração & dosagem , Animais , Linhagem Celular , Vírus da Encefalite Equina Venezuelana/efeitos dos fármacos , Vírus da Encefalite Equina Venezuelana/imunologia , Vírus da Encefalite Equina Venezuelana/patogenicidade , Fibroblastos , Regulação da Expressão Gênica , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunoglobulina G/biossíntese , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Janus Quinases/antagonistas & inibidores , Janus Quinases/genética , Janus Quinases/imunologia , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , NF-kappa B/genética , NF-kappa B/imunologia , Vírus da Parainfluenza 5/efeitos dos fármacos , Vírus da Parainfluenza 5/imunologia , Vírus da Parainfluenza 5/patogenicidade , Pirazóis/farmacologia , Coelhos , Vírus da Raiva/efeitos dos fármacos , Vírus da Raiva/imunologia , Vírus da Raiva/patogenicidade , Ratos , Fatores de Transcrição STAT/antagonistas & inibidores , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/imunologia , Transdução de Sinais , Vacinas Sintéticas/biossíntese , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
16.
J Biomed Sci ; 27(1): 104, 2020 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-33341119

RESUMO

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a new type of coronavirus that causes the Coronavirus Disease 2019 (COVID-19), which has been the most challenging pandemic in this century. Considering its high mortality and rapid spread, an effective vaccine is urgently needed to control this pandemic. As a result, the academia, industry, and government sectors are working tightly together to develop and test a variety of vaccines at an unprecedented pace. In this review, we outline the essential coronavirus biological characteristics that are important for vaccine design. In addition, we summarize key takeaways from previous vaccination studies of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), highlighting the pros and cons of each immunization strategy. Finally, based on these prior vaccination experiences, we discuss recent progress and potential challenges of COVID-19 vaccine development.


Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Pandemias/prevenção & controle , Vírus da SARS/imunologia , Vacinação , /epidemiologia , /prevenção & controle , /uso terapêutico , Humanos
17.
Cell Rep ; 33(5): 108345, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33147460

RESUMO

Bat cells and tissue have elevated basal expression levels of antiviral genes commonly associated with interferon alpha (IFNα) signaling. Here, we show Interferon Regulatory Factor 1 (IRF1), 3, and 7 levels are elevated in most bat tissues and that, basally, IRFs contribute to the expression of type I IFN ligands and high expression of interferon regulated genes (IRGs). CRISPR knockout (KO) of IRF 1/3/7 in cells reveals distinct subsets of genes affected by each IRF in an IFN-ligand signaling-dependent and largely independent manner. As the master regulators of innate immunity, the IRFs control the kinetics and maintenance of the IRG response and play essential roles in response to influenza A virus (IAV), herpes simplex virus 1 (HSV-1), Melaka virus/Pteropine orthoreovirus 3 Melaka (PRV3M), and Middle East respiratory syndrome-related coronavirus (MERS-CoV) infection. With its differential expression in bats compared to that in humans, this highlights a critical role for basal IRF expression in viral responses and potentially immune cell development in bats with relevance for IRF function in human biology.


Assuntos
Quirópteros/imunologia , Regulação da Expressão Gênica/imunologia , Fator Regulador 1 de Interferon/imunologia , Fator Regulador 7 de Interferon/imunologia , Viroses/imunologia , Animais , Herpesvirus Humano 1/imunologia , Vírus da Influenza A/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Orthoreovirus/imunologia
18.
Bol Med Hosp Infant Mex ; 77(5): 252-261, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33064679

RESUMO

Since the emergence of the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China at the end of 2019, when its characteristics were practically unknown, one aspect was evident: its high contagion rate. This high infection rate resulted in the spread of the virus in China, Europe, and, eventually, the rest of the world, including Mexico. At present, around 9 million people are infected, and around 470,000 have died worldwide. In this context, the need to generate protective immunity, and especially the generation of a vaccine that can protect the world population against infection in the shortest possible time, is a challenge that is being addressed in different countries using different strategies in multiple clinical trials. This opinion article will present the evidence of the induction of immune response in some of the viruses of the coronavirus family before COVID-19, such as SARS-CoV and MERS-CoV (Middle East respiratory syndrome coronavirus). The information collected about the induction of an immune response by SARS-CoV-2 will be presented, as well as a description of the vaccine candidates reported to date in the various ongoing clinical trials. Finally, an opinion based on the evidence presented will be issued on the potential success of developing vaccine prototypes.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Vacinas Virais , Animais , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Vírus da SARS/imunologia , Vírus da SARS/isolamento & purificação , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle
19.
Sci Rep ; 10(1): 16615, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024203

RESUMO

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.


Assuntos
Anticorpos Antivirais/análise , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Infecções por Coronavirus/imunologia , Testes Imunológicos de Citotoxicidade/métodos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/virologia , Genes Reporter , Células HEK293 , Humanos , Células Jurkat , Luciferases/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Fatores de Transcrição NFATC/genética , Receptores de IgG/genética , Receptores de IgG/imunologia , Elementos de Resposta , Glicoproteína da Espícula de Coronavírus/metabolismo , Transfecção
20.
Sci Rep ; 10(1): 16561, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024213

RESUMO

As the Coronavirus Disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses (CoVs). The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Proteínas do Nucleocapsídeo/imunologia , Pneumonia Viral/diagnóstico , Soroconversão , Testes Sorológicos/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Betacoronavirus/genética , Estudos de Coortes , Infecções por Coronavirus/virologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Pandemias , Fosfoproteínas , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Adulto Jovem
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