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1.
PLoS Pathog ; 18(9): e1010782, 2022 09.
Article in English | MEDLINE | ID: covidwho-2039444

ABSTRACT

Safe, passive immunization methods are required against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants. Immunization of chickens with antigen is known to induce specific IgY antibodies concentrated in the egg yolk and has a good safety profile, high yield of IgY per egg, can be topically applied, not requiring parenteral delivery. Our data provide the first evidence of the prophylactic efficacy of Immunoglobulin Y antibodies against SARS-CoV-2 in mice. Lohmann hens were injected with recombinant SARS-CoV-2 RBD protein; IgY-Abs were extracted from the eggs and characterized using SDS-PAGE. Antiviral activity was evaluated using plaque reduction neutralization tests. In additional experiments, IgY-RBD efficacy was examined in mice sensitized to SARS-CoV-2 infection by transduction with Ad5-hACE2 (mild disease) or by using mouse-adapted virus (severe disease). In both cases, prophylactic intranasal administration of IgY-Abs reduced SARS-CoV-2 replication, and reduced morbidity, inflammatory cell infiltration, hemorrhage, and edema in the lungs and increased survival compared to control groups that received non-specific IgY-Abs. These results indicate that further evaluation of IgY-RBD antibodies in humans is warranted.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Antibodies, Viral , Antiviral Agents , COVID-19/prevention & control , Chickens , Female , Humans , Immunoglobulins , Mice
2.
Cell Res ; 2022 Sep 21.
Article in English | MEDLINE | ID: covidwho-2036802
3.
Nature ; 605(7908): 146-151, 2022 05.
Article in English | MEDLINE | ID: covidwho-1815561

ABSTRACT

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern2. Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D2 (PGD2)) and a phospholipase (phospholipase A2 group 2D (PLA2G2D)) contributed to poor outcomes in aged mice3,4. mRNA expression of PLA2G2D and prostaglandin D2 receptor (PTGDR), and production of PGD2 also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA2G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D-PGD2/PTGDR pathway is a useful target for therapeutic interventions.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Eicosanoids , Leukocytes, Mononuclear , Mice , Organic Chemicals , Oxazoles , Piperazines , Polyesters , Prostaglandins , Spike Glycoprotein, Coronavirus , Sulfonamides
4.
Nature ; 603(7902): 687-692, 2022 03.
Article in English | MEDLINE | ID: covidwho-1641974

ABSTRACT

The recent emergence of B.1.1.529, the Omicron variant1,2, has raised concerns of escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in preclinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of several B.1.1.529 isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. Despite modelling data indicating that B.1.1.529 spike can bind more avidly to mouse ACE2 (refs. 3,4), we observed less infection by B.1.1.529 in 129, C57BL/6, BALB/c and K18-hACE2 transgenic mice than by previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease and pathology with B.1.1.529 were also milder than with historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from the SAVE/NIAID network with several B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.


Subject(s)
COVID-19/pathology , COVID-19/virology , Disease Models, Animal , SARS-CoV-2/pathogenicity , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Cricetinae , Female , Humans , Lung/pathology , Lung/virology , Male , Mesocricetus , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Viral Load
6.
Nat Rev Immunol ; 22(1): 47-56, 2022 01.
Article in English | MEDLINE | ID: covidwho-1541216

ABSTRACT

Human coronaviruses cause a wide spectrum of disease, ranging from mild common colds to acute respiratory distress syndrome and death. Three highly pathogenic human coronaviruses - severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus and SARS-CoV-2 - have illustrated the epidemic and pandemic potential of human coronaviruses, and a better understanding of their disease-causing mechanisms is urgently needed for the rational design of therapeutics. Analyses of patients have revealed marked dysregulation of the immune system in severe cases of human coronavirus infection, and there is ample evidence that aberrant immune responses to human coronaviruses are typified by impaired induction of interferons, exuberant inflammatory responses and delayed adaptive immune responses. In addition, various viral proteins have been shown to impair interferon induction and signalling and to induce inflammasome activation. This suggests that severe disease associated with human coronaviruses is mediated by both dysregulated host immune responses and active viral interference. Here we discuss our current understanding of the mechanisms involved in each of these scenarios.


Subject(s)
COVID-19/immunology , COVID-19/pathology , Common Cold/virology , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Animals , COVID-19/virology , Common Cold/immunology , Common Cold/pathology , Drug Design , Humans , Inflammasomes , Interferons
7.
Proc Natl Acad Sci U S A ; 118(29)2021 07 20.
Article in English | MEDLINE | ID: covidwho-1294550

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small-molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme- and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and were proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 h postinfection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2-treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Protease Inhibitors/therapeutic use , Pyrrolidines/therapeutic use , SARS-CoV-2/drug effects , Angiotensin-Converting Enzyme 2/genetics , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/pathology , Coronavirus 3C Proteases/chemistry , Coronavirus Papain-Like Proteases/chemistry , Crystallography, X-Ray , Deuterium , Disease Models, Animal , Drug Evaluation, Preclinical , Female , Lung/pathology , Mice , Mice, Transgenic , Models, Molecular , Molecular Structure , Protease Inhibitors/chemical synthesis , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Conformation , Pyrrolidines/chemistry , SARS-CoV-2/enzymology , Sulfonic Acids , Transgenes
8.
J Clin Invest ; 131(11)2021 06 01.
Article in English | MEDLINE | ID: covidwho-1249495

ABSTRACT

Worse outcomes occur in aged compared with young populations after infections with respiratory viruses, including pathogenic coronaviruses (SARS-CoV, MERS-CoV, and SARS-CoV-2), and are associated with a suboptimal lung milieu ("inflammaging"). We previously showed that a single inducible phospholipase, PLA2G2D, is associated with a proresolving/antiinflammatory response in the lungs, and increases with age. Survival was increased in naive Pla2g2d-/- mice infected with SARS-CoV resulting from augmented respiratory dendritic cell (rDC) activation and enhanced priming of virus-specific T cells. Here, in contrast, we show that intranasal immunization provided no additional protection in middle-aged Pla2g2d-/- mice infected with any of the 3 pathogenic human coronaviruses because virtually no virus-specific antibodies or follicular helper CD4+ T (Tfh) cells were produced. Using MERS-CoV-infected mice, we found that these effects did not result from T or B cell intrinsic factors. Rather, they resulted from enhanced, and ultimately, pathogenic rDC activation, as manifested most prominently by enhanced IL-1ß expression. Wild-type rDC transfer to Pla2g2d-/- mice in conjunction with partial IL-1ß blockade reversed this defect and resulted in increased virus-specific antibody and Tfh responses. Together, these results indicate that PLA2G2D has an unexpected role in the lungs, serving as an important modulator of rDC activation, with protective and pathogenic effects in respiratory coronavirus infections and immunization, respectively.


Subject(s)
Antibodies, Viral/immunology , Antibody Formation , COVID-19/immunology , Group II Phospholipases A2/immunology , Middle East Respiratory Syndrome Coronavirus/immunology , SARS Virus/immunology , SARS-CoV-2/immunology , Severe Acute Respiratory Syndrome/immunology , Animals , COVID-19/enzymology , COVID-19/genetics , Chlorocebus aethiops , Group II Phospholipases A2/deficiency , Mice , Mice, Knockout , Severe Acute Respiratory Syndrome/enzymology , Severe Acute Respiratory Syndrome/genetics , Vero Cells
9.
J Immunol ; 205(6): 1564-1579, 2020 09 15.
Article in English | MEDLINE | ID: covidwho-694818

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human coronavirus causing severe disease and mortality. MERS-CoV infection failed to elicit robust IFN response, suggesting that the virus might have evolved strategies to evade host innate immune surveillance. In this study, we identified and characterized type I IFN antagonism of MERS-CoV open reading frame (ORF) 8b accessory protein. ORF8b was abundantly expressed in MERS-CoV-infected Huh-7 cells. When ectopically expressed, ORF8b inhibited IRF3-mediated IFN-ß expression induced by Sendai virus and poly(I:C). ORF8b was found to act at a step upstream of IRF3 to impede the interaction between IRF3 kinase IKKε and chaperone protein HSP70, which is required for the activation of IKKε and IRF3. An infection study using recombinant wild-type and ORF8b-deficient MERS-CoV further confirmed the suppressive role of ORF8b in type I IFN induction and its disruption of the colocalization of HSP70 with IKKε. Ectopic expression of HSP70 relieved suppression of IFN-ß expression by ORF8b in an IKKε-dependent manner. Enhancement of IFN-ß induction in cells infected with ORF8b-deficient virus was erased when HSP70 was depleted. Taken together, HSP70 chaperone is important for IKKε activation, and MERS-CoV ORF8b suppresses type I IFN expression by competing with IKKε for interaction with HSP70.


Subject(s)
Enzyme Activation/immunology , I-kappa B Kinase/immunology , Interferon Type I/immunology , Middle East Respiratory Syndrome Coronavirus/immunology , Viral Proteins/immunology , Betacoronavirus , COVID-19 , Cell Line , Coronavirus Infections , HSP70 Heat-Shock Proteins/immunology , HSP70 Heat-Shock Proteins/metabolism , Humans , I-kappa B Kinase/metabolism , Interferon Type I/metabolism , Middle East Respiratory Syndrome Coronavirus/metabolism , Pandemics , Pneumonia, Viral , SARS-CoV-2 , Viral Proteins/metabolism
10.
Nature ; 589(7843): 603-607, 2021 01.
Article in English | MEDLINE | ID: covidwho-917539

ABSTRACT

The ongoing coronavirus disease 2019 (COVID-19) pandemic is associated with substantial morbidity and mortality. Although much has been learned in the first few months of the pandemic, many features of COVID-19 pathogenesis remain to be determined. For example, anosmia is a common presentation, and many patients with anosmia show no or only minor respiratory symptoms1. Studies in animals infected experimentally with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID-19, provide opportunities to study aspects of the disease that are not easily investigated in human patients. Although the severity of COVID-19 ranges from asymptomatic to lethal2, most experimental infections provide insights into mild disease3. Here, using K18-hACE2 transgenic mice that were originally developed for SARS studies4, we show that infection with SARS-CoV-2 causes severe disease in the lung and, in some mice, the brain. Evidence of thrombosis and vasculitis was detected in mice with severe pneumonia. Furthermore, we show that infusion of convalescent plasma from a recovered patient with COVID-19 protected against lethal disease. Mice developed anosmia at early time points after infection. Notably, although pre-treatment with convalescent plasma prevented most signs of clinical disease, it did not prevent anosmia. Thus, K18-hACE2 mice provide a useful model for studying the pathological basis of both mild and lethal COVID-19 and for assessing therapeutic interventions.


Subject(s)
Anosmia/virology , COVID-19/physiopathology , COVID-19/therapy , Disease Models, Animal , SARS-CoV-2/pathogenicity , Animals , Anosmia/physiopathology , Anosmia/therapy , Brain/immunology , Brain/pathology , Brain/virology , COVID-19/immunology , COVID-19/virology , Epithelium/immunology , Epithelium/virology , Female , Humans , Immunization, Passive , Inflammation/pathology , Inflammation/therapy , Inflammation/virology , Lung Diseases/pathology , Lung Diseases/therapy , Lung Diseases/virology , Male , Mice , Paranasal Sinuses/immunology , Paranasal Sinuses/virology , SARS-CoV-2/growth & development , SARS-CoV-2/immunology , Treatment Outcome
11.
STAR Protoc ; 1(3): 100169, 2020 12 18.
Article in English | MEDLINE | ID: covidwho-917458

ABSTRACT

Common laboratory mice such as BALB/c and C57BL/6 mice are not permissive to SARS-CoV2 infection. Sensitization of laboratory mice with Adenovirus expressing human ACE2 (Ad5-hACE2) provides a rapid model for testing viral intervention in vivo. Despite the lack of lethal outcome, Ad5-hACE2-sensitized mice show 20% weight loss on average upon viral challenge with infectious virus being detected at the site of sensitization. This protocol describes the sensitization and subsequent infection of common laboratory mice for use in testing anti-viral interventions. For complete details on the use and execution of this protocol, please refer to Sun et al. (2020).


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Disease Models, Animal , Mice, Transgenic , SARS-CoV-2 , Adenoviridae/genetics , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , Mice , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity
12.
Mol Cell Proteomics ; 19(8): 1303-1309, 2020 08.
Article in English | MEDLINE | ID: covidwho-360321

ABSTRACT

MERS is a life-threatening disease and MERS-CoV has the potential to cause the next pandemic. Protein acetylation is known to play a crucial role in host response to viral infection. Acetylation of viral proteins encoded by other RNA viruses have been reported to affect viral replication. It is therefore of interest to see whether MERS-CoV proteins are also acetylated. Viral proteins obtained from infected cells were trypsin-digested into peptides. Acetylated peptides were enriched by immunoprecipitation and subject to nano-LC-Orbitrap analysis. Bioinformatic analysis was performed to assess the conservation level of identified acetylation sites and to predict the upstream regulatory factors. A total of 12 acetylation sites were identified from 7 peptides, which all belong to the replicase polyprotein pp1ab. All identified acetylation sites were found to be highly conserved across MERS-CoV sequences in NCBI database. Upstream factors, including deacetylases of the SIRT1 and HDAC families as well as acetyltransferases of the TIP60 family, were predicted to be responsible for regulating the acetylation events identified. Western blotting confirms that acetylation events indeed occur on pp1ab protein by expressing NSP4 in HEK293 cells. Acetylation events on MERS-CoV viral protein pp1ab were identified for the first time, which indicate that MERS-CoV might use the host acetylation machinery to regulate its enzyme activity and to achieve optimal replication. Upstream factors were predicted, which might facilitate further analysis of the regulatory mechanism of MERS-CoV replication.


Subject(s)
Lysine/metabolism , Middle East Respiratory Syndrome Coronavirus/metabolism , Viral Proteins/metabolism , Acetylation , HEK293 Cells , Humans
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