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1.
J Immunol ; 211(2): 252-260, 2023 07 15.
Article in English | MEDLINE | ID: covidwho-20241408

ABSTRACT

SARS-CoV-2 has caused an estimated 7 million deaths worldwide to date. A secreted SARS-CoV-2 accessory protein, known as open reading frame 8 (ORF8), elicits inflammatory pulmonary cytokine responses and is associated with disease severity in COVID-19 patients. Recent reports proposed that ORF8 mediates downstream signals in macrophages and monocytes through the IL-17 receptor complex (IL-17RA, IL-17RC). However, generally IL-17 signals are found to be restricted to the nonhematopoietic compartment, thought to be due to rate-limiting expression of IL-17RC. Accordingly, we revisited the capacity of IL-17 and ORF8 to induce cytokine gene expression in mouse and human macrophages and monocytes. In SARS-CoV-2-infected human and mouse lungs, IL17RC mRNA was undetectable in monocyte/macrophage populations. In cultured mouse and human monocytes and macrophages, ORF8 but not IL-17 led to elevated expression of target cytokines. ORF8-induced signaling was fully preserved in the presence of anti-IL-17RA/RC neutralizing Abs and in Il17ra-/- cells. ORF8 signaling was also operative in Il1r1-/- bone marrow-derived macrophages. However, the TLR/IL-1R family adaptor MyD88, which is dispensable for IL-17R signaling, was required for ORF8 activity yet MyD88 is not required for IL-17 signaling. Thus, we conclude that ORF8 transduces inflammatory signaling in monocytes and macrophages via MyD88 independently of the IL-17R.


Subject(s)
COVID-19 , Monocytes , Humans , Mice , Animals , Monocytes/metabolism , SARS-CoV-2/genetics , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Receptors, Interleukin-17/genetics , Receptors, Interleukin-17/metabolism , Open Reading Frames , COVID-19/genetics , Macrophages/metabolism , Cytokines/metabolism
2.
Viruses ; 15(1)2022 Dec 30.
Article in English | MEDLINE | ID: covidwho-2227484

ABSTRACT

Increasing evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection impacts neurological function both acutely and chronically, even in the absence of pronounced respiratory distress. Developing clinically relevant laboratory mouse models of the neuropathogenesis of SARS-CoV-2 infection is an important step toward elucidating the underlying mechanisms of SARS-CoV-2-induced neurological dysfunction. Although various transgenic models and viral delivery methods have been used to study the infection potential of SARS-CoV-2 in mice, the use of commonly available laboratory mice would facilitate the study of SARS-CoV-2 neuropathology. Herein we show neuroinflammatory profiles of immunologically intact mice, C57BL/6J and BALB/c, as well as immunodeficient (Rag2-/-) mice, to a mouse-adapted strain of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2 (MA10)). Our findings indicate that brain IL-6 levels are significantly higher in BALB/c male mice infected with SARS-CoV-2 MA10. Additionally, blood-brain barrier integrity, as measured by the vascular tight junction protein claudin-5, was reduced by SARS-CoV-2 MA10 infection in all three strains. Brain glial fibrillary acidic protein (GFAP) mRNA was also elevated in male C57BL/6J infected mice compared with the mock group. Lastly, immune-vascular effects of SARS-CoV-2 (MA10), as measured by H&E scores, demonstrate an increase in perivascular lymphocyte cuffing (PLC) at 30 days post-infection among infected female BALB/c mice with a significant increase in PLC over time only in SARS-CoV-2 MA10) infected mice. Our study is the first to demonstrate that SARS-CoV-2 (MA10) infection induces neuroinflammation in laboratory mice and could be used as a novel model to study SARS-CoV-2-mediated cerebrovascular pathology.


Subject(s)
COVID-19 , SARS-CoV-2 , Mice , Male , Female , Animals , COVID-19/pathology , Lung , Neuroinflammatory Diseases , Mice, Inbred C57BL , Disease Models, Animal , Mice, Transgenic
3.
Cell Rep ; 42(1): 112014, 2023 01 31.
Article in English | MEDLINE | ID: covidwho-2177165

ABSTRACT

The SARS-CoV-2 Omicron variant of concern (VoC) and its sublineages contain 31-36 mutations in spike and escape neutralization by most therapeutic antibodies. In a pseudovirus neutralization assay, 66 of the nearly 400 candidate therapeutics in the Coronavirus Immunotherapeutic Consortium (CoVIC) panel neutralize Omicron and multiple Omicron sublineages. Among natural immunoglobulin Gs (IgGs), especially those in the receptor-binding domain (RBD)-2 epitope community, nearly all Omicron neutralizers recognize spike bivalently, with both antigen-binding fragments (Fabs) simultaneously engaging adjacent RBDs on the same spike. Most IgGs that do not neutralize Omicron bind either entirely monovalently or have some (22%-50%) monovalent occupancy. Cleavage of bivalent-binding IgGs to Fabs abolishes neutralization and binding affinity, with disproportionate loss of activity against Omicron pseudovirus and spike. These results suggest that VoC-resistant antibodies overcome mutagenic substitution via avidity. Hence, vaccine strategies targeting future SARS-CoV-2 variants should consider epitope display with spacing and organization identical to trimeric spike.


Subject(s)
COVID-19 , Humans , SARS-CoV-2 , Ethnicity , Epitopes , Antibodies, Viral , Antibodies, Neutralizing , Neutralization Tests
4.
Am J Respir Crit Care Med ; 206(9): 1178-1179, 2022 11 01.
Article in English | MEDLINE | ID: covidwho-2098113
5.
Viruses ; 14(5)2022 05 05.
Article in English | MEDLINE | ID: covidwho-1820422

ABSTRACT

SARS-CoV-2 variants, including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) variants, have displayed increased transmissibility and, therefore, have been categorized as variants of concern (VOCs). The pervasiveness of VOCs suggests a high probability of future mutations that may lead to increased virulence. Prior reports have shown that VOC infection without expression of human angiotensin converting enzyme-2 receptor (hACE2) in mice is possible. We sought to understand if the increased transmissibility of VOCs can infect C57BL/6 mice without expression of hACE2 receptor required for entry of SARS-CoV-2 normally. We examined the ability of infection with Beta and Gamma variants to infect and cause both pathological and clinical changes consistent with severe COVID-19, including body weight changes, survival, subgenomic viral titer, lung histology on Hematoxylin and Eosin (H&E) staining, and viral protein expression as measured by immunohistochemistry staining of viral antigen (IHC). These methods were used to examine three groups of mice: C57BL6, Rag2-/-, and Ccr2-/- mice. We observed that these mice, infected with Beta and Gamma variants of SARS-CoV-2, did not show pathological changes as indicated by weight loss, altered survival, or significant lung pathology on H&E staining. Subgenomic qPCR and IHC staining for viral protein indicated that there was some evidence of infection but far below ACE2 transgenic mice, which showed clinical disease and pathologic changes consistent with ARDS. These data suggest that these variants replicate poorly even in the setting of profound immune deficiency.


Subject(s)
COVID-19 , Disease Models, Animal , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Animals , COVID-19/virology , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , SARS-CoV-2/genetics , Viral Proteins
6.
iScience ; 25(1): 103670, 2022 Jan 21.
Article in English | MEDLINE | ID: covidwho-1654625

ABSTRACT

SARS-CoV-2, the etiologic agent of COVID-19, uses ACE2 as a cell entry receptor. Soluble ACE2 has been shown to have neutralizing antiviral activity but has a short half-life and no active transport mechanism from the circulation into the alveolar spaces of the lung. To overcome this, we constructed an ACE2-human IgG1 fusion protein with mutations in the catalytic domain of ACE2. A mutation in the catalytic domain of ACE2, MDR504, significantly increased binding to SARS-CoV-2 spike protein, as well as to a spike variant, in vitro with more potent viral neutralization in plaque assays. Parental administration of the protein showed stable serum concentrations with excellent bioavailability in the epithelial lining fluid of the lung, and ameliorated lung SARS-CoV-2 infection in vivo. These data support that the MDR504 hACE2-Fc is an excellent candidate for treatment or prophylaxis of COVID-19 and potentially emerging variants.

8.
Front Immunol ; 12: 754642, 2021.
Article in English | MEDLINE | ID: covidwho-1485059

ABSTRACT

Understanding SARS-CoV-2 immune pathology is critical for the development of effective vaccines and treatments. Here, we employed unbiased serial whole-blood transcriptome profiling by weighted gene network correlation analysis (WGCNA) at pre-specified timepoints of infection to understand SARS-CoV-2-related immune alterations in a cohort of rhesus macaques (RMs) and African green monkeys (AGMs) presenting with varying degrees of pulmonary pathology. We found that the bulk of transcriptional changes occurred at day 3 post-infection and normalized to pre-infection levels by 3 weeks. There was evidence of coordination of transcriptional networks in blood (defined by WGCNA) and the nasopharyngeal SARS-CoV-2 burden as well as the absolute monocyte count. Pathway analysis of gene modules revealed prominent regulation of type I and type II interferon stimulated genes (ISGs) in both RMs and AGMs, with the latter species exhibiting a greater breadth of ISG upregulation. Notably, pathways relating to neutrophil degranulation were enriched in blood of SARS-CoV-2 infected AGMs, but not RMs. Our results elude to hallmark similarities as well as differences in the RM and AGM acute response to SARS-CoV-2 infection, and may help guide the selection of particular NHP species in modeling aspects of COVID-19 disease outcome.


Subject(s)
COVID-19/immunology , Cell Degranulation , Neutrophils/immunology , SARS-CoV-2/immunology , Animals , COVID-19/blood , Chlorocebus aethiops , Disease Models, Animal , Macaca mulatta , Neutrophils/metabolism , SARS-CoV-2/metabolism , Species Specificity
9.
Front Immunol ; 12: 672523, 2021.
Article in English | MEDLINE | ID: covidwho-1389182

ABSTRACT

Lower respiratory infections are among the leading causes of morbidity and mortality worldwide. These potentially deadly infections are further exacerbated due to the growing incidence of antimicrobial resistance. To combat these infections there is a need to better understand immune mechanisms that promote microbial clearance. This need in the context of lung infections has been further heightened with the emergence of SARS-CoV-2. Group 3 innate lymphoid cells (ILC3s) are a recently discovered tissue resident innate immune cell found at mucosal sites that respond rapidly in the event of an infection. ILC3s have clear roles in regulating mucosal immunity and tissue homeostasis in the intestine, though the immunological functions in lungs remain unclear. It has been demonstrated in both viral and bacterial pneumonia that stimulated ILC3s secrete the cytokines IL-17 and IL-22 to promote both microbial clearance as well as tissue repair. In this review, we will evaluate regulation of ILC3s during inflammation and discuss recent studies that examine ILC3 function in the context of both bacterial and viral pulmonary infections.


Subject(s)
COVID-19/immunology , Immunity, Mucosal/immunology , Lymphocytes/immunology , Pneumonia, Bacterial/immunology , Respiratory Mucosa/immunology , SARS-CoV-2/immunology , Bacteria/immunology , COVID-19/mortality , COVID-19/pathology , Immunity, Innate/immunology , Inflammation/immunology , Interleukin-17/metabolism , Interleukins/metabolism , Lung/immunology , Lymphocyte Activation/immunology , Respiratory Mucosa/cytology
10.
JCI Insight ; 6(16)2021 08 23.
Article in English | MEDLINE | ID: covidwho-1369460

ABSTRACT

Evidence suggests an association between severe acute respiratory syndrome-cornavirus-2 (SARS-CoV-2) infection and the occurrence of new-onset diabetes. We examined pancreatic expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), the cell entry factors for SARS-CoV-2, using publicly available single-cell RNA sequencing data sets, and pancreatic tissue from control male and female nonhuman primates (NHPs) and humans. We also examined SARS-CoV-2 immunolocalization in pancreatic cells of SARS-CoV-2-infected NHPs and patients who had died from coronavirus disease 2019 (COVID-19). We report expression of ACE2 in pancreatic islet, ductal, and endothelial cells in NHPs and humans. In pancreata from SARS-CoV-2-infected NHPs and COVID-19 patients, SARS-CoV-2 infected ductal, endothelial, and islet cells. These pancreata also exhibited generalized fibrosis associated with multiple vascular thrombi. Two out of 8 NHPs developed new-onset diabetes following SARS-CoV-2 infection. Two out of 5 COVID-19 patients exhibited new-onset diabetes at admission. These results suggest that SARS-CoV-2 infection of the pancreas may promote acute and especially chronic pancreatic dysfunction that could potentially lead to new-onset diabetes.


Subject(s)
COVID-19/complications , Diabetes Mellitus/etiology , Pancreas/virology , SARS-CoV-2/isolation & purification , Thrombosis/etiology , Angiotensin-Converting Enzyme 2/analysis , Animals , Chlorocebus aethiops , Female , Fibrosis , Humans , Macaca mulatta , Male , Serine Endopeptidases/analysis
11.
Theranostics ; 11(16): 8076-8091, 2021.
Article in English | MEDLINE | ID: covidwho-1337802

ABSTRACT

Rationale: Pulmonary vascular endotheliitis, perivascular inflammation, and immune activation are observed in COVID-19 patients. While the initial SARS-CoV-2 infection mainly infects lung epithelial cells, whether it also infects endothelial cells (ECs) and to what extent SARS-CoV-2-mediated pulmonary vascular endotheliitis is associated with immune activation remain to be determined. Methods: To address these questions, we studied SARS-CoV-2-infected K18-hACE2 (K18) mice, a severe COVID-19 mouse model, as well as lung samples from SARS-CoV-2-infected nonhuman primates (NHP) and patient deceased from COVID-19. We used immunostaining, RNAscope, and electron microscopy to analyze the organs collected from animals and patient. We conducted bulk and single cell (sc) RNA-seq analyses, and cytokine profiling of lungs or serum of the severe COVID-19 mice. Results: We show that SARS-CoV-2-infected K18 mice develop severe COVID-19, including progressive body weight loss and fatality at 7 days, severe lung interstitial inflammation, edema, hemorrhage, perivascular inflammation, systemic lymphocytopenia, and eosinopenia. Body weight loss in K18 mice correlated with the severity of pneumonia, but not with brain infection. We also observed endothelial activation and dysfunction in pulmonary vessels evidenced by the up-regulation of VCAM1 and ICAM1 and the downregulation of VE-cadherin. We detected SARS-CoV-2 in capillary ECs, activation and adhesion of platelets and immune cells to the vascular wall of the alveolar septa, and increased complement deposition in the lungs, in both COVID-19-murine and NHP models. We also revealed that pathways of coagulation, complement, K-ras signaling, and genes of ICAM1 and VCAM1 related to EC dysfunction and injury were upregulated, and were associated with massive immune activation in the lung and circulation. Conclusion: Together, our results indicate that SARS-CoV-2 causes endotheliitis via both infection and infection-mediated immune activation, which may contribute to the pathogenesis of severe COVID-19 disease.


Subject(s)
COVID-19/immunology , COVID-19/pathology , Animals , COVID-19/metabolism , Disease Models, Animal , Endothelial Cells/immunology , Endothelial Cells/virology , Epithelial Cells/immunology , Epithelial Cells/virology , Lung/pathology , Mice , Mice, Inbred Strains , Mice, Transgenic , SARS-CoV-2/isolation & purification
12.
Front Cell Infect Microbiol ; 11: 701278, 2021.
Article in English | MEDLINE | ID: covidwho-1325517

ABSTRACT

SARS-CoV-2 infection can cause fatal inflammatory lung pathology, including thrombosis and increased pulmonary vascular permeability leading to edema and hemorrhage. In addition to the lung, cytokine storm-induced inflammatory cascade also affects other organs. SARS-CoV-2 infection-related vascular inflammation is characterized by endotheliopathy in the lung and other organs. Whether SARS-CoV-2 causes endotheliopathy by directly infecting endothelial cells is not known and is the focus of the present study. We observed 1) the co-localization of SARS-CoV-2 with the endothelial cell marker CD31 in the lungs of SARS-CoV-2-infected mice expressing hACE2 in the lung by intranasal delivery of adenovirus 5-hACE2 (Ad5-hACE2 mice) and non-human primates at both the protein and RNA levels, and 2) SARS-CoV-2 proteins in endothelial cells by immunogold labeling and electron microscopic analysis. We also detected the co-localization of SARS-CoV-2 with CD31 in autopsied lung tissue obtained from patients who died from severe COVID-19. Comparative analysis of RNA sequencing data of the lungs of infected Ad5-hACE2 and Ad5-empty (control) mice revealed upregulated KRAS signaling pathway, a well-known pathway for cellular activation and dysfunction. Further, we showed that SARS-CoV-2 directly infects mature mouse aortic endothelial cells (AoECs) that were activated by performing an aortic sprouting assay prior to exposure to SARS-CoV-2. This was demonstrated by co-localization of SARS-CoV-2 and CD34 by immunostaining and detection of viral particles in electron microscopic studies. Moreover, the activated AoECs became positive for ACE-2 but not quiescent AoECs. Together, our results indicate that in addition to pneumocytes, SARS-CoV-2 also directly infects mature vascular endothelial cells in vivo and ex vivo, which may contribute to cardiovascular complications in SARS-CoV-2 infection, including multipleorgan failure.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Disease Models, Animal , Endothelial Cells , Humans , Lung , Mice , Mice, Transgenic
14.
Cell Rep ; 35(5): 109055, 2021 05 04.
Article in English | MEDLINE | ID: covidwho-1179291

ABSTRACT

Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Although infection initiates in the proximal airways, severe and sometimes fatal symptoms of the disease are caused by infection of the alveolar type 2 (AT2) cells of the distal lung and associated inflammation. In this study, we develop primary human lung epithelial infection models to understand initial responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface (ALI) cultures of proximal airway epithelium and alveosphere cultures of distal lung AT2 cells are readily infected by SARS-CoV-2, leading to an epithelial cell-autonomous proinflammatory response with increased expression of interferon signaling genes. Studies to validate the efficacy of selected candidate COVID-19 drugs confirm that remdesivir strongly suppresses viral infection/replication. We provide a relevant platform for study of COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and emergent respiratory pathogens.


Subject(s)
Alveolar Epithelial Cells/virology , COVID-19 Drug Treatment , COVID-19/pathology , Lung/virology , SARS-CoV-2/drug effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Adult , Aged , Alanine/analogs & derivatives , Alanine/pharmacology , Alveolar Epithelial Cells/metabolism , COVID-19/metabolism , COVID-19/virology , Child, Preschool , Drug Discovery/methods , Epithelial Cells/virology , Epithelium/metabolism , Epithelium/virology , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Lung/pathology , Male , Middle Aged , Models, Biological , Primary Cell Culture , Respiratory Mucosa/virology , SARS-CoV-2/physiology , Virus Replication/drug effects
15.
Am J Respir Cell Mol Biol ; 64(1): 79-88, 2021 01.
Article in English | MEDLINE | ID: covidwho-1004249

ABSTRACT

Preclinical mouse models that recapitulate some characteristics of coronavirus disease (COVID-19) will facilitate focused study of pathogenesis and virus-host responses. Human agniotensin-converting enzyme 2 (hACE2) serves as an entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to infect people via binding to envelope spike proteins. Herein we report development and characterization of a rapidly deployable COVID-19 mouse model. C57BL/6J (B6) mice expressing hACE2 in the lung were transduced by oropharyngeal delivery of the recombinant human adenovirus type 5 that expresses hACE2 (Ad5-hACE2). Mice were infected with SARS-CoV-2 at Day 4 after transduction and developed interstitial pneumonia associated with perivascular inflammation, accompanied by significantly higher viral load in lungs at Days 3, 6, and 12 after infection compared with Ad5-empty control group. SARS-CoV-2 was detected in pneumocytes in alveolar septa. Transcriptomic analysis of lungs demonstrated that the infected Ad5-hACE mice had a significant increase in IFN-dependent chemokines Cxcl9 and Cxcl10, and genes associated with effector T-cell populations including Cd3 g, Cd8a, and Gzmb. Pathway analysis showed that several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched in the data set, including cytokine-cytokine receptor interaction, the chemokine signaling pathway, the NOD-like receptor signaling pathway, the measles pathway, and the IL-17 signaling pathway. This response is correlative to clinical response in lungs of patients with COVID-19. These results demonstrate that expression of hACE2 via adenovirus delivery system sensitized the mouse to SARS-CoV-2 infection and resulted in the development of a mild COVID-19 phenotype, highlighting the immune and inflammatory host responses to SARS-CoV-2 infection. This rapidly deployable COVID-19 mouse model is useful for preclinical and pathogenesis studies of COVID-19.


Subject(s)
Alveolar Epithelial Cells/immunology , COVID-19/immunology , Gene Expression , SARS-CoV-2/immunology , Signal Transduction/immunology , Adenoviridae/genetics , Adenoviridae/metabolism , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/virology , Angiotensin-Converting Enzyme 2/biosynthesis , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Animals , COVID-19/genetics , COVID-19/metabolism , COVID-19/pathology , Cytokines/genetics , Cytokines/immunology , Disease Models, Animal , Humans , Mice , Mice, Transgenic , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Signal Transduction/genetics , Transduction, Genetic
16.
JACC Basic Transl Sci ; 6(1): 1-8, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-866798

ABSTRACT

Many efforts to design and screen therapeutics for the current severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic have focused on inhibiting viral host cell entry by disrupting angiotensin-converting enzyme-2 (ACE2) binding with the SARS-CoV-2 spike protein. This work focuses on the potential to inhibit SARS-CoV-2 entry through a hypothesized α5ß1 integrin-based mechanism and indicates that inhibiting the spike protein interaction with α5ß1 integrin (+/- ACE2) and the interaction between α5ß1 integrin and ACE2 using a novel molecule (ATN-161) represents a promising approach to treat coronavirus disease-19.

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