ABSTRACT
Background: Aberrant activation of the classic renin-angiotensin system (RAS) and intestinal micro dysbiosis adversely affect insulin resistance (IR), dyslipidemia, and other metabolic syndrome markers. However, the action of angiotensin-converting enzyme 2 (ACE2) and gut health in systemic homeostasis vary, and their interaction is not completely understood. Methods: We adopted a combinatory approach of metabolomics and fecal 16S rRNA analysis to investigate gut microbiota and metabolite in two different mouse models, ACE2 knockout (ACE2 KO) mice and the ACE2-overexpressing obese mice. Results: 16S rRNA gene sequencing revealed that ACE2 influences microbial community composition and function, and ACE2 KO mice had increased Deferribacteres, Alcaligenaceae, Parasutterella, Catenibacterium, and Anaerotruncus, with decreased short-chain fatty acid (SCFA)-producing bacteria (Marvinbryantia and Alistipes). In contrast, ACE2-overexpressed mice exhibited increased anti-inflammatory probiotic (Oscillospiraceae, Marinifilaceae, and Bifidobacteriaceae) and SCFA-producing microbes (Rikenellaceae, Muribaculaceae, Ruminococcaceae, Odoribacter, and Alistipes) and decreased Firmicutes/Bacteroidetes, Lactobacillaceae, Erysipelotrichaceae, and Lachnospiraceae. Metabolome analysis indicated differential metabolites in ACE2 KO and ACE2-overexpression mice, especially the glucolipid metabolism-related compounds. Furthermore, correlation analysis between gut microbiota and metabolites showed a dynamic mutual influence affecting host health. Conclusion: Our study confirms for the first time a significant association between ACE2 status and gut microbiome and metabolome profiles, providing a novel mechanism for the positive effect of ACE2 on energy homeostasis.
Subject(s)
Angiotensin-Converting Enzyme 2 , Bacteria , Gastrointestinal Microbiome , Mice, Knockout , RNA, Ribosomal, 16S , Animals , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Mice , RNA, Ribosomal, 16S/genetics , Bacteria/classification , Bacteria/genetics , Bacteria/metabolism , Bacteria/isolation & purification , Feces/microbiology , Metabolomics , Dysbiosis/microbiology , Male , Metabolome , Peptidyl-Dipeptidase A/metabolism , Peptidyl-Dipeptidase A/genetics , Obesity/metabolism , Obesity/microbiology , Mice, Inbred C57BL , Probiotics , Fatty Acids, Volatile/metabolismABSTRACT
The involvement of neutrophils in the lungs during the recovery phase of coronavirus disease 2019 (COVID-19) is not well defined mainly due to the limited accessibility of lung tissues from COVID-19 survivors. The lack of an appropriate small animal model has affected the development of effective therapeutic strategies. We here developed a long COVID mouse model to study changes in neutrophil phenotype and association with lung injury. Our data shows persistent neutrophil recruitment and neutrophil extracellular trap formation in the lungs for up to 30 days post-infection which correlates with lung fibrosis and inflammation.
Subject(s)
COVID-19 , Disease Models, Animal , Extracellular Traps , Lung , Neutrophils , SARS-CoV-2 , Animals , Extracellular Traps/immunology , COVID-19/immunology , COVID-19/complications , Mice , Neutrophils/immunology , SARS-CoV-2/immunology , SARS-CoV-2/physiology , Lung/pathology , Lung/immunology , Lung/virology , Lung Injury/immunology , Lung Injury/virology , Lung Injury/pathology , Lung Injury/etiology , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Neutrophil Infiltration/immunology , Humans , Pulmonary Fibrosis/immunology , Pulmonary Fibrosis/pathology , Pulmonary Fibrosis/etiologyABSTRACT
Activation of the renin-angiotensin-aldosterone system (RAAS) plays an important pathophysiological role in hypertension. Increased mRNA levels of the angiotensinogen angiotensin-converting enzyme, angiotensin type 1 receptor gene, Agtr1a, and the aldosterone synthase gene, CYP11B2, have been reported in the heart, blood vessels, and kidneys in salt-sensitive hypertension. However, the mechanism of gene regulation in each component of the RAAS in cardiovascular and renal tissues is unclear. Epigenetic mechanisms, which are important for regulating gene expression, include DNA methylation, histone post-translational modifications, and microRNA (miRNA) regulation. A close association exists between low DNA methylation at CEBP-binding sites and increased AGT expression in visceral adipose tissue and the heart of salt-sensitive hypertensive rats. Several miRNAs influence AGT expression and are associated with cardiovascular diseases. Expression of both ACE and ACE2 genes is regulated by DNA methylation, histone modifications, and miRNAs. Expression of both angiotensinogen and CYP11B2 is reversibly regulated by epigenetic modifications and is related to salt-sensitive hypertension. The mineralocorticoid receptor (MR) exists in cardiovascular and renal tissues, in which many miRNAs influence expression and contribute to the pathogenesis of hypertension. Expression of the 11beta-hydroxysteroid dehydrogenase type 2 (HSD11B2) gene is also regulated by methylation and miRNAs. Epigenetic regulation of renal and vascular HSD11B2 is an important pathogenetic mechanism for salt-sensitive hypertension.
Subject(s)
DNA Methylation , Epigenesis, Genetic , Hypertension , Renin-Angiotensin System , Renin-Angiotensin System/genetics , Hypertension/genetics , Hypertension/metabolism , Animals , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Angiotensinogen/genetics , Angiotensinogen/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Receptor, Angiotensin, Type 1/genetics , Receptor, Angiotensin, Type 1/metabolism , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Cytochrome P-450 CYP11B2/genetics , Cytochrome P-450 CYP11B2/metabolismABSTRACT
The COVID-19 pandemic has uncovered the high genetic variability of the SARS-CoV-2 virus and its ability to evade the immune responses that were induced by earlier viral variants. Only a few monoclonal antibodies that have been reported to date are capable of neutralizing a broad spectrum of SARS-CoV-2 variants. Here, we report the isolation of a new broadly neutralizing human monoclonal antibody, iC1. The antibody was identified through sorting the SARS-CoV-1 RBD-stained individual B cells that were isolated from the blood of a vaccinated donor following a breakthrough infection. In vitro, iC1 potently neutralizes pseudoviruses expressing a wide range of SARS-CoV-2 Spike variants, including those of the XBB sublineage. In an hACE2-transgenic mouse model, iC1 provided effective protection against the Wuhan strain of the virus as well as the BA.5 and XBB.1.5 variants. Therefore, iC1 can be considered as a potential component of the broadly neutralizing antibody cocktails resisting the SARS-CoV-2 mutation escape.
Subject(s)
Angiotensin-Converting Enzyme 2 , Antibodies, Monoclonal , Antibodies, Neutralizing , Antibodies, Viral , COVID-19 , Mice, Transgenic , SARS-CoV-2 , Animals , SARS-CoV-2/immunology , Humans , COVID-19/immunology , COVID-19/prevention & control , COVID-19/virology , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Mice , Antibodies, Viral/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/genetics , Pandemics/prevention & control , Betacoronavirus/immunology , Betacoronavirus/genetics , Broadly Neutralizing Antibodies/immunology , Disease Models, Animal , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Pneumonia, Viral/prevention & control , Coronavirus Infections/immunology , Coronavirus Infections/virology , Coronavirus Infections/prevention & controlABSTRACT
Despite the high efficiency of current SARS-CoV-2 mRNA vaccines in reducing COVID-19 morbidity and mortality, waning immunity and the emergence of resistant variants underscore the need for novel vaccination strategies. This study explores a heterologous mRNA/Modified Vaccinia virus Ankara (MVA) prime/boost regimen employing a trimeric form of the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein compared to a homologous MVA/MVA regimen. In C57BL/6 mice, the RBD was delivered during priming via an mRNA vector encapsulated in nanoemulsions (NE) or lipid nanoparticles (LNP), followed by a booster with a replication-deficient MVA-based recombinant virus (MVA-RBD). This heterologous mRNA/MVA regimen elicited strong anti-RBD binding and neutralizing antibodies (BAbs and NAbs) against both the ancestral SARS-CoV-2 strain and different variants of concern (VoCs). Additionally, this protocol induced robust and polyfunctional RBD-specific CD4 and CD8 T cell responses, particularly in animals primed with mLNP-RBD. In K18-hACE2 transgenic mice, the LNP-RBD/MVA combination provided complete protection from morbidity and mortality following a live SARS-CoV-2 challenge compared with the partial protection observed with mNE-RBD/MVA or MVA/MVA regimens. Although the mNE-RBD/MVA regimen only protects half of the animals, it was able to induce antibodies with Fc-mediated effector functions besides NAbs. Moreover, viral replication and viral load in the respiratory tract were markedly reduced and decreased pro-inflammatory cytokine levels were observed. These results support the efficacy of heterologous mRNA/MVA vaccine combinations over homologous MVA/MVA regimen, using alternative nanocarriers that circumvent intellectual property restrictions of current mRNA vaccine formulations.
Subject(s)
Antibodies, Neutralizing , Antibodies, Viral , COVID-19 Vaccines , COVID-19 , Mice, Inbred C57BL , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Vaccinia virus , Animals , COVID-19 Vaccines/immunology , COVID-19 Vaccines/administration & dosage , SARS-CoV-2/immunology , SARS-CoV-2/genetics , Mice , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/genetics , COVID-19/prevention & control , COVID-19/immunology , Antibodies, Viral/immunology , Antibodies, Viral/blood , Antibodies, Neutralizing/immunology , Vaccinia virus/genetics , Vaccinia virus/immunology , Humans , Female , Nanoparticles/administration & dosage , Vaccination , mRNA Vaccines/administration & dosage , Mice, Transgenic , Vaccines, Synthetic/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , CD8-Positive T-Lymphocytes/immunology , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/genetics , LiposomesABSTRACT
Coronavirus disease-2019 (COVID-19) has caused continuous effects on the global public, especially for susceptible and vulnerable populations like pregnant women. COVID-19-related studies and publications have shown blowout development, making it challenging to identify development trends and hot areas by using traditional review methods for such massive data. Aimed to perform a bibliometric analysis to explore the status and hotspots of COVID-19 in obstetrics. An online search was conducted in the Web of Science Core Collection (WOSCC) database from January 01, 2020 to November 31, 2022, using the following search expression: (((TS= ("COVID 19" OR "coronavirus 2019" OR "coronavirus disease 2019" OR "SARS-CoV-2" OR "2019-nCoV" OR "2019 novel coronavirus" OR "SARS coronavirus 2" OR "Severe Acute Respiratory Syndrome Coronavirus-2" OR "SARS-COV2")) AND TS= ("obstetric*" OR "pregnancy*" OR "pregnant" OR "parturition*" OR "puerperium"))). VOSviewer version 1.6.18, CiteSpace version 6.1.R6, R version 4.2.0, and Rstudio were used for the bibliometric and visualization analyses. 4144 articles were included in further analysis, including authors, titles, number of citations, countries, and author affiliations. The United States has contributed the most significant publications with the leading position. "Sahin, Dilek" has the largest output, and "Khalil, Asma" was the most influential author with the highest citations. Keywords of "Cov," "Experience," and "Neonate" with the highest frequency, and "Systematic Review" might be the new research hotspots and frontiers. The top 3 concerned genes included ACE2, CRP, and IL6. The new research hotspot is gradually shifting from the COVID-19 mechanism and its related clinical research to reviewing treatment options for pregnant women. This research uniquely delves into specific genes related to COVID-19's effects on obstetrics, a focus that has not been previously explored in other reviews. Our research enables clinicians and researchers to summarize the overall point of view of the existing literature and obtain more accurate conclusions.
Subject(s)
COVID-19 Drug Treatment , COVID-19 , Obstetrics , Pandemics , COVID-19/epidemiology , COVID-19/genetics , Bibliometrics , Obstetrics/trends , Humans , Female , Pregnancy , Angiotensin-Converting Enzyme 2/genetics , C-Reactive Protein/genetics , Interleukin-6/geneticsABSTRACT
Despite a substantial body of research, we lack fundamental understanding of the pathophysiology of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) including pulmonary and cardiovascular outcomes, in part due to limitations of murine models. Most models use transgenic mice (K18) that express the human (h) angiotensin converting enzyme 2 (ACE2), ACE2 knock-in (KI) mice, or mouse-adapted strains of SARS-CoV-2. Further, many SARS-CoV-2 variants produce fatal neurologic disease in K18 mice and most murine studies focus only on acute disease in the first 14 days post inoculation (dpi). To better enable understanding of both acute (<14 dpi) and post-acute (>14 dpi) infection phases, we describe the development and characterization of a novel non-lethal KI mouse that expresses both the ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (hACE2/hTMPRSS2). The human genes were engineered to replace the orthologous mouse gene loci but remain under control of their respective murine promoters, resulting in expression of ACE2 and TMPRSS2 instead of their murine counterparts. After intranasal inoculation with an omicron strain of SARS-CoV-2, hACE2/hTMPRSS2 KI mice transiently lost weight but recovered by 7 dpi. Infectious SARS-CoV-2 was detected in nasopharyngeal swabs 1-2 dpi and in lung tissues 2-6 dpi, peaking 4 dpi. These outcomes were similar to those in K18 mice that were inoculated in parallel. To determine the extent to which hACE2/hTMPRSS2 KI mice are suitable to model pulmonary and cardiovascular outcomes, physiological assessments measuring locomotion, behavior and reflexes, biomonitoring to measure cardiac activity and respiration, and micro computed tomography to assess lung function were conducted frequently to 6 months post inoculation. Male but not female SARS-CoV-2 inoculated hACE2/hTMPRSS2 KI mice showed a transient reduction in locomotion compared to control saline treated mice. No significant changes in respiration, oxygen saturation, heart rate variability, or conductivity were detected in SARS-CoV-2 inoculated mice of either sex. When re-inoculated 6 months after the first inoculation, hACE2/hTMPRSS2 KI became re-infected with disease signs similar to after the first inoculation. Together these data show that a newly generated hACE2/hTMPRSS2 KI mouse can be used to study mild COVID-19.
Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Disease Models, Animal , Gene Knock-In Techniques , Mice, Transgenic , SARS-CoV-2 , Serine Endopeptidases , Animals , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/genetics , COVID-19/virology , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Mice , Humans , Serine Endopeptidases/genetics , Female , Male , Lung/virology , Lung/pathology , Mice, Inbred C57BLABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has acquired multiple mutations since its emergence. Analyses of the SARS-CoV-2 genomes from infected patients exhibit a bias toward C-to-U mutations, which are suggested to be caused by the apolipoprotein B mRNA editing enzyme polypeptide-like 3 (APOBEC3, A3) cytosine deaminase proteins. However, the role of A3 enzymes in SARS-CoV-2 replication remains unclear. To address this question, we investigated the effect of A3 family proteins on SARS-CoV-2 replication in the myeloid leukemia cell line THP-1 lacking A3A to A3G genes. The Wuhan, BA.1, and BA.5 variants had comparable viral replication in parent and A3A-to-A3G-null THP-1 cells stably expressing angiotensin-converting enzyme 2 (ACE2) protein. On the other hand, the replication and infectivity of these variants were abolished in A3A-to-A3G-null THP-1-ACE2 cells in a series of passage experiments over 20 days. In contrast to previous reports, we observed no evidence of A3-induced SARS-CoV-2 mutagenesis in the passage experiments. Furthermore, our analysis of a large number of publicly available SARS-CoV-2 genomes did not reveal conclusive evidence for A3-induced mutagenesis. Our studies suggest that A3 family proteins can positively contribute to SARS-CoV-2 replication; however, this effect is deaminase-independent.
Subject(s)
APOBEC Deaminases , COVID-19 , Cytidine Deaminase , SARS-CoV-2 , Virus Replication , Humans , SARS-CoV-2/genetics , SARS-CoV-2/physiology , SARS-CoV-2/metabolism , APOBEC Deaminases/metabolism , APOBEC Deaminases/genetics , COVID-19/virology , COVID-19/metabolism , Cytidine Deaminase/metabolism , Cytidine Deaminase/genetics , THP-1 Cells , Mutation , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Genome, ViralABSTRACT
Virus-induced cell death is a key contributor to COVID-19 pathology. Cell death induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is well studied in myeloid cells but less in its primary host cell type, angiotensin-converting enzyme 2 (ACE2)-expressing human airway epithelia (HAE). SARS-CoV-2 induces apoptosis, necroptosis, and pyroptosis in HAE organotypic cultures. Single-cell and limiting-dilution analysis revealed that necroptosis is the primary cell death event in infected cells, whereas uninfected bystanders undergo apoptosis, and pyroptosis occurs later during infection. Mechanistically, necroptosis is induced by viral Z-RNA binding to Z-DNA-binding protein 1 (ZBP1) in HAE and lung tissues from patients with COVID-19. The Delta (B.1.617.2) variant, which causes more severe disease than Omicron (B1.1.529) in humans, is associated with orders of magnitude-greater Z-RNA/ZBP1 interactions, necroptosis, and disease severity in animal models. Thus, Delta induces robust ZBP1-mediated necroptosis and more disease severity.
Subject(s)
COVID-19 , Necroptosis , Pyroptosis , RNA-Binding Proteins , Respiratory Mucosa , SARS-CoV-2 , Humans , SARS-CoV-2/immunology , COVID-19/immunology , COVID-19/pathology , Necroptosis/immunology , Animals , Respiratory Mucosa/virology , Respiratory Mucosa/immunology , Respiratory Mucosa/pathology , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Mice , Cell Death/immunology , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Apoptosis/immunologyABSTRACT
Understanding how proteins evolve under selective pressure is a longstanding challenge. The immensity of the search space has limited efforts to systematically evaluate the impact of multiple simultaneous mutations, so mutations have typically been assessed individually. However, epistasis, or the way in which mutations interact, prevents accurate prediction of combinatorial mutations based on measurements of individual mutations. Here, we use artificial intelligence to define the entire functional sequence landscape of a protein binding site in silico, and we call this approach Complete Combinatorial Mutational Enumeration (CCME). By leveraging CCME, we are able to construct a comprehensive map of the evolutionary connectivity within this functional sequence landscape. As a proof of concept, we applied CCME to the ACE2 binding site of the SARS-CoV-2 spike protein receptor binding domain. We selected representative variants from across the functional sequence landscape for testing in the laboratory. We identified variants that retained functionality to bind ACE2 despite changing over 40% of evaluated residue positions, and the variants now escape binding and neutralization by monoclonal antibodies. This work represents a crucial initial stride toward achieving precise predictions of pathogen evolution, opening avenues for proactive mitigation.
Subject(s)
Angiotensin-Converting Enzyme 2 , Mutation , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , SARS-CoV-2/genetics , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Humans , Binding Sites , COVID-19/virology , COVID-19/genetics , Protein Binding , Artificial IntelligenceABSTRACT
The upper respiratory tract is the initial site of SARS-CoV-2 infection. Nasal spike-specific secretory immunoglobulin A (sIgA) correlates with protection against Omicron breakthrough infection. We report that intranasal vaccination using human adenovirus serotype 5 (Ad5) vectored Omicron spike in people who previously vaccinated with ancestral vaccine could induce robust neutralizing sIgA in the nasal passage. Nasal sIgA was predominantly present in dimeric and multimeric forms and accounted for nearly 40% of total proteins in nasal mucosal lining fluids (NMLFs). A low-level IgG could also be detected in NMLFs but not IgM, IgD, and IgE. After a complete nasal wash, sIgA in the nasal passage could be replenished rapidly within a few hours. A comparison of purified paired serum IgA, serum IgG, and nasal sIgA from the same individuals showed that sIgA was up to 3-logs more potent than serum antibodies in binding to spikes and in neutralizing Omicron subvariants. Serum IgG and IgA failed to neutralize XBB and BA.2.86, while nasal sIgA retained potent neutralization against these newly emerged variants. Further analysis showed that sIgA was more effective than IgG or IgA in blocking spike-mediated cell-to-cell transmission and protecting hACE2 mice from XBB challenge. Using a sIgA monoclonal antibody as a reference, we estimated that the total nasal sIgA contains about 2.6-3.9% spike-specific sIgA in NMLFs collected approximately one month after intranasal vaccination. Our study provided insights for developing intranasal vaccines that can induce sIgA to build an effective and mutation-resistant first-line immune barrier against constantly emerging variants.
Subject(s)
Administration, Intranasal , Antibodies, Neutralizing , Antibodies, Viral , COVID-19 Vaccines , COVID-19 , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Humans , SARS-CoV-2/immunology , SARS-CoV-2/genetics , COVID-19/prevention & control , COVID-19/immunology , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/blood , Animals , Mice , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/genetics , Antibodies, Viral/immunology , Antibodies, Viral/blood , COVID-19 Vaccines/immunology , COVID-19 Vaccines/genetics , COVID-19 Vaccines/administration & dosage , Immunoglobulin A/immunology , Immunoglobulin A/blood , Immunoglobulin A/genetics , Nasal Mucosa/immunology , Nasal Mucosa/virology , Female , Genetic Vectors/immunology , Genetic Vectors/genetics , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Immunoglobulin A, Secretory/immunology , Adenoviridae/genetics , Adenoviridae/immunology , Immunoglobulin G/immunology , Immunoglobulin G/blood , MaleABSTRACT
BACKGROUND: Severe COVID-19 is uncommon, restricted to 19% of the total population. In response to the first virus wave (alpha variant of SARS-CoV-2), we investigated whether a biomarker indicated severity of disease and, in particular, if variable expression of angiotensin converting enzyme 2 (ACE2) in blood might clarify this difference in risk and of post COVID -19 conditions (PCC). METHODS: The IRB-approved study compared patients hospitalized with severe COVID-19 to healthy controls. Severe infection was defined requiring oxygen or increased oxygen need from baseline at admission with positive COVID-19 PCR. A single blood sample was obtained from patients within a day of admission. ACE2 RNA expression in blood cells was measured by an RT-PCR assay. Plasma ACE1 and ACE2 enzyme activities were quantified by fluorescent peptides. Plasma TIMP-1, PIIINP and MMP-9 antigens were quantified by ELISA. Data were entered into REDCap and analyzed using STATA v 14 and GraphPad Prism v 10. RESULTS: Forty-eight patients and 72 healthy controls were recruited during the pandemic. ACE2 RNA expression in peripheral blood mononuclear cells (PBMC) was rarely detected acutely during severe COVID-19 but common in controls (OR for undetected ACE2: 12.4 [95% CI: 2.62-76.1]). ACE2 RNA expression in PBMC did not determine plasma ACE1 and ACE2 activity, suggesting alternative cell-signaling pathways. Markers of fibrosis (TIMP-1 and PIIINP) and vasculopathy (MMP-9) were additionally elevated. ACE2 RNA expression during severe COVID-19 often responded within hours to convalescent plasma. Analogous to oncogenesis, we speculate that potent, persistent, cryptic processes following COVID-19 (the renin-angiotensin system (RAS), fibrosis and vasculopathy) initiate or promote post-COVID-19 conditions (PCC) in susceptible individuals. CONCLUSIONS: This work elucidates biological and temporal plausibility for ACE2, TIMP1, PIIINP and MMP-9 in the pathogenesis of PCC. Intersection of these independent systems is uncommon and may in part explain the rarity of PCC.
Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Leukocytes, Mononuclear , SARS-CoV-2 , Humans , COVID-19/blood , Angiotensin-Converting Enzyme 2/blood , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Male , Female , Middle Aged , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/virology , Aged , Adult , Biomarkers/blood , Tissue Inhibitor of Metalloproteinase-1/blood , Tissue Inhibitor of Metalloproteinase-1/genetics , Matrix Metalloproteinase 9/blood , Matrix Metalloproteinase 9/genetics , Severity of Illness Index , Case-Control Studies , Peptidyl-Dipeptidase A/blood , Peptidyl-Dipeptidase A/geneticsABSTRACT
As SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor (ACE2-OE) that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. Interferon-lambda was upregulated in cells with low-level infection while the NF-kB inhibitor alpha gene (encoding IkBa) was consistently upregulated in infected cells, and its expression positively correlated with infection levels. Confocal microscopy showed more IkBa expression in infected than bystander cells, but found concurrent nuclear translocation of NF-kB that IkBa usually prevents. Overexpressing a nondegradable IkBa mutant reduced NF-kB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and underscore that the strength of the NF-kB feedback loop in infected cells controls viral replication.
Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , NF-KappaB Inhibitor alpha , Organoids , SARS-CoV-2 , Virus Replication , Humans , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/genetics , COVID-19/virology , COVID-19/metabolism , COVID-19/genetics , NF-kappa B/metabolism , NF-KappaB Inhibitor alpha/metabolism , NF-KappaB Inhibitor alpha/genetics , Organoids/virology , Organoids/metabolism , SARS-CoV-2/physiologyABSTRACT
Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.
Subject(s)
Angiotensin-Converting Enzyme 2 , Cryoelectron Microscopy , Proline , SARS-CoV-2 , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Proline/metabolism , Humans , SARS-CoV-2/metabolism , SARS-CoV-2/genetics , COVID-19/virology , COVID-19/metabolism , Amino Acid Transport Systems, Neutral/metabolism , Amino Acid Transport Systems, Neutral/genetics , Amino Acid Transport Systems, Neutral/chemistry , Models, MolecularABSTRACT
Extracellular vesicles (EVs) are shown to be a novel viral transmission model capable of increasing a virus's tropism. According to our earlier research, cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or transfected with envelope protein plasmids generate a novel type of EVs that are micrometer-sized and able to encase virus particles. Here, we showed the capacity of these EVs to invade various animals both in vitro and in vivo independent of the angiotensin-converting enzyme 2 receptor. First, via macropinocytosis, intact EVs produced from Vero E6 (monkey) cells were able to enter cells from a variety of animals, including cats, dogs, bats, hamsters, and minks, and vice versa. Second, when given to zebrafish with cutaneous wounds, the EVs showed favorable stability in aqueous environments and entered the fish. Moreover, infection of wild-type (WT) mice with heterogeneous EVs carrying SARS-CoV-2 particles led to a strong cytokine response and a notable amount of lung damage. Conversely, free viral particles did not infect WT mice. These results highlight the variety of processes behind viral transmission and cross-species evolution by indicating that EVs may be possible vehicles for SARS-CoV-2 spillover and raising risk concerns over EVs' potential for viral gene transfer.
Subject(s)
COVID-19 , Extracellular Vesicles , SARS-CoV-2 , Animals , Extracellular Vesicles/virology , Extracellular Vesicles/metabolism , SARS-CoV-2/physiology , SARS-CoV-2/pathogenicity , SARS-CoV-2/genetics , COVID-19/transmission , COVID-19/virology , Mice , Chlorocebus aethiops , Vero Cells , Humans , Cricetinae , Coronavirus Envelope Proteins/metabolism , Coronavirus Envelope Proteins/genetics , Dogs , Zebrafish/virology , Cats , Chiroptera/virology , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/geneticsABSTRACT
BACKGROUND: Alzheimer's disease (AD) is the highest risk of COVID-19 infection, hospitalization, and mortality. However, it remains largely unclear about the link between AD and COVID-19 outcomes. ACE2 is an entry receptor for SARS-CoV-2. Circulating ACE2 is a novel biomarker of death and associated with COVID-19 outcomes. METHODS: Here, we explored the shared genetics and causal association between AD and plasma ACE2 levels using large-scale genome-wide association study, gene expression, expression quantitative trait loci, and high-throughput plasma proteomic profiling datasets. RESULTS: We found a significant causal effect of genetically increased circulating ACE2 on increased risk of AD. Cross-trait association analysis identified 19 shared genetic variants, and three variants rs3104412, rs2395166, and rs3135344 at chromosome 6p21.32 were associated with COVID-19 infection, hospitalization, and severity. We mapped 19 variants to 117 genes, which were significantly upregulated in lung, spleen, and small intestine, downregulated in brain tissues, and involved in immune system, immune disease, and infectious disease pathways. The plasma proteins corresponding to LST1, AGER, TNXB, and APOC1 were predominantly associated with COVID-19 infection, ventilation, and death. CONCLUSION: Together, our findings suggest the shared genetics and causal association between AD and plasma ACE2 levels, which may partially explain the link between AD and COVID-19.
Subject(s)
Alzheimer Disease , Angiotensin-Converting Enzyme 2 , COVID-19 , Genome-Wide Association Study , Humans , Alzheimer Disease/genetics , Alzheimer Disease/blood , COVID-19/genetics , COVID-19/blood , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/blood , SARS-CoV-2 , Polymorphism, Single Nucleotide , Quantitative Trait Loci , Male , Female , Aged , Genetic Predisposition to Disease/genetics , Biomarkers/bloodABSTRACT
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection inhibits mitochondrial oxidative phosphorylation (OXPHOS) and elevates mitochondrial reactive oxygen species (ROS, mROS) which activates hypoxia-inducible factor-1alpha (HIF-1α), shifting metabolism toward glycolysis to drive viral biogenesis but also causing the release of mitochondrial DNA (mtDNA) and activation of innate immunity. To determine whether mitochondrially targeted antioxidants could mitigate these viral effects, we challenged mice expressing human angiotensin-converting enzyme 2 (ACE2) with SARS-CoV-2 and intervened using transgenic and pharmacological mitochondrially targeted catalytic antioxidants. Transgenic expression of mitochondrially targeted catalase (mCAT) or systemic treatment with EUK8 decreased weight loss, clinical severity, and circulating levels of mtDNA; as well as reduced lung levels of HIF-1α, viral proteins, and inflammatory cytokines. RNA-sequencing of infected lungs revealed that mCAT and Eukarion 8 (EUK8) up-regulated OXPHOS gene expression and down-regulated HIF-1α and its target genes as well as innate immune gene expression. These data demonstrate that SARS-CoV-2 pathology can be mitigated by catalytically reducing mROS, potentially providing a unique host-directed pharmacological therapy for COVID-19 which is not subject to viral mutational resistance.
Subject(s)
Antioxidants , COVID-19 , Mice, Transgenic , Mitochondria , Oxidative Phosphorylation , SARS-CoV-2 , Animals , Mice , COVID-19/virology , COVID-19/metabolism , COVID-19/immunology , COVID-19/pathology , Antioxidants/metabolism , Antioxidants/pharmacology , Mitochondria/metabolism , Mitochondria/drug effects , SARS-CoV-2/drug effects , Oxidative Phosphorylation/drug effects , Humans , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Lung/virology , Lung/pathology , Lung/metabolism , Reactive Oxygen Species/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , Catalase/metabolism , Catalase/genetics , COVID-19 Drug Treatment , Disease Models, Animal , Immunity, InnateABSTRACT
The recently emerged BA.2.86, JN.1, EG.5, EG.5.1, and HV.1 variants have a growth advantage. In this study, we explore the structural bases of receptor binding and immune evasion for the Omicron BA.2.86, JN.1, EG.5, EG.5.1, and HV.1 sub-variants. Our findings reveal that BA.2.86 exhibits strong receptor binding, whereas its JN.1 sub-lineage displays a decreased binding affinity to human ACE2 (hACE2). Through complex structure analyses, we observed that the reversion of R493Q in BA.2.86 receptor binding domain (RBD) plays a facilitating role in receptor binding, while the L455S substitution in JN.1 RBD restores optimal affinity. Furthermore, the structure of monoclonal antibody (mAb) S309 complexed with BA.2.86 RBD highlights the importance of the K356T mutation, which brings a new N-glycosylation motif, altering the binding pattern of mAbs belonging to RBD-5 represented by S309. These findings emphasize the importance of closely monitoring BA.2.86 and its sub-lineages to prevent another wave of SARS-CoV-2 infections.
Subject(s)
Angiotensin-Converting Enzyme 2 , Antibodies, Monoclonal , COVID-19 , Immune Evasion , Protein Binding , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Humans , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , COVID-19/immunology , COVID-19/virology , COVID-19/metabolism , Binding Sites , Models, Molecular , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/metabolism , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , MutationABSTRACT
In December 2023, we observed a notable shift in the COVID-19 landscape, when JN.1 omicron emerged as the predominant SARS-CoV-2 variant with a 95% incidence. We characterized the clinical profile, and genetic changes in JN.1, an emerging SARS-CoV-2 variant of interest. Whole genome sequencing was performed on SARS-CoV-2 positive clinical specimens, followed by sequence analysis. Mutations within the spike protein sequences were analysed and compared with the previously reported lineages and sub-lineages, to identify the potential impact of the unique mutations on protein structure and possible alterations in the functionality. Several unique and dynamic mutations were identified herein. Molecular docking analysis showed changes in the binding affinity, and key interacting residues of wild-type and mutated structures with key host cell receptors of SARS-CoV-2 entry viz., ACE2, CD147, CD209L and AXL. Our data provides key insights on the emergence of newer variants and highlights the necessity for robust and sustained global genomic surveillance of SARS-CoV-2.
Subject(s)
COVID-19 , Mutation , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Humans , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , India/epidemiology , COVID-19/virology , COVID-19/epidemiology , COVID-19/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/chemistry , Male , Female , Adult , Middle Aged , Whole Genome Sequencing , Molecular Docking Simulation , Genome, Viral , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Aged , PhylogenyABSTRACT
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with neurological sequelae including haemorrhage, thrombosis and ischaemic necrosis and encephalitis. However, the mechanism by which this occurs is unclear. Neurological disease associated with COVID-19 has been proposed to occur following direct infection of the central nervous system and/or indirectly by local or systemic immune activation. We evaluated the expression of angiotensin-converting enzyme-2 and transmembrane protease, serine 2 (TMPRSS2) in brain tissue from five healthy human donors and observed low-level expression of these proteins in cells morphologically consistent with astrocytes, neurons and choroidal ependymal cells within the frontal cortex and medulla oblongata. Primary human astrocytes, neurons, choroid plexus epithelial cells and pericytes supported productive SARS-CoV-2 infection with ancestral, Alpha, Delta and Omicron variants. Infected cells supported the full viral life cycle, releasing infectious virus particles. In contrast, primary brain microvascular endothelial cells and microglia were refractory to SARS-CoV-2 infection. These data support a model whereby SARS-CoV-2 can infect human brain cells, and the mechanism of viral entry warrants further investigation.