RESUMO
The global spread of SARS-CoV-2/COVID-19 is devastating health systems and economies worldwide. Recombinant or vaccine-induced neutralizing antibodies are used to combat the COVID-19 pandemic. However, the recently emerged SARS-CoV-2 variants B.1.1.7 (UK), B.1.351 (South Africa), and P.1 (Brazil) harbor mutations in the viral spike (S) protein that may alter virus-host cell interactions and confer resistance to inhibitors and antibodies. Here, using pseudoparticles, we show that entry of all variants into human cells is susceptible to blockade by the entry inhibitors soluble ACE2, Camostat, EK-1, and EK-1-C4. In contrast, entry of the B.1.351 and P.1 variant was partially (Casirivimab) or fully (Bamlanivimab) resistant to antibodies used for COVID-19 treatment. Moreover, entry of these variants was less efficiently inhibited by plasma from convalescent COVID-19 patients and sera from BNT162b2-vaccinated individuals. These results suggest that SARS-CoV-2 may escape neutralizing antibody responses, which has important implications for efforts to contain the pandemic.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , SARS-CoV-2/imunologia , Animais , COVID-19/imunologia , COVID-19/terapia , COVID-19/virologia , Linhagem Celular , Farmacorresistência Viral , Humanos , Imunização Passiva , Cinética , Fusão de Membrana , Modelos Moleculares , Testes de Neutralização , Serina Endopeptidases/metabolismo , Solubilidade , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação , Internalização do Vírus , Soroterapia para COVID-19RESUMO
The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been associated with more than 780,000 deaths worldwide (as of 20 August 2020). To develop antiviral interventions quickly, drugs used for the treatment of unrelated diseases are currently being repurposed to treat COVID-19. Chloroquine is an anti-malaria drug that is used for the treatment of COVID-19 as it inhibits the spread of SARS-CoV-2 in the African green monkey kidney-derived cell line Vero1-3. Here we show that engineered expression of TMPRSS2, a cellular protease that activates SARS-CoV-2 for entry into lung cells4, renders SARS-CoV-2 infection of Vero cells insensitive to chloroquine. Moreover, we report that chloroquine does not block infection with SARS-CoV-2 in the TMPRSS2-expressing human lung cell line Calu-3. These results indicate that chloroquine targets a pathway for viral activation that is not active in lung cells and is unlikely to protect against the spread of SARS-CoV-2 in and between patients.
Assuntos
Cloroquina/farmacologia , Cloroquina/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Animais , Betacoronavirus/efeitos dos fármacos , COVID-19 , Linhagem Celular , Chlorocebus aethiops , Humanos , Técnicas In Vitro , Pulmão/virologia , Pandemias , SARS-CoV-2 , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Falha de Tratamento , Células Vero , Internalização do Vírus , Tratamento Farmacológico da COVID-19RESUMO
BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has caused a pandemic with tens of millions of cases and hundreds of thousands of deaths. The infection causes coronavirus disease 2019 (COVID-19), a disease of the respiratory system of divergent severity. In the current study, humoral immune responses were characterized in a cohort of 143 patients with COVID-19 from the University Hospital Frankfurt am Main, Germany. METHODS: SARS-CoV-2-specific-antibodies were detected by enzyme-linked immunosorbent assay (ELISA). SARS-CoV-2 and human coronavirus NL63 neutralization activity was analyzed with pseudotyped lentiviral vectors. RESULTS: The severity of COVID-19 increased with age, and male patients encountered more serious symptoms than female patients. Disease severity was correlated with the amount of SARS-CoV-2-specific immunoglobulin (Ig) G and IgA and the neutralization activity of the antibodies. The amount of SARS-CoV-2-specific IgG antibodies decreased with time after polymerase chain reaction conformation of the infection, and antibodies directed against the nucleoprotein waned faster than spike protein-directed antibodies. In contrast, for the common flu coronavirus NL63, COVID-19 disease severity seemed to be correlated with low NL63-neutralizing activities, suggesting the possibility of cross-reactive protection. CONCLUSION: The results describe the humoral immune responses against SARS-CoV-2 and might aid the identification of correlates of protection needed for vaccine development.
Assuntos
Anticorpos Antivirais/imunologia , COVID-19/imunologia , Imunidade Humoral , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/imunologia , Estudos de Coortes , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Feminino , Alemanha , Células HEK293 , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Vaccine-induced neutralizing antibodies are key in combating the coronavirus disease 2019 (COVID-19) pandemic. However, delays of boost immunization due to limited availability of vaccines may leave individuals vulnerable to infection and prolonged or severe disease courses. The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC)-B.1.1.7 (United Kingdom), B.1.351 (South Africa), and P.1 (Brazil)-may exacerbate this issue, as the latter two are able to evade control by antibodies. METHODS: We assessed humoral and T-cell responses against SARS-CoV-2 wild-type (WT), VOC, and endemic human coronaviruses (hCoVs) that were induced after single and double vaccination with BNT162b2. RESULTS: Despite readily detectable immunoglobulin G (IgG) against the receptor-binding domain of the SARS-CoV-2 S protein at day 14 after a single vaccination, inhibition of SARS-CoV-2 S-driven host cell entry was weak and particularly low for the B.1.351 variant. Frequencies of SARS-CoV-2 WT and VOC-specific T cells were low in many vaccinees after application of a single dose and influenced by immunity against endemic hCoV. The second vaccination significantly boosted T-cell frequencies reactive for WT and B.1.1.7 and B.1.351 variants. CONCLUSIONS: These results call into question whether neutralizing antibodies significantly contribute to protection against COVID-19 upon single vaccination and suggest that cellular immunity is central for the early defenses against COVID-19.
Assuntos
Vacina BNT162/imunologia , COVID-19 , Imunidade Celular , Imunidade Humoral , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/imunologia , COVID-19/prevenção & controle , Humanos , Imunoglobulina G/sangue , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T/imunologia , VacinaçãoRESUMO
Low plasma levels of the signaling lipid metabolite sphingosine 1-phosphate (S1P) are associated with disrupted endothelial cell (EC) barriers, lymphopenia and reduced responsivity to hypoxia. Total S1P levels were also reduced in 23 critically ill patients with coronavirus disease 2019 (COVID-19), and the two main S1P carriers, serum albumin (SA) and high-density lipoprotein (HDL) were dramatically low. Surprisingly, we observed a carrier-changing shift from SA to HDL, which probably prevented an even further drop in S1P levels. Furthermore, intracellular S1P levels in red blood cells (RBCs) were significantly increased in COVID-19 patients compared with healthy controls due to up-regulation of S1P producing sphingosine kinase 1 and down-regulation of S1P degrading lyase expression. Cell culture experiments supported increased sphingosine kinase activity and unchanged S1P release from RBC stores of COVID-19 patients. These observations suggest adaptive mechanisms for maintenance of the vasculature and immunity as well as prevention of tissue hypoxia in COVID-19 patients.
Assuntos
COVID-19/sangue , COVID-19/fisiopatologia , Eritrócitos/metabolismo , Lisofosfolipídeos/sangue , Esfingosina/análogos & derivados , Idoso , Células Cultivadas , Humanos , Lipoproteínas HDL/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , SARS-CoV-2 , Albumina Sérica/metabolismo , Esfingosina/sangueRESUMO
Human cytomegalovirus (HCMV) is a widespread human pathogen that causes asymptomatic infection in healthy individuals but poses a serious threat to immunocompromised patients. During the late phase of HCMV infection, the viral capsid is transported to the cytoplasmic viral assembly center (cVAC), where it is enclosed by the tegument protein layer and the viral envelope. The cVAC consists of circularly arranged vesicles from the trans-Golgi and endosomal networks. The HCMV gene UL35 encodes ppUL35 and its shorter form, ppUL35A. We have previously shown that the UL35 gene is involved in HCMV assembly, but it is unknown how UL35 proteins regulate viral assembly. Here we show that sorting nexin 5 (SNX5), a component of the retromer and part of the retrograde transport pathway, interacts with UL35 proteins. Expression of wild-type proteins but not mutants defective in SNX5 binding resulted in the cellular redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR), indicating that UL35 proteins bind and negatively regulate SNX5 to modulate cellular transport pathways. Furthermore, binding of UL35 proteins to SNX5 was required for efficient viral replication and for transport of the most abundant HCMV glycoprotein B (gB; gpUL55) to the cVAC. These results indicate that ppUL35 and ppUL35A control the localization of the essential gB through the regulation of a retrograde transport pathway. Thus, this work is the first to define a molecular interaction between a tegument protein and a vesicular transport factor to regulate glycoprotein localization.IMPORTANCE Human cytomegalovirus is ubiquitously present in the healthy population, but reactivation or reinfection can cause serious, life-threatening infections in immunocompromised patients. For completion of its lytic cycle, human cytomegalovirus induces formation of an assembly center where mature virus particles are formed from multiple viral proteins. Viral glycoproteins use separate vesicular pathways for transport to the assembly center, which are incompletely understood. Our research identified a viral structural protein which affects the localization of one of the major glycoproteins. We could link this change in glycoprotein localization to an interaction of the structural protein with a cellular protein involved in regulation of vesicle transport. This increases our understanding of how the virus intersects into cellular regulatory pathways to enhance its own replication.
Assuntos
Citomegalovirus/fisiologia , Nexinas de Classificação/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Montagem de Vírus/fisiologia , Células A549 , Linhagem Celular Tumoral , Regulação Viral da Expressão Gênica , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Ligação Proteica , Transporte Proteico/fisiologia , Receptor IGF Tipo 2/metabolismo , Replicação ViralRESUMO
UNLABELLED: The expression of the antiviral host cell factor tetherin is induced by interferon and can inhibit the release of enveloped viruses from infected cells. The Vpu protein of HIV-1 antagonizes the antiviral activity of tetherin, and tetherin antagonists with Vpu-like activity have been identified in other viruses. In contrast, it is incompletely understood whether tetherin inhibits influenza A virus (FLUAV) release and whether FLUAV encodes tetherin antagonists. Here, we show that release of several laboratory-adapted FLUAV strains and a seasonal FLUAV strain is inhibited by tetherin, while pandemic FLUAV A/Hamburg/4/2009 is resistant. Studies with a virus-like particle system and analysis of reassortant viruses provided evidence that the viral hemagglutinin (HA) is an important determinant of tetherin antagonism but requires the presence of its cognate neuraminidase (NA) to inhibit tetherin. Finally, tetherin antagonism by FLUAV was dependent on the virion context, since retrovirus release from tetherin-positive cells was not rescued, and correlated with an HA- and NA-dependent reduction in tetherin expression. In sum, our study identifies HA and NA proteins of certain pandemic FLUAV as tetherin antagonists, which has important implications for understanding FLUAV pathogenesis. IMPORTANCE: Influenza A virus (FLUAV) infection is responsible for substantial global morbidity and mortality, and understanding how the virus evades the immune defenses of the host may uncover novel targets for antiviral intervention. Tetherin is an antiviral effector molecule of the innate immune system which can contribute to control of viral invasion. However, it has been unclear whether FLUAV is inhibited by tetherin and whether these viruses encode tetherin-antagonizing proteins. Our observation that several pandemic FLUAV strains can counteract tetherin via their HA and NA proteins identifies these proteins as novel tetherin antagonists and indicates that HA/NA-dependent inactivation of innate defenses may contribute to the efficient spread of pandemic FLUAV.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Neuraminidase/imunologia , Antígenos CD/genética , Antígenos CD/imunologia , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Células HEK293 , HIV-1/genética , HIV-1/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Proteínas do Vírus da Imunodeficiência Humana/genética , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Neuraminidase/genética , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/imunologiaRESUMO
The ongoing Ebola virus (EBOV) disease (EVD) epidemic in Western Africa is the largest EVD outbreak recorded to date and requires the rapid development and deployment of antiviral measures. The viral glycoprotein (GP) facilitates host cell entry and, jointly with cellular interaction partners, constitutes a potential target for antiviral intervention. However, it is unknown whether the GPs of the currently and previously circulating EBOVs use the same mechanisms for cellular entry and are thus susceptible to inhibition by the same antivirals and cellular defenses. Here, we show that the GPs of the EBOVs circulating in 1976 and 2014 transduce the same spectrum of target cells, use the same cellular factors for host cell entry, and are comparably susceptible to blockade by antiviral interferon-induced transmembrane proteins and neutralizing antibody KZ52. Thus, the viruses responsible for the ongoing EVD epidemic should be fully susceptible to established antiviral strategies targeting GP and cellular entry factors.
Assuntos
Ebolavirus/patogenicidade , Doença pelo Vírus Ebola/virologia , Animais , Anticorpos Neutralizantes/imunologia , Antivirais/farmacologia , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , República Democrática do Congo/epidemiologia , Ebolavirus/efeitos dos fármacos , Ebolavirus/imunologia , Ebolavirus/metabolismo , Euphorbiaceae , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Células HEK293 , Células HeLa , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/imunologia , Humanos , Células Jurkat , Macaca mulatta , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Serra Leoa/epidemiologia , Células Vero , Proteínas Virais/imunologia , Proteínas Virais/metabolismo , Internalização do VírusRESUMO
Ebolaviruses constitute a public health threat, particularly in Central and Western Africa. Host cell factors required for spread of ebolaviruses may serve as targets for antiviral intervention. Lectins, TAM receptor tyrosine kinases (Tyro3, Axl, Mer), T cell immunoglobulin and mucin domain (TIM) proteins, integrins, and Niemann-Pick C1 (NPC1) have been reported to promote entry of ebolaviruses into certain cellular systems. However, the factors used by ebolaviruses to invade macrophages, major viral targets, are poorly defined. Here, we show that mannose-specific lectins, TIM-1 and Axl augment entry into certain cell lines but do not contribute to Ebola virus (EBOV)-glycoprotein (GP)-driven transduction of macrophages. In contrast, expression of Mer, integrin αV, and NPC1 was required for efficient GP-mediated transduction and EBOV infection of macrophages. These results define cellular factors hijacked by EBOV for entry into macrophages and, considering that Mer and integrin αV promote phagocytosis of apoptotic cells, support the concept that EBOV relies on apoptotic mimicry to invade target cells.
Assuntos
Ebolavirus/metabolismo , Ebolavirus/patogenicidade , Doença pelo Vírus Ebola/virologia , Macrófagos/virologia , Fatores de Virulência/metabolismo , Linhagem Celular , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Lectinas/metabolismo , Internalização do VírusRESUMO
The type II transmembrane serine proteases TMPRSS2 and HAT can cleave and activate the spike protein (S) of the severe acute respiratory syndrome coronavirus (SARS-CoV) for membrane fusion. In addition, these proteases cleave the viral receptor, the carboxypeptidase angiotensin-converting enzyme 2 (ACE2), and it was proposed that ACE2 cleavage augments viral infectivity. However, no mechanistic insights into this process were obtained and the relevance of ACE2 cleavage for SARS-CoV S protein (SARS-S) activation has not been determined. Here, we show that arginine and lysine residues within ACE2 amino acids 697 to 716 are essential for cleavage by TMPRSS2 and HAT and that ACE2 processing is required for augmentation of SARS-S-driven entry by these proteases. In contrast, ACE2 cleavage was dispensable for activation of the viral S protein. Expression of TMPRSS2 increased cellular uptake of soluble SARS-S, suggesting that protease-dependent augmentation of viral entry might be due to increased uptake of virions into target cells. Finally, TMPRSS2 was found to compete with the metalloprotease ADAM17 for ACE2 processing, but only cleavage by TMPRSS2 resulted in augmented SARS-S-driven entry. Collectively, our results in conjunction with those of previous studies indicate that TMPRSS2 and potentially related proteases promote SARS-CoV entry by two separate mechanisms: ACE2 cleavage, which might promote viral uptake, and SARS-S cleavage, which activates the S protein for membrane fusion. These observations have interesting implications for the development of novel therapeutics. In addition, they should spur efforts to determine whether receptor cleavage promotes entry of other coronaviruses, which use peptidases as entry receptors.
Assuntos
Proteínas ADAM/metabolismo , Peptidil Dipeptidase A/metabolismo , Serina Endopeptidases/metabolismo , Síndrome Respiratória Aguda Grave/enzimologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do Vírus , Proteínas ADAM/genética , Proteína ADAM17 , Motivos de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Linhagem Celular , Humanos , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/genética , Processamento de Proteína Pós-Traducional , Proteólise , Receptores Virais/genética , Receptores Virais/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Serina Endopeptidases/genética , Síndrome Respiratória Aguda Grave/genética , Síndrome Respiratória Aguda Grave/metabolismo , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Infection with human coronavirus 229E (HCoV-229E) is associated with the common cold and may result in pneumonia in immunocompromised patients. The viral spike (S) protein is incorporated into the viral envelope and mediates infectious entry of HCoV-229E into host cells, a process that depends on the activation of the S-protein by host cell proteases. However, the proteases responsible for HCoV-229E activation are incompletely defined. Here we show that the type II transmembrane serine proteases TMPRSS2 and HAT cleave the HCoV-229E S-protein (229E-S) and augment 229E-S-driven cell-cell fusion, suggesting that TMPRSS2 and HAT can activate 229E-S. Indeed, engineered expression of TMPRSS2 and HAT rendered 229E-S-driven virus-cell fusion insensitive to an inhibitor of cathepsin L, a protease previously shown to facilitate HCoV-229E infection. Inhibition of endogenous cathepsin L or TMPRSS2 demonstrated that both proteases can activate 229E-S for entry into cells that are naturally susceptible to infection. In addition, evidence was obtained that activation by TMPRSS2 rescues 229E-S-dependent cell entry from inhibition by IFITM proteins. Finally, immunohistochemistry revealed that TMPRSS2 is coexpressed with CD13, the HCoV-229E receptor, in human airway epithelial (HAE) cells, and that CD13(+) TMPRSS2(+) cells are preferentially targeted by HCoV-229E, suggesting that TMPRSS2 can activate HCoV-229E in infected humans. In sum, our results indicate that HCoV-229E can employ redundant proteolytic pathways to ensure its activation in host cells. In addition, our observations and previous work suggest that diverse human respiratory viruses are activated by TMPRSS2, which may constitute a target for antiviral intervention.
Assuntos
Catepsinas/metabolismo , Coronavirus Humano 229E/fisiologia , Infecções por Coronavirus/enzimologia , Mucosa Respiratória/enzimologia , Serina Endopeptidases/metabolismo , Internalização do Vírus , Catepsinas/genética , Linhagem Celular , Coronavirus Humano 229E/genética , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Expressão Gênica , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Mucosa Respiratória/virologia , Serina Endopeptidases/genéticaRESUMO
We used a lentiviral vector bearing the viral spike protein to detect neutralizing antibodies against Middle East respiratory syndrome coronavirus (MERS-CoV) in persons from the Eastern Province of Saudi Arabia. None of the 268 samples tested displayed neutralizing activity, which suggests that MERS-CoV infections in humans are infrequent in this province.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Coronavirus/imunologia , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Infecções por Coronavirus/transmissão , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Arábia Saudita/epidemiologia , Estudos Soroepidemiológicos , Adulto JovemRESUMO
Primate simplexviruses are closely related neurotropic herpesviruses, which are largely apathogenic in their respective host species. However, cross-species transmission of Macacine alphaherpesvirus 1 (McHV1, also termed herpes B virus) from rhesus macaques to humans can cause fatal encephalomyelitis. In contrast, closely related viruses, such as Cercopithecine alphaherpesvirus 2 (CeHV2, also termed simian agent 8) or Papiine alphaherpesvirus 2 (PaHV2, also termed herpesvirus papio 2), have not been linked to human disease and are believed to be largely apathogenic in humans. Here, we investigated whether McHV1, PaHV2 and CeHV2 differ in their capacity to infect human and non-human primate (NHP) cells. For comparison, we included the human simplexviruses HSV1 and HSV2 in our analyses. All five viruses replicated efficiently in cell lines of human and African green monkey origin, and McHV1 and PaHV2 also showed robust replication in rhesus macaque cell lines. In contrast, the replication of CeHV2 and particularly HSV1 and HSV2 in cell lines of rhesus macaque origin were reduced or inefficient. Similarly, McHV1, but not CeHV2, efficiently infected rhesus macaque brain organoids. These results point towards the previously unappreciated partial resistance of certain rhesus macaque cells to HSV1/HSV2/CeHV2 infection and reveal similarities between the cell tropism of McHV1 and PaHV2 that might be relevant for risk assessment.
RESUMO
Transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to farmed mink has been observed in Europe and the US. In the infected animals, viral variants arose that harbored mutations in the spike (S) protein, the target of neutralizing antibodies, and these variants were transmitted back to humans. This raised concerns that mink might become a constant source of human infection with SARS-CoV-2 variants associated with an increased threat to human health and resulted in mass culling of mink. Here, we report that mutations frequently found in the S proteins of SARS-CoV-2 from mink are mostly compatible with efficient entry into human cells and its inhibition by soluble angiotensin-converting enzyme 2 (ACE2). In contrast, mutation Y453F reduces neutralization by an antibody with emergency use authorization for coronavirus disease 2019 (COVID-19) therapy and sera/plasma from COVID-19 patients. These results suggest that antibody responses induced upon infection or certain antibodies used for treatment might offer insufficient protection against SARS-CoV-2 variants from mink.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19 , Vison , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Células A549 , Enzima de Conversão de Angiotensina 2/imunologia , Animais , COVID-19/genética , COVID-19/imunologia , Chlorocebus aethiops , Cricetinae , Humanos , Vison/imunologia , Vison/virologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Células VeroRESUMO
Background: Acute kidney injury (AKI) is very common in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) disease 2019 (COVID-19) and considered as a risk factor for COVID-19 severity. SARS-CoV-2 renal tropism has been observed in COVID-19 patients, suggesting that direct viral injury of the kidneys may contribute to AKI. We examined 20 adult cases with confirmed SARS-CoV-2 infection requiring ICU supportive care in a single-center prospective observational study and investigated whether urinary markers for viral infection (SARS-CoV-2 N) and shedded cellular membrane proteins (ACE2, TMPRSS2) allow identification of patients at risk for AKI and outcome of COVID-19. Objectives: The objective of the study was to evaluate whether urinary markers for viral infection (SARS-CoV-2 N) and shedded cellular membrane proteins (ACE2, TMPRSS2) allow identification of patients at risk for AKI and outcome of COVID-19. Results: Urinary SARS-CoV-2 N measured at ICU admission identified patients at risk for AKI in COVID-19 (HR 5.9, 95% CI 1.4-26, p = 0.0095). In addition, the combination of urinary SARS-CoV-2 N and plasma albumin measurements further improved the association with AKI (HR 11.4, 95% CI 2.7-48, p = 0.0016). Finally, combining urinary SARS-CoV-2 N and plasma albumin measurements associated with the length of ICU supportive care (HR 3.3, 95% CI 1.1-9.9, p = 0.0273) and premature death (HR 7.6, 95% CI 1.3-44, p = 0.0240). In contrast, urinary ACE2 and TMPRSS2 did not correlate with AKI in COVID-19. Conclusions: In conclusion, urinary SARS-CoV-2 N levels associate with risk for AKI and correlate with COVID-19 severity.
RESUMO
Effective treatment strategies for severe coronavirus disease (COVID-19) remain scarce. Hydrolysis of membrane-embedded, inert sphingomyelin by stress responsive sphingomyelinases is a hallmark of adaptive responses and cellular repair. As demonstrated in experimental and observational clinical studies, the transient and stress-triggered release of a sphingomyelinase, SMPD1, into circulation and subsequent ceramide generation provides a promising target for FDA-approved drugs. Here, we report the activation of sphingomyelinase-ceramide pathway in 23 intensive care patients with severe COVID-19. We observed an increase of circulating activity of sphingomyelinase with subsequent derangement of sphingolipids in serum lipoproteins and from red blood cells (RBC). Consistent with increased ceramide levels derived from the inert membrane constituent sphingomyelin, increased activity of acid sphingomyelinase (ASM) accurately distinguished the patient cohort undergoing intensive care from healthy controls. Positive correlational analyses with biomarkers of severe clinical phenotype support the concept of an essential pathophysiological role of ASM in the course of SARS-CoV-2 infection as well as of a promising role for functional inhibition with anti-inflammatory agents in SARS-CoV-2 infection as also proposed in independent observational studies. We conclude that large-sized multicenter, interventional trials are now needed to evaluate the potential benefit of functional inhibition of this sphingomyelinase in critically ill patients with COVID-19.
Assuntos
COVID-19/metabolismo , Ceramidas/metabolismo , Transdução de Sinais , Esfingomielina Fosfodiesterase/metabolismo , Anti-Inflamatórios/uso terapêutico , COVID-19/virologia , Ceramidas/sangue , Ativação Enzimática , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Ácidos Graxos/metabolismo , Humanos , Unidades de Terapia Intensiva , Gravidade do Paciente , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Esfingomielina Fosfodiesterase/sangue , Esfingomielinas/metabolismo , Tratamento Farmacológico da COVID-19RESUMO
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants threatens efforts to contain the coronavirus disease 2019 (COVID-19) pandemic. The number of COVID-19 cases and deaths in India has risen steeply, and a SARS-CoV-2 variant, B.1.617, is believed to be responsible for many of these cases. The spike protein of B.1.617 harbors two mutations in the receptor binding domain, which interacts with the angiotensin converting enzyme 2 (ACE2) receptor and constitutes the main target of neutralizing antibodies. Therefore, we analyze whether B.1.617 is more adept in entering cells and/or evades antibody responses. B.1.617 enters two of eight cell lines tested with roughly 50% increased efficiency and is equally inhibited by two entry inhibitors. In contrast, B.1.617 is resistant against bamlanivimab, an antibody used for COVID-19 treatment. B.1.617 evades antibodies induced by infection or vaccination, although less so than the B.1.351 variant. Collectively, our study reveals that antibody evasion of B.1.617 may contribute to the rapid spread of this variant.
Assuntos
Enzima de Conversão de Angiotensina 2/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Ésteres/farmacologia , Guanidinas/farmacologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Linhagem Celular , Humanos , Inibidores de Proteases/farmacologia , Ligação Proteica , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , VacinaçãoRESUMO
BACKGROUND: Antivirals are needed to combat the COVID-19 pandemic, which is caused by SARS-CoV-2. The clinically-proven protease inhibitor Camostat mesylate inhibits SARS-CoV-2 infection by blocking the virus-activating host cell protease TMPRSS2. However, antiviral activity of Camostat mesylate metabolites and potential viral resistance have not been analyzed. Moreover, antiviral activity of Camostat mesylate in human lung tissue remains to be demonstrated. METHODS: We used recombinant TMPRSS2, reporter particles bearing the spike protein of SARS-CoV-2 or authentic SARS-CoV-2 to assess inhibition of TMPRSS2 and viral entry, respectively, by Camostat mesylate and its metabolite GBPA. FINDINGS: We show that several TMPRSS2-related proteases activate SARS-CoV-2 and that two, TMPRSS11D and TMPRSS13, are robustly expressed in the upper respiratory tract. However, entry mediated by these proteases was blocked by Camostat mesylate. The Camostat metabolite GBPA inhibited recombinant TMPRSS2 with reduced efficiency as compared to Camostat mesylate. In contrast, both inhibitors exhibited similar antiviral activity and this correlated with the rapid conversion of Camostat mesylate into GBPA in the presence of serum. Finally, Camostat mesylate and GBPA blocked SARS-CoV-2 spread in human lung tissue ex vivo and the related protease inhibitor Nafamostat mesylate exerted augmented antiviral activity. INTERPRETATION: Our results suggest that SARS-CoV-2 can use TMPRSS2 and closely related proteases for spread in the upper respiratory tract and that spread in the human lung can be blocked by Camostat mesylate and its metabolite GBPA. FUNDING: NIH, Damon Runyon Foundation, ACS, NYCT, DFG, EU, Berlin Mathematics center MATH+, BMBF, Lower Saxony, Lundbeck Foundation, Novo Nordisk Foundation.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Ésteres/farmacologia , Guanidinas/farmacologia , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Células HEK293 , Humanos , Pulmão/patologia , Pulmão/virologia , Proteínas de Membrana/biossíntese , Simulação de Dinâmica Molecular , Serina Endopeptidases/biossíntese , Serina Proteases/biossíntese , Células Vero , Ativação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacosRESUMO
The major transactivator protein IE2p86 of human cytomegalovirus (HCMV) has previously been shown to undergo posttranslational modification by the covalent attachment of SUMO proteins, termed SUMOylation, which occurs at two lysine residues located at amino acid positions 175 and 180. Mutation of the acceptor lysines resulted in the abrogation of IE2p86 SUMOylation in mammalian cells and a strong reduction of IE2p86-mediated transactivation. In this paper, we identify an additional SUMO interaction motif (SIM) within IE2p86, which mediates noncovalent binding to SUMO, as shown by yeast two-hybrid analyses. Transient-expression experiments revealed that an IE2p86 SIM mutant exhibited significantly reduced SUMOylation, strongly suggesting that noncovalent SUMO interactions affect the efficacy of covalent SUMO coupling. In order to define the relevance of IE2p86 SUMO interactions for viral replication, recombinant viruses originating from two different HCMV strains (AD169 and VR1814) were generated. Analysis of viruses expressing SUMOylation-negative IE2p86 revealed strongly impaired replication due to reduced viral DNA and protein accumulation, as well as diminished initiation of immediate-early gene expression. The additional introduction of the SIM mutation into the viral genome did not further compromise viral replication but resulted in altered expression of viral proteins at late times postinfection. In summary, this paper clearly shows that IE2p86 SUMOylation is necessary for efficient replication of the HCMV laboratory strain AD169 and the clinical isolate VR1814 and thus for the in vivo function of this viral transcription factor.
Assuntos
Citomegalovirus/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Transativadores/metabolismo , Células Cultivadas , DNA Viral/metabolismo , Éxons , Humanos , Proteínas Imediatamente Precoces/genética , Transativadores/genética , Replicação ViralRESUMO
Severe acute respiratory syndrome coronavirus 2 cell entry depends on angiotensin-converting enzyme 2 and transmembrane serine protease 2 and is blocked in cell culture by camostat mesylate, a clinically proven protease inhibitor. Whether camostat mesylate is able to lower disease burden in coronavirus disease 2019 sepsis is currently unknown. DESIGN: Retrospective observational case series. SETTING: Patient treated in ICU of University hospital Göttingen, Germany. PATIENTS: Eleven critical ill coronavirus disease 2019 patients with organ failure were treated in ICU. INTERVENTIONS: Compassionate use of camostat mesylate (six patients, camostat group) or hydroxychloroquine (five patients, hydroxychloroquine group). MEASUREMENTS AND MAIN RESULTS: Clinical courses were assessed by Sepsis-related Organ Failure Assessment score at days 1, 3, and 8. Further, viral load, oxygenation, and inflammatory markers were determined. Sepsis-related Organ Failure Assessment score was comparable between camostat and hydroxychloroquine groups upon ICU admission. During observation, the Sepsis-related Organ Failure Assessment score decreased in the camostat group but remained elevated in the hydroxychloroquine group. The decline in disease severity in camostat mesylate treated patients was paralleled by a decline in inflammatory markers and improvement of oxygenation. CONCLUSIONS: The severity of coronavirus disease 2019 decreased upon camostat mesylate treatment within a period of 8 days and a similar effect was not observed in patients receiving hydroxychloroquine. Camostat mesylate thus warrants further evaluation within randomized clinical trials.