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1.
Front Immunol ; 12: 791753, 2021.
Article in English | MEDLINE | ID: covidwho-1613553

ABSTRACT

Background: Infection of SARS-CoV-2 may cause acute respiratory syndrome. It has been reported that SARS-CoV-2 nucleocapsid protein (N-protein) presents early in body fluids during infection. The direct involvement of N-protein in lung injury is poorly understood. Methods: Recombinant N-protein was pretreated with polymyxin B, a lipopolysaccharide (LPS)-neutralizing agent. C57BL/6, C3H/HeJ (resistant to LPS), and C3H/HeN (control for C3H/HeJ) mice were exposed to N-protein via intratracheal administration to examine acute lung injury. In vitro, bone marrow-derived macrophages (BMDMs) were cultured with N-protein to study phosphorylation of nuclear factor kappa B (NF-ĸB) p65, macrophage polarization, and expression of proinflammatory cytokines. Results: N-protein produced acute lung injury in C57BL/6 mice, with elevated protein permeability, total cell count, neutrophil infiltration, and proinflammatory cytokines in the bronchioalveolar lavage. N-protein also induced lung injury in both C3H/HeJ and C3H/HeN mice, indicating that the effect could not be attributed to the LPS contamination. N-protein triggered phosphorylation of NF-ĸB p65 in vitro, which was abolished by both N-protein denaturation and treatment with an antibody for N-protein, demonstrating that the effect is N-protein specific. In addition, N-protein promoted M1 macrophage polarization and the expression of proinflammatory cytokines, which was also blocked by N-protein denaturation and antibody for N-protein. Furthermore, N-protein induced NF-ĸB p65 phosphorylation in the lung, while pyrrolidine dithiocarbamate, an NF-ĸB inhibitor, alleviated the effect of N-protein on acute lung injury. Conclusions: SARS-CoV-2 N-protein itself is toxic and induces acute lung injury in mice. Both N-protein and NF-ĸB pathway may be therapeutic targets for treating multi-organ injuries in Coronavirus disease 2019 (COVID-19).


Subject(s)
Acute Lung Injury/virology , COVID-19 , Coronavirus Nucleocapsid Proteins/toxicity , NF-kappa B/metabolism , Acute Lung Injury/metabolism , Animals , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Phosphoproteins/toxicity , SARS-CoV-2
2.
BMC Mol Cell Biol ; 23(1): 2, 2022 Jan 07.
Article in English | MEDLINE | ID: covidwho-1613226

ABSTRACT

BACKGROUND: SARS-CoV-2, the causative agent of COVID-19 pandemic is a RNA virus prone to mutations. Formation of a stable binding interface between the Receptor Binding Domain (RBD) of SARS-CoV-2 Spike (S) protein and Angiotensin-Converting Enzyme 2 (ACE2) of host is pivotal for viral entry. RBD has been shown to mutate frequently during pandemic. Although, a few mutations in RBD exhibit enhanced transmission rates leading to rise of new variants of concern, most RBD mutations show sustained ACE2 binding and virus infectivity. Yet, how all these mutations make the binding interface constantly favourable for virus remain enigmatic. This study aims to delineate molecular rearrangements in the binding interface of SARS-CoV-2 RBD mutants. RESULTS: Here, we have generated a mutational and structural landscape of SARS-CoV-2 RBD in first six months of the pandemic. We analyzed 31,403 SARS-CoV-2 genomes randomly across the globe, and identified 444 non-synonymous mutations in RBD that cause 49 distinct amino acid substitutions in contact and non-contact amino acid residues. Molecular phylogenetic analysis suggested independent emergence of RBD mutants. Structural mapping of these mutations on the SARS-CoV-2 Wuhan reference strain RBD and structural comparison with RBDs from bat-CoV, SARS-CoV, and pangolin-CoV, all bound to human or mouse ACE2, revealed several changes in the interfacial interactions in all three binding clusters. Interestingly, interactions mediated via N487 residue in cluster-I and Y449, G496, T500, G502 residues in cluster-III remained largely unchanged in all RBD mutants. Further analysis showed that these interactions are evolutionarily conserved in sarbecoviruses which use ACE2 for entry. Importantly, despite extensive changes in the interface, RBD-ACE2 stability and binding affinities were maintained in all the analyzed mutants. Taken together, these findings reveal how SARS-CoV-2 uses its RBD residues to constantly remodel the binding interface. CONCLUSION: Our study broadly signifies understanding virus-host binding interfaces and their alterations during pandemic. Our findings propose a possible interface remodelling mechanism used by SARS-CoV-2 to escape deleterious mutations. Future investigations will focus on functional validation of in-silico findings and on investigating interface remodelling mechanisms across sarbecoviruses. Thus, in long run, this study may provide novel clues to therapeutically target RBD-ACE2 interface for pan-sarbecovirus infections.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Host Microbial Interactions , Humans , Mice , Mutation , Pandemics , Phylogeny , Spike Glycoprotein, Coronavirus/genetics
3.
Signal Transduct Target Ther ; 7(1): 7, 2022 01 04.
Article in English | MEDLINE | ID: covidwho-1606287

ABSTRACT

Activation-induced cytidine deaminase (AID) initiates class-switch recombination and somatic hypermutation (SHM) in antibody genes. Protein expression and activity are tightly controlled by various mechanisms. However, it remains unknown whether a signal from the extracellular environment directly affects the AID activity in the nucleus where it works. Here, we demonstrated that a deubiquitinase USP10, which specifically stabilizes nuclear AID protein, can translocate into the nucleus after AKT-mediated phosphorylation at its T674 within the NLS domain. Interestingly, the signals from BCR and TLR1/2 synergistically promoted this phosphorylation. The deficiency of USP10 in B cells significantly decreased AID protein levels, subsequently reducing neutralizing antibody production after immunization with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or human immunodeficiency virus type 1 (HIV-1) nanoparticle vaccines. Collectively, we demonstrated that USP10 functions as an integrator for both BCR and TLR signals and directly regulates nuclear AID activity. Its manipulation could be used for the development of vaccines and adjuvants.


Subject(s)
AIDS Vaccines/immunology , B-Cell Activating Factor/immunology , COVID-19 Vaccines/immunology , Cytidine Deaminase/immunology , HIV-1/immunology , Nanoparticles , SARS-CoV-2/immunology , Signal Transduction/immunology , Ubiquitin Thiolesterase/immunology , Ubiquitination/immunology , AIDS Vaccines/genetics , Animals , B-Cell Activating Factor/genetics , COVID-19 Vaccines/genetics , Cytidine Deaminase/genetics , HEK293 Cells , HIV-1/genetics , Humans , Mice , Mice, Knockout , SARS-CoV-2/genetics , Signal Transduction/genetics , Ubiquitin Thiolesterase/genetics
5.
J Neuroinflammation ; 19(1): 2, 2022 Jan 04.
Article in English | MEDLINE | ID: covidwho-1603365

ABSTRACT

BACKGROUND: Anxiety disorders are the most prevalent mental illnesses in the U.S. and are estimated to consume one-third of the country's mental health treatment cost. Although anxiolytic therapies are available, many patients still exhibit treatment resistance, relapse, or substantial side effects. Further, due to the COVID-19 pandemic and stay-at-home order, social isolation, fear of the pandemic, and unprecedented times, the incidence of anxiety has dramatically increased. Previously, we have demonstrated dihydromyricetin (DHM), the major bioactive flavonoid extracted from Ampelopsis grossedentata, exhibits anxiolytic properties in a mouse model of social isolation-induced anxiety. Because GABAergic transmission modulates the immune system in addition to the inhibitory signal transmission, we investigated the effects of short-term social isolation on the neuroimmune system. METHODS: Eight-week-old male C57BL/6 mice were housed under absolute social isolation for 4 weeks. The anxiety-like behaviors after DHM treatment were examined using elevated plus-maze and open field behavioral tests. Gephyrin protein expression, microglial profile changes, NF-κB pathway activation, cytokine level, and serum corticosterone were measured. RESULTS: Socially isolated mice showed increased anxiety levels, reduced exploratory behaviors, and reduced gephyrin levels. Also, a dynamic alteration in hippocampal microglia were detected illustrated as a decline in microglia number and overactivation as determined by significant morphological changes including decreases in lacunarity, perimeter, and cell size and increase in cell density. Moreover, social isolation induced an increase in serum corticosterone level and activation in NF-κB pathway. Notably, DHM treatment counteracted these changes. CONCLUSION: The results suggest that social isolation contributes to neuroinflammation, while DHM has the ability to improve neuroinflammation induced by anxiety.


Subject(s)
Flavonols/pharmacology , Inflammation Mediators/antagonists & inhibitors , Inflammation Mediators/metabolism , Microglia/drug effects , Microglia/metabolism , Social Isolation/psychology , Animals , Anxiety/metabolism , Anxiety/prevention & control , Anxiety/psychology , Flavonols/therapeutic use , Male , Maze Learning/drug effects , Maze Learning/physiology , Mice , Mice, Inbred C57BL
6.
Virol J ; 19(1): 2, 2022 01 04.
Article in English | MEDLINE | ID: covidwho-1608023

ABSTRACT

The current COVID-19 pandemic caused by constantly emerging SARS-CoV-2 variants still poses a threat to public health worldwide. Effective next-generation vaccines and optimized booster vaccination strategies are urgently needed. Here, we sequentially immunized mice with a SARS-CoV-2 wild-type inactivated vaccine and a heterologous mutant RBD vaccine, and then evaluated their neutralizing antibody responses against variants including Beta, Delta, Alpha, Iota, Kappa, and A.23.1. These data showed that a third booster dose of heterologous RBD vaccine especially after two doses of inactivated vaccines significantly enhanced the GMTs of nAbs against all SARS-CoV-2 variants we tested. In addition, the WT and variants all displayed good cross-immunogenicity and might be applied in the design of booster vaccines to induce broadly neutralizing antibodies.


Subject(s)
COVID-19 Vaccines , COVID-19 , SARS-CoV-2 , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19/prevention & control , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/immunology , Mice , SARS-CoV-2/immunology
7.
Int J Biol Sci ; 18(1): 386-408, 2022.
Article in English | MEDLINE | ID: covidwho-1607858

ABSTRACT

Responding to the coronavirus disease 2019 (COVID-19) pandemic has been an unexpected and unprecedented global challenge for humanity in this century. During this crisis, specialists from the laboratories and frontline clinical personnel have made great efforts to prevent and treat COVID-19 by revealing the molecular biological characteristics and epidemic characteristics of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, SARS-CoV-2 has severe consequences for public health, including human respiratory system, immune system, blood circulation system, nervous system, motor system, urinary system, reproductive system and digestive system. In the review, we summarize the physiological and pathological damage of SARS-CoV-2 to these systems and its molecular mechanisms followed by clinical manifestation. Concurrently, the prevention and treatment strategies of COVID-19 will be discussed in preclinical and clinical studies. With constantly unfolding and expanding scientific understanding about COVID-19, the updated information can help applied researchers understand the disease to build potential antiviral drugs or vaccines, and formulate creative therapeutic ideas for combating COVID-19 at speed.


Subject(s)
COVID-19/pathology , COVID-19/therapy , Immunotherapy/methods , SARS-CoV-2 , Animals , Antiviral Agents/therapeutic use , COVID-19/drug therapy , COVID-19 Vaccines , Cytokines/metabolism , Female , Humans , Immune System , Immunity, Innate , Immunologic Memory , Male , Mice
8.
Nucleic Acids Res ; 50(D1): D1-D10, 2022 01 07.
Article in English | MEDLINE | ID: covidwho-1607482

ABSTRACT

The 2022 Nucleic Acids Research Database Issue contains 185 papers, including 87 papers reporting on new databases and 85 updates from resources previously published in the Issue. Thirteen additional manuscripts provide updates on databases most recently published elsewhere. Seven new databases focus specifically on COVID-19 and SARS-CoV-2, including SCoV2-MD, the first of the Issue's Breakthrough Articles. Major nucleic acid databases reporting updates include MODOMICS, JASPAR and miRTarBase. The AlphaFold Protein Structure Database, described in the second Breakthrough Article, is the stand-out in the protein section, where the Human Proteoform Atlas and GproteinDb are other notable new arrivals. Updates from DisProt, FuzDB and ELM comprehensively cover disordered proteins. Under the metabolism and signalling section Reactome, ConsensusPathDB, HMDB and CAZy are major returning resources. In microbial and viral genomes taxonomy and systematics are well covered by LPSN, TYGS and GTDB. Genomics resources include Ensembl, Ensembl Genomes and UCSC Genome Browser. Major returning pharmacology resource names include the IUPHAR/BPS guide and the Therapeutic Target Database. New plant databases include PlantGSAD for gene lists and qPTMplants for post-translational modifications. The entire Database Issue is freely available online on the Nucleic Acids Research website (https://academic.oup.com/nar). Our latest update to the NAR online Molecular Biology Database Collection brings the total number of entries to 1645. Following last year's major cleanup, we have updated 317 entries, listing 89 new resources and trimming 80 discontinued URLs. The current release is available at http://www.oxfordjournals.org/nar/database/c/.


Subject(s)
Databases, Factual , Molecular Biology , Animals , COVID-19 , Databases, Nucleic Acid , Databases, Protein , Genome, Microbial , Genome, Viral , Humans , Mice , Plants/genetics , Protein Processing, Post-Translational , Proteome , SARS-CoV-2/genetics , Signal Transduction
9.
Proc Natl Acad Sci U S A ; 119(1)2022 01 04.
Article in English | MEDLINE | ID: covidwho-1599475

ABSTRACT

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in tremendous loss worldwide. Although viral spike (S) protein binding of angiotensin-converting enzyme 2 (ACE2) has been established, the functional consequences of the initial receptor binding and the stepwise fusion process are not clear. By utilizing a cell-cell fusion system, in complement with a pseudoviral infection model, we found that the spike engagement of ACE2 primed the generation of S2' fragments in target cells, a key proteolytic event coupled with spike-mediated membrane fusion. Mutagenesis of an S2' cleavage site at the arginine (R) 815, but not an S2 cleavage site at arginine 685, was sufficient to prevent subsequent syncytia formation and infection in a variety of cell lines and primary cells isolated from human ACE2 knock-in mice. The requirement for S2' cleavage at the R815 site was also broadly shared by other SARS-CoV-2 spike variants, such as the Alpha, Beta, and Delta variants of concern. Thus, our study highlights an essential role for host receptor engagement and the key residue of spike for proteolytic activation, and uncovers a targetable mechanism for host cell infection by SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Membrane Fusion , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Animals , COVID-19/virology , HEK293 Cells , Host-Pathogen Interactions , Humans , Mice , Protein Binding , Proteolysis , Virus Internalization
10.
mBio ; 12(6): e0334721, 2021 12 21.
Article in English | MEDLINE | ID: covidwho-1599212

ABSTRACT

The world was unprepared for coronavirus disease 2019 (COVID-19) and remains ill-equipped for future pandemics. While unprecedented strides have been made developing vaccines and treatments for COVID-19, there remains a need for highly effective and widely available regimens for ambulatory use for novel coronaviruses and other viral pathogens. We posit that a priority is to develop pan-family drug cocktails to enhance potency, limit toxicity, and avoid drug resistance. We urge cocktail development for all viruses with pandemic potential both in the short term (<1 to 2 years) and longer term with pairs of drugs in advanced clinical testing or repurposed agents approved for other indications. While significant efforts were launched against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in vitro and in the clinic, many studies employed solo drugs and had disappointing results. Here, we review drug combination studies against SARS-CoV-2 and other viruses and introduce a model-driven approach to assess drug pairs with the highest likelihood of clinical efficacy. Where component agents lack sufficient potency, we advocate for synergistic combinations to achieve therapeutic levels. We also discuss issues that stymied therapeutic progress against COVID-19, including testing of agents with low likelihood of efficacy late in clinical disease and lack of focus on developing virologic surrogate endpoints. There is a need to expedite efficient clinical trials testing drug combinations that could be taken at home by recently infected individuals and exposed contacts as early as possible during the next pandemic, whether caused by a coronavirus or another viral pathogen. The approach herein represents a proactive plan for global viral pandemic preparedness.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus/drug effects , Drug Combinations , Animals , COVID-19/drug therapy , Coronavirus/classification , Coronavirus/pathogenicity , Coronavirus Infections/drug therapy , Humans , Mice , Pandemics/prevention & control , SARS-CoV-2/drug effects
11.
Microbiol Spectr ; 9(3): e0165921, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1598527

ABSTRACT

COVID-19 vaccines are currently being administered worldwide and playing a critical role in controlling the pandemic. They have been designed to elicit neutralizing antibodies against Spike protein of the original SARS-CoV-2, and hence they are less effective against SARS-CoV-2 variants with mutated Spike than the original virus. It is possible that novel variants with abilities of enhanced transmissibility and/or immunoevasion will appear in the near future and perfectly escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Several lines of evidence suggest the contribution of CTLs on the viral control in COVID-19, and CTLs target a wide range of proteins involving comparatively conserved nonstructural proteins. Here, we identified 22 HLA-A*24:02-restricted CTL candidate epitopes derived from the nonstructural polyprotein 1a (pp1a) of SARS-CoV-2 using computational algorithms, HLA-A*24:02 transgenic mice and the peptide-encapsulated liposomes. We focused on pp1a and HLA-A*24:02 because pp1a is relatively conserved and HLA-A*24:02 is predominant in East Asians such as Japanese. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by a number of mutations in the Sequence Read Archive database of SARS-CoV-2 variants. The information of such conserved epitopes might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any SARS-CoV-2 variants by the induction of both anti-Spike neutralizing antibodies and CTLs specific for conserved epitopes. IMPORTANCE COVID-19 vaccines have been designed to elicit neutralizing antibodies against the Spike protein of the original SARS-CoV-2, and hence they are less effective against variants. It is possible that novel variants will appear and escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Here, we identified 22 HLA-A*24:02-restricted CTL candidate epitopes derived from the nonstructural polyprotein 1a (pp1a) of SARS-CoV-2. We focused on pp1a and HLA-A*24:02 because pp1a is conserved and HLA-A*24:02 is predominant in East Asians. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by mutations in the database of SARS-CoV-2 variants. The information might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any variants.


Subject(s)
COVID-19/immunology , Epitopes/immunology , HLA-A24 Antigen/genetics , HLA-A24 Antigen/immunology , Mutation , Polyproteins/genetics , SARS-CoV-2/genetics , T-Lymphocytes, Cytotoxic/immunology , Amino Acid Sequence , Animals , Antibodies, Neutralizing/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/immunology , Epitopes/genetics , HLA-A24 Antigen/isolation & purification , Humans , Mice , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology
12.
J Microbiol Biotechnol ; 31(12): 1601-1614, 2021 12 28.
Article in English | MEDLINE | ID: covidwho-1595833

ABSTRACT

To overcome the ongoing COVID-19 pandemic, vaccination campaigns are the highest priority of majority of countries. Limited supply and worldwide disproportionate availability issues for the approved vaccines, together with concerns about rare side-effects have recently initiated the switch to heterologous vaccination, commonly known as mixing of vaccines. The COVID-19 vaccines are highly effective in the general population. However, none of the vaccines is 100% efficacious or effective, with variants posing more challenges, resulting in breakthrough cases. This review summarizes the current knowledge of immune responses to variants of concern (VOC) and breakthrough infections. Furthermore, we discuss the scope of heterologous vaccination and future strategies to tackle the COVID-19 pandemic, including fractionation of vaccine doses and alternative route of vaccination.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Immunity, Heterologous , SARS-CoV-2/immunology , Vaccination/methods , Vaccination/trends , Animals , COVID-19/immunology , COVID-19 Vaccines/classification , Clinical Trials as Topic , Humans , Mice
13.
Science ; 372(6547): 1169-1175, 2021 06 11.
Article in English | MEDLINE | ID: covidwho-1583231

ABSTRACT

Emergent resistance to all clinical antibiotics calls for the next generation of therapeutics. Here we report an effective antimicrobial strategy targeting the bacterial hydrogen sulfide (H2S)-mediated defense system. We identified cystathionine γ-lyase (CSE) as the primary generator of H2S in two major human pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, and discovered small molecules that inhibit bacterial CSE. These inhibitors potentiate bactericidal antibiotics against both pathogens in vitro and in mouse models of infection. CSE inhibitors also suppress bacterial tolerance, disrupting biofilm formation and substantially reducing the number of persister bacteria that survive antibiotic treatment. Our results establish bacterial H2S as a multifunctional defense factor and CSE as a drug target for versatile antibiotic enhancers.


Subject(s)
Anti-Bacterial Agents/pharmacology , Cystathionine gamma-Lyase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Hydrogen Sulfide/metabolism , Pseudomonas aeruginosa/drug effects , Staphylococcus aureus/drug effects , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Biofilms , Crystallography, X-Ray , Cystathionine gamma-Lyase/chemistry , Cystathionine gamma-Lyase/genetics , Cystathionine gamma-Lyase/metabolism , Drug Discovery , Drug Resistance, Bacterial , Drug Synergism , Drug Tolerance , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Mice , Microbial Sensitivity Tests , Models, Molecular , Molecular Docking Simulation , Molecular Structure , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/growth & development , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Staphylococcal Infections/drug therapy , Staphylococcal Infections/microbiology , Staphylococcus aureus/enzymology , Staphylococcus aureus/genetics , Staphylococcus aureus/growth & development
14.
Signal Transduct Target Ther ; 6(1): 420, 2021 12 14.
Article in English | MEDLINE | ID: covidwho-1585885

ABSTRACT

COVID-19 is identified as a zoonotic disease caused by SARS-CoV-2, which also can cross-transmit to many animals but not mice. Genetic modifications of SARS-CoV-2 or mice enable the mice susceptible to viral infection. Although neither is the natural situation, they are currently utilized to establish mouse infection models. Here we report a direct contact transmission of SARS-CoV-2 variant B.1.351 in wild-type mice. The SARS-CoV-2 (B.1.351) replicated efficiently and induced significant pathological changes in lungs and tracheas, accompanied by elevated proinflammatory cytokines in the lungs and sera. Mechanistically, the receptor-binding domain (RBD) of SARS-CoV-2 (B.1.351) spike protein turned to a high binding affinity to mouse angiotensin-converting enzyme 2 (mACE2), allowing the mice highly susceptible to SARS-CoV-2 (B.1.351) infection. Our work suggests that SARS-CoV-2 (B.1.351) expands the host range and therefore increases its transmission route without adapted mutation. As the wild house mice live with human populations quite closely, this possible transmission route could be potentially risky. In addition, because SARS-CoV-2 (B.1.351) is one of the major epidemic strains and the mACE2 in laboratory-used mice is naturally expressed and regulated, the SARS-CoV-2 (B.1.351)/mice could be a much convenient animal model system to study COVID-19 pathogenesis and evaluate antiviral inhibitors and vaccines.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/transmission , Host-Pathogen Interactions/genetics , Receptors, Virus/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/immunology , Animals , COVID-19/immunology , COVID-19/virology , Cytokines/genetics , Cytokines/immunology , Disease Models, Animal , Gene Expression , HEK293 Cells , Host-Pathogen Interactions/immunology , Humans , Lung/pathology , Lung/virology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Protein Binding , Protein Domains , Receptors, Virus/immunology , SARS-CoV-2/classification , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/immunology , Virus Replication
15.
Signal Transduct Target Ther ; 6(1): 439, 2021 12 24.
Article in English | MEDLINE | ID: covidwho-1585883

ABSTRACT

The development of animal models for COVID-19 is essential for basic research and drug/vaccine screening. Previously reported COVID-19 animal models need to be established under a high biosafety level condition for the utilization of live SARS-CoV-2, which greatly limits its application in routine research. Here, we generate a mouse model of COVID-19 under a general laboratory condition that captures multiple characteristics of SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) observed in humans. Briefly, human ACE2-transgenic (hACE2) mice were intratracheally instilled with the formaldehyde-inactivated SARS-CoV-2, resulting in a rapid weight loss and detrimental changes in lung structure and function. The pulmonary pathologic changes were characterized by diffuse alveolar damage with pulmonary consolidation, hemorrhage, necrotic debris, and hyaline membrane formation. The production of fatal cytokines (IL-1ß, TNF-α, and IL-6) and the infiltration of activated neutrophils, inflammatory monocyte-macrophages, and T cells in the lung were also determined, suggesting the activation of an adaptive immune response. Therapeutic strategies, such as dexamethasone or passive antibody therapy, could effectively ameliorate the disease progression in this model. Therefore, the established mouse model for SARS-CoV-2-induced ARDS in the current study may provide a robust tool for researchers in the standard open laboratory to investigate the pathological mechanisms or develop new therapeutic strategies for COVID-19 and ARDS.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , COVID-19/immunology , Lung/immunology , Respiratory Distress Syndrome/immunology , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/genetics , Disease Models, Animal , Humans , Mice , Mice, Transgenic , Respiratory Distress Syndrome/genetics
16.
Sci Rep ; 11(1): 23993, 2021 12 14.
Article in English | MEDLINE | ID: covidwho-1585801

ABSTRACT

Previous work indicates that SARS-CoV-2 virus entry proteins angiotensin-converting enzyme 2 (ACE-2) and the cell surface transmembrane protease serine 2 (TMPRSS-2) are regulated by sex hormones. However, clinical studies addressing this association have yielded conflicting results. We sought to analyze the impact of sex hormones, age, and cardiovascular disease on ACE-2 and TMPRSS-2 expression in different mouse models. ACE-2 and TMPRSS-2 expression was analyzed by immunostaining in a variety of tissues obtained from FVB/N mice undergoing either gonadectomy or sham-surgery and being subjected to ischemia-reperfusion injury or transverse aortic constriction surgery. In lung tissues sex did not have a significant impact on the expression of ACE-2 and TMPRSS-2. On the contrary, following myocardial injury, female sex was associated to a lower expression of ACE-2 at the level of the kidney tubules. In addition, after myocardial injury, a significant correlation between younger age and higher expression of both ACE-2 and TMPRSS-2 was observed for lung alveoli and bronchioli, kidney tubules, and liver sinusoids. Our experimental data indicate that gonadal hormones and biological sex do not alter ACE-2 and TMPRSS-2 expression in the respiratory tract in mice, independent of disease state. Thus, sex differences in ACE-2 and TMPRSS-2 protein expression observed in mice may not explain the higher disease burden of COVID-19 among men.


Subject(s)
Aging/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Cardiomyopathies/metabolism , Castration/adverse effects , Serine Endopeptidases/metabolism , Animals , Bronchioles/metabolism , Disease Models, Animal , Female , Gene Expression Regulation , Kidney Tubules/metabolism , Liver/metabolism , Male , Mice , Pulmonary Alveoli/metabolism , Virus Internalization
17.
Sci Rep ; 11(1): 24432, 2021 12 24.
Article in English | MEDLINE | ID: covidwho-1585772

ABSTRACT

Despite the initial success of some drugs and vaccines targeting COVID-19, understanding the mechanism underlying SARS-CoV-2 disease pathogenesis remains crucial for the development of further approaches to treatment. Some patients with severe Covid-19 experience a cytokine storm and display evidence of inflammasome activation leading to increased levels of IL-1ß and IL-18; however, other reports have suggested reduced inflammatory responses to Sars-Cov-2. In this study we have examined the effects of the Sars-Cov-2 envelope (E) protein, a virulence factor in coronaviruses, on inflammasome activation and pulmonary inflammation. In cultured macrophages the E protein suppressed inflammasome priming and NLRP3 inflammasome activation. Similarly, in mice transfected with E protein and treated with poly(I:C) to simulate the effects of viral RNA, the E protein, in an NLRP3-dependent fashion, reduced expression of pro-IL-1ß, levels of IL-1ß and IL-18 in broncho-alveolar lavage fluid, and macrophage infiltration in the lung. To simulate the effects of more advanced infection, macrophages were treated with both LPS and poly(I:C). In this setting the E protein increased NLRP3 inflammasome activation in both murine and human macrophages. Thus, the Sars-Cov-2 E protein may initially suppress the host NLRP3 inflammasome response to viral RNA while potentially increasing NLRP3 inflammasome responses in the later stages of infection. Targeting the Sars-Cov-2 E protein especially in the early stages of infection may represent a novel approach to Covid-19 therapy.


Subject(s)
Coronavirus Envelope Proteins/metabolism , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , SARS-CoV-2/metabolism , Animals , Bronchoalveolar Lavage Fluid/chemistry , COVID-19/pathology , COVID-19/virology , Coronavirus Envelope Proteins/genetics , Down-Regulation/drug effects , Endoplasmic Reticulum Stress , Humans , Inflammasomes/drug effects , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Janus Kinases/genetics , Janus Kinases/metabolism , Lipopolysaccharides/pharmacology , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein/deficiency , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Poly I-C/pharmacology , RNA, Viral/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/isolation & purification
18.
PLoS Pathog ; 17(12): e1010092, 2021 12.
Article in English | MEDLINE | ID: covidwho-1581718

ABSTRACT

The development of safe and effective vaccines to prevent SARS-CoV-2 infections remains an urgent priority worldwide. We have used a recombinant vesicular stomatitis virus (rVSV)-based prime-boost immunization strategy to develop an effective COVID-19 vaccine candidate. We have constructed VSV genomes carrying exogenous genes resulting in the production of avirulent rVSV carrying the full-length spike protein (SF), the S1 subunit, or the receptor-binding domain (RBD) plus envelope (E) protein of SARS-CoV-2. Adding the honeybee melittin signal peptide (msp) to the N-terminus enhanced the protein expression, and adding the VSV G protein transmembrane domain and the cytoplasmic tail (Gtc) enhanced protein incorporation into pseudotype VSV. All rVSVs expressed three different forms of SARS-CoV-2 spike proteins, but chimeras with VSV-Gtc demonstrated the highest rVSV-associated expression. In immunized mice, rVSV with chimeric S protein-Gtc derivatives induced the highest level of potent neutralizing antibodies and T cell responses, and rVSV harboring the full-length msp-SF-Gtc proved to be the superior immunogen. More importantly, rVSV-msp-SF-Gtc vaccinated animals were completely protected from a subsequent SARS-CoV-2 challenge. Overall, we have developed an efficient strategy to induce a protective response in SARS-CoV-2 challenged immunized mice. Vaccination with our rVSV-based vector may be an effective solution in the global fight against COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Vesicular stomatitis Indiana virus/genetics , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/genetics , Chlorocebus aethiops , Humans , Immunization , Mice , Mice, Inbred C57BL , Mice, Transgenic , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Viral Proteins/genetics , Viral Proteins/immunology
19.
Front Immunol ; 12: 761949, 2021.
Article in English | MEDLINE | ID: covidwho-1581340

ABSTRACT

The decline in mucosal immunity during aging increases susceptibility, morbidity and mortality to infections acquired via the gastrointestinal and respiratory tracts in the elderly. We previously showed that this immunosenescence includes a reduction in the functional maturation of M cells in the follicle-associated epithelia (FAE) covering the Peyer's patches, diminishing the ability to sample of antigens and pathogens from the gut lumen. Here, co-expression analysis of mRNA-seq data sets revealed a general down-regulation of most FAE- and M cell-related genes in Peyer's patches from aged mice, including key transcription factors known to be essential for M cell differentiation. Conversely, expression of ACE2, the cellular receptor for SARS-Cov-2 virus, was increased in the aged FAE. This raises the possibility that the susceptibility of aged Peyer's patches to infection with the SARS-Cov-2 virus is increased. Expression of key Paneth cell-related genes was also reduced in the ileum of aged mice, consistent with the adverse effects of aging on their function. However, the increased expression of these genes in the villous epithelium of aged mice suggested a disturbed distribution of Paneth cells in the aged intestine. Aging effects on Paneth cells negatively impact on the regenerative ability of the gut epithelium and could indirectly impede M cell differentiation. Thus, restoring Paneth cell function may represent a novel means to improve M cell differentiation in the aging intestine and increase mucosal vaccination efficacy in the elderly.


Subject(s)
COVID-19 , Immunity, Mucosal/immunology , Immunosenescence/immunology , Paneth Cells/immunology , Peyer's Patches/immunology , Animals , Cell Differentiation/immunology , Mice , Mice, Inbred C57BL , SARS-CoV-2
20.
Front Immunol ; 12: 765211, 2021.
Article in English | MEDLINE | ID: covidwho-1581337

ABSTRACT

Saturation suppressor mutagenesis was used to generate thermostable mutants of the SARS-CoV-2 spike receptor-binding domain (RBD). A triple mutant with an increase in thermal melting temperature of ~7°C with respect to the wild-type B.1 RBD and was expressed in high yield in both mammalian cells and the microbial host, Pichia pastoris, was downselected for immunogenicity studies. An additional derivative with three additional mutations from the B.1.351 (beta) isolate was also introduced into this background. Lyophilized proteins were resistant to high-temperature exposure and could be stored for over a month at 37°C. In mice and hamsters, squalene-in-water emulsion (SWE) adjuvanted formulations of the B.1-stabilized RBD were considerably more immunogenic than RBD lacking the stabilizing mutations and elicited antibodies that neutralized all four current variants of concern with similar neutralization titers. However, sera from mice immunized with the stabilized B.1.351 derivative showed significantly decreased neutralization titers exclusively against the B.1.617.2 (delta) VOC. A cocktail comprising stabilized B.1 and B.1.351 RBDs elicited antibodies with qualitatively improved neutralization titers and breadth relative to those immunized solely with either immunogen. Immunized hamsters were protected from high-dose viral challenge. Such vaccine formulations can be rapidly and cheaply produced, lack extraneous tags or additional components, and can be stored at room temperature. They are a useful modality to combat COVID-19, especially in remote and low-resource settings.


Subject(s)
Antibodies, Neutralizing/immunology , COVID-19 Vaccines/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Viral/immunology , Cricetinae , Immunogenicity, Vaccine/immunology , Mice , Spike Glycoprotein, Coronavirus/genetics
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