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1.
J Am Soc Nephrol ; 32(9): 2242-2254, 2021 09.
Article in English | MEDLINE | ID: covidwho-1702796

ABSTRACT

BACKGROUND: Although coronavirus disease 2019 (COVID-19) causes significan t morbidity, mainly from pulmonary involvement, extrapulmonary symptoms are also major componen ts of the disease. Kidney disease, usually presenting as AKI, is particularly severe among patients with COVID-19. It is unknown, however, whether such injury results from direct kidney infection with COVID-19's causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or from indirect mechanisms. METHODS: Using ex vivo cell models, we sought to analyze SARS-CoV-2 interactions with kidney tubular cells and assess direct tubular injury. These models comprised primary human kidney epithelial cells (derived from nephrectomies) and grown as either proliferating monolayers or quiescent three-dimensional kidney spheroids. RESULTS: We demonstrated that viral entry molecules and high baseline levels of type 1 IFN-related molecules were present in monolayers and kidney spheroids. Although both models support viral infection and replication, they did not exhibit a cytopathic effect and cell death, outcomes that were strongly present in SARS-CoV-2-infected controls (African green monkey kidney clone E6 [Vero E6] cultures). A comparison of monolayer and spheroid cultures demonstrated higher infectivity and replication of SARS-CoV-2 in actively proliferating monolayers, although the spheroid cultures exhibited high er levels of ACE2. Monolayers exhibited elevation of some tubular injury molecules-including molecules related to fibrosis (COL1A1 and STAT6) and dedifferentiation (SNAI2)-and a loss of cell identity, evident by reduction in megalin (LRP2). The three-dimensional spheroids were less prone to such injury. CONCLUSIONS: SARS-CoV-2 can infect kidney cells without a cytopathic effect. AKI-induced cellular proliferation may potentially intensify infectivity and tubular damage by SARS-CoV-2, suggesting that early intervention in AKI is warranted to help minimize kidney infection.


Subject(s)
Acute Kidney Injury/etiology , Acute Kidney Injury/virology , COVID-19/complications , SARS-CoV-2/pathogenicity , Spheroids, Cellular/virology , Animals , Cells, Cultured , Chlorocebus aethiops , Cohort Studies , Cytopathogenic Effect, Viral , Epithelial Cells/pathology , Epithelial Cells/virology , Host Microbial Interactions , Humans , Interferon Type I/metabolism , Kidney/immunology , Kidney/pathology , Kidney/virology , Mice , Mice, Inbred NOD , Mice, SCID , Models, Biological , Pandemics , Receptors, Virus/metabolism , Retrospective Studies , SARS-CoV-2/physiology , Spheroids, Cellular/pathology , Vero Cells , Virus Replication
2.
PLoS Pathog ; 17(12): e1010175, 2021 12.
Article in English | MEDLINE | ID: covidwho-1592244

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.


Subject(s)
Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/metabolism , Protein Domains , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , Binding Sites , Binding Sites, Antibody , COVID-19/prevention & control , Chlorocebus aethiops , Female , HEK293 Cells , Humans , Immunoglobulin Fc Fragments/therapeutic use , Immunoglobulin G/therapeutic use , Mice, Transgenic , Neutralization Tests , Protein Binding , Recombinant Fusion Proteins/therapeutic use , SARS-CoV-2/drug effects , Vero Cells
3.
Microorganisms ; 9(7)2021 Jul 19.
Article in English | MEDLINE | ID: covidwho-1323311

ABSTRACT

A bioterror event using an infectious bacterium may lead to catastrophic outcomes involving morbidity and mortality as well as social and psychological stress. Moreover, a bioterror event using an antibiotic resistance engineered bacterial agent may raise additional concerns. Thus, preparedness is essential to preclude and control the dissemination of the bacterial agent as well as to appropriately and promptly treat potentially exposed individuals or patients. Rates of morbidity, death, and social anxiety can be drastically reduced if the rapid delivery of antimicrobial agents for post-exposure prophylaxis and treatment is initiated as soon as possible. Availability of rapid antibiotic susceptibility tests that may provide key recommendations to targeted antibiotic treatment is mandatory, yet, such tests are only at the development stage. In this review, we describe the recently published rapid antibiotic susceptibility tests implemented on bioterror bacterial agents and discuss their assimilation in clinical and environmental samples.

4.
J Am Soc Nephrol ; 32(9): 2242-2254, 2021 09.
Article in English | MEDLINE | ID: covidwho-1266593

ABSTRACT

BACKGROUND: Although coronavirus disease 2019 (COVID-19) causes significan t morbidity, mainly from pulmonary involvement, extrapulmonary symptoms are also major componen ts of the disease. Kidney disease, usually presenting as AKI, is particularly severe among patients with COVID-19. It is unknown, however, whether such injury results from direct kidney infection with COVID-19's causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or from indirect mechanisms. METHODS: Using ex vivo cell models, we sought to analyze SARS-CoV-2 interactions with kidney tubular cells and assess direct tubular injury. These models comprised primary human kidney epithelial cells (derived from nephrectomies) and grown as either proliferating monolayers or quiescent three-dimensional kidney spheroids. RESULTS: We demonstrated that viral entry molecules and high baseline levels of type 1 IFN-related molecules were present in monolayers and kidney spheroids. Although both models support viral infection and replication, they did not exhibit a cytopathic effect and cell death, outcomes that were strongly present in SARS-CoV-2-infected controls (African green monkey kidney clone E6 [Vero E6] cultures). A comparison of monolayer and spheroid cultures demonstrated higher infectivity and replication of SARS-CoV-2 in actively proliferating monolayers, although the spheroid cultures exhibited high er levels of ACE2. Monolayers exhibited elevation of some tubular injury molecules-including molecules related to fibrosis (COL1A1 and STAT6) and dedifferentiation (SNAI2)-and a loss of cell identity, evident by reduction in megalin (LRP2). The three-dimensional spheroids were less prone to such injury. CONCLUSIONS: SARS-CoV-2 can infect kidney cells without a cytopathic effect. AKI-induced cellular proliferation may potentially intensify infectivity and tubular damage by SARS-CoV-2, suggesting that early intervention in AKI is warranted to help minimize kidney infection.


Subject(s)
Acute Kidney Injury/etiology , Acute Kidney Injury/virology , COVID-19/complications , SARS-CoV-2/pathogenicity , Spheroids, Cellular/virology , Animals , Cells, Cultured , Chlorocebus aethiops , Cohort Studies , Cytopathogenic Effect, Viral , Epithelial Cells/pathology , Epithelial Cells/virology , Host Microbial Interactions , Humans , Interferon Type I/metabolism , Kidney/immunology , Kidney/pathology , Kidney/virology , Mice , Mice, Inbred NOD , Mice, SCID , Models, Biological , Pandemics , Receptors, Virus/metabolism , Retrospective Studies , SARS-CoV-2/physiology , Spheroids, Cellular/pathology , Vero Cells , Virus Replication
5.
JCI Insight ; 6(12)2021 06 22.
Article in English | MEDLINE | ID: covidwho-1223641

ABSTRACT

Mice are normally unaffected by SARS coronavirus 2 (SARS-CoV-2) infection since the virus does not bind effectively to the murine version of the angiotensin-converting enzyme 2 (ACE2) receptor molecule. Here, we report that induced mild pulmonary morbidities rendered SARS-CoV-2-refractive CD-1 mice susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low doses of the acute lung injury stimulants bleomycin or ricin caused severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates greater than 50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart, and serum of low-dose ricin-pretreated mice compared with non-pretreated mice. Furthermore, lung extracts prepared 2-3 days after viral infection contained subgenomic mRNA and virus particles capable of replication only when derived from the pretreated mice. The deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against the SARS-CoV-2 receptor binding domain (RBD). Thus, viral cell entry in the sensitized mice seems to depend on viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. This unique mode of viral entry, observed over a mildly injured tissue background, may contribute to the exacerbation of coronavirus disease 2019 (COVID-19) pathologies in patients with preexisting morbidities.


Subject(s)
Bleomycin/toxicity , COVID-19/pathology , Lung Injury , Ricin/toxicity , Animals , Chlorocebus aethiops , Comorbidity , Disease Models, Animal , Female , Lung Injury/chemically induced , Lung Injury/virology , Mice , Vero Cells , Virus Attachment , Virus Internalization/drug effects
6.
Viruses ; 13(4)2021 03 26.
Article in English | MEDLINE | ID: covidwho-1154538

ABSTRACT

Monoclonal antibodies represent an important avenue for COVID-19 therapy and are routinely used for rapid and accessible diagnosis of SARS-CoV-2 infection. The recent emergence of SARS-CoV-2 genetic variants emphasized the need to enlarge the repertoire of antibodies that target diverse epitopes, the combination of which may improve immune-diagnostics, augment the efficiency of the immunotherapy and prevent selection of escape-mutants. Antigen-specific controlled immunization of experimental animals may elicit antibody repertoires that significantly differ from those generated in the context of the immune response mounted in the course of disease. Accordingly, rabbits were immunized by several recombinant antigens representing distinct domains of the viral spike protein and monoclonal antibodies were isolated from single cells obtained by cell sorting. Characterization of a panel of successfully isolated anti-receptor binding domain (RBD) and anti-N-terminal domain (NTD) antibodies demonstrated that they exhibit high specificity and affinity profiles. Anti-RBD antibodies revealing significant neutralizing potency against SARS-CoV-2 in vitro were found to target at least three distinct epitopes. Epitope mapping established that two of these antibodies recognized a novel epitope located on the surface of the RBD. We suggest that the antibodies isolated in this study are useful for designing SARS-CoV-2 diagnosis and therapy approaches.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/immunology , COVID-19/virology , Epitope Mapping , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Female , Humans , Neutralization Tests , Rabbits , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics
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