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1.
iScience ; 25(1): 103553, 2022 Jan 21.
Article in English | MEDLINE | ID: covidwho-1757439

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019. Few studies have compared replication dynamics and host responses to SARS-CoV-2 in cell lines from different tissues and species. Therefore, we investigated the role of tissue type and antiviral genes during SARS-CoV-2 infection in nonhuman primate (kidney) and human (liver, respiratory epithelial, gastric) cell lines. We report different viral growth kinetics and release among the cell lines despite comparable ACE2 expression. Transcriptomics revealed that absence of STAT1 in nonhuman primate cells appeared to enhance inflammatory responses without effecting infectious viral titer. Deletion of RL-6 in respiratory epithelial cells increased viral replication. Impaired infectious virus release was detected in Huh7 but not Huh7.5 cells, suggesting a role for RIG1. Gastric cells MKN45 exhibited robust antiviral gene expression and supported viral replication. Data here provide insight into molecular pathogenesis of and alternative cell lines for studying SARS-CoV-2 infection.

2.
iScience ; 25(1): 103553, 2022 Jan 21.
Article in English | MEDLINE | ID: covidwho-1556368

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019. Few studies have compared replication dynamics and host responses to SARS-CoV-2 in cell lines from different tissues and species. Therefore, we investigated the role of tissue type and antiviral genes during SARS-CoV-2 infection in nonhuman primate (kidney) and human (liver, respiratory epithelial, gastric) cell lines. We report different viral growth kinetics and release among the cell lines despite comparable ACE2 expression. Transcriptomics revealed that absence of STAT1 in nonhuman primate cells appeared to enhance inflammatory responses without effecting infectious viral titer. Deletion of RL-6 in respiratory epithelial cells increased viral replication. Impaired infectious virus release was detected in Huh7 but not Huh7.5 cells, suggesting a role for RIG1. Gastric cells MKN45 exhibited robust antiviral gene expression and supported viral replication. Data here provide insight into molecular pathogenesis of and alternative cell lines for studying SARS-CoV-2 infection.

3.
[Unspecified Source]; 2020.
Preprint in English | [Unspecified Source] | ID: ppcovidwho-292766

ABSTRACT

The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic resulting in nearly 20 million infections across the globe, as of August 2020. Critical to the rapid evaluation of vaccines and antivirals is the development of tractable animal models of infection. The use of common laboratory strains of mice to this end is hindered by significant divergence of the angiotensin-converting enzyme 2 (ACE2), which is the receptor required for entry of SARS-CoV-2. In the current study, we designed and utilized an mRNA-based transfection system to induce expression of the hACE2 receptor in order to confer entry of SARS-CoV-2 in otherwise non-permissive cells. By employing this expression system in an in vivo setting, we were able to interrogate the adaptive immune response to SARS-CoV-2 in type 1 interferon receptor deficient mice. In doing so, we showed that the T cell response to SARS-CoV-2 is enhanced when hACE2 is expressed during infection. Moreover, we demonstrated that these responses are preserved in memory and are boosted upon secondary infection. Interestingly, we did not observe an enhancement of SARS-CoV-2 specific antibody responses with hACE2 induction. Importantly, using this system, we functionally identified the CD4+ and CD8+ peptide epitopes targeted during SARS-CoV-2 infection in H2b restricted mice. Antigen-specific CD8+ T cells in mice of this MHC haplotype primarily target peptides of the spike and membrane proteins, while the antigen-specific CD4+ T cells target peptides of the nucleocapsid, membrane, and spike proteins. The functional identification of these T cell epitopes will be critical for evaluation of vaccine efficacy in murine models of SARS-CoV-2. The use of this tractable expression system has the potential to be used in other instances of emerging infections in which the rapid development of an animal model is hindered by a lack of host susceptibility factors.

4.
J Leukoc Biol ; 110(6): 1225-1239, 2021 12.
Article in English | MEDLINE | ID: covidwho-1499280

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-2019 (COVID-19), a respiratory disease that varies in severity from mild to severe/fatal. Several risk factors for severe disease have been identified, notably age, male sex, and pre-existing conditions such as diabetes, obesity, and hypertension. Several advancements in clinical care have been achieved over the past year, including the use of corticosteroids (e.g., corticosteroids) and other immune-modulatory treatments that have now become standard of care for patients with acute severe COVID-19. While the understanding of the mechanisms that underlie increased disease severity with age has improved over the past few months, it remains incomplete. Furthermore, the molecular impact of corticosteroid treatment on host response to acute SARS-CoV-2 infection has not been investigated. In this study, a cross-sectional and longitudinal analysis of Ab, soluble immune mediators, and transcriptional responses in young (65 ≤ years) and aged (≥ 65 years) diabetic males with obesity hospitalized with acute severe COVID-19 was conducted. Additionally, the transcriptional profiles in samples obtained before and after corticosteroids became standard of care were compared. The analysis indicates that severe COVID-19 is characterized by robust Ab responses, heightened systemic inflammation, increased expression of genes related to inflammatory and pro-apoptotic processes, and reduced expression of those important for adaptive immunity regardless of age. In contrast, COVID-19 patients receiving steroids did not show high levels of systemic immune mediators and lacked transcriptional indicators of heightened inflammatory and apoptotic responses. Overall, these data suggest that inflammation and cell death are key drivers of severe COVID-19 pathogenesis in the absence of corticosteroid therapy.


Subject(s)
Adrenal Cortex Hormones/therapeutic use , COVID-19/drug therapy , COVID-19/immunology , Inflammation/immunology , Transcriptome/drug effects , Adult , Aged , Cross-Sectional Studies , Humans , Longitudinal Studies , Male , Middle Aged , SARS-CoV-2 , Transcriptome/immunology
5.
Viruses ; 13(10)2021 09 30.
Article in English | MEDLINE | ID: covidwho-1444331

ABSTRACT

Both the SARS-CoV-2 pandemic and emergence of variants of concern have highlighted the need for functional antibody assays to monitor the humoral response over time. Antibodies directed against the spike (S) protein of SARS-CoV-2 are an important component of the neutralizing antibody response. In this work, we report that in a subset of patients-despite a decline in total S-specific antibodies-neutralizing antibody titers remain at a similar level for an average of 98 days in longitudinal sampling of a cohort of 59 Hispanic/Latino patients exposed to SARS-CoV-2. Our data suggest that 100% of seroconverting patients make detectable neutralizing antibody responses which can be quantified by a surrogate viral neutralization test. Examination of sera from ten out of the 59 subjects which received mRNA-based vaccination revealed that both IgG titers and neutralizing activity of sera were higher after vaccination compared to a cohort of 21 SARS-CoV-2 naïve subjects. One dose was sufficient for the induction of a neutralizing antibody, but two doses were necessary to reach 100% surrogate virus neutralization in subjects irrespective of previous SARS-CoV-2 natural infection status. Like the pattern observed after natural infection, the total anti-S antibodies titers declined after the second vaccine dose; however, neutralizing activity remained relatively constant for more than 80 days after the first vaccine dose. Furthermore, our data indicates that-compared with mRNA vaccination-natural infection induces a more robust humoral immune response in unexposed subjects. This work is an important contribution to understanding the natural immune response to the novel coronavirus in a population severely impacted by SARS-CoV-2. Furthermore, by comparing the dynamics of the immune response after the natural infection vs. the vaccination, these findings suggest that functional neutralizing antibody tests are more relevant indicators than the presence or absence of binding antibodies.


Subject(s)
Immunity, Humoral/physiology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/physiology , Adult , Aged , Antibodies, Neutralizing/analysis , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , COVID-19/immunology , COVID-19/physiopathology , COVID-19 Vaccines/immunology , Female , Follow-Up Studies , Humans , Immunity, Humoral/genetics , Immunity, Humoral/immunology , Male , Middle Aged , Protein Binding/genetics , Protein Domains/genetics , Puerto Rico/epidemiology , SARS-CoV-2/pathogenicity , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/immunology , Vaccination
6.
Front Immunol ; 12: 739025, 2021.
Article in English | MEDLINE | ID: covidwho-1417086

ABSTRACT

A rise in adiposity in the United States has resulted in more than 70% of adults being overweight or obese, and global obesity rates have tripled since 1975. Following the 2009 H1N1 pandemic, obesity was characterized as a risk factor that could predict severe infection outcomes to viral infection. Amidst the SARS-CoV-2 pandemic, obesity has remained a significant risk factor for severe viral disease as obese patients have a higher likelihood for developing severe symptoms and requiring hospitalization. However, the mechanism by which obesity enhances viral disease is unknown. In this study, we utilized a diet-induced obesity mouse model of West Nile virus (WNV) infection, a flavivirus that cycles between birds and mosquitoes and incidentally infects both humans and mice. Likelihood for severe WNV disease is associated with risk factors such as diabetes that are comorbidities also linked to obesity. Utilizing this model, we showed that obesity-associated chronic inflammation increased viral disease severity as obese female mice displayed higher mortality rates and elevated viral titers in the central nervous system. In addition, our studies highlighted that obesity also dysregulates host acute adaptive immune responses, as obese female mice displayed significant dysfunction in neutralizing antibody function. These studies highlight that obesity-induced immunological dysfunction begins at early time points post infection and is sustained through memory phase, thus illuminating a potential for obesity to alter the differentiation landscape of adaptive immune cells.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Cytokines/blood , Obesity/immunology , West Nile Fever/mortality , West Nile virus/immunology , Animals , COVID-19/pathology , Disease Models, Animal , Female , Humans , Inflammation/pathology , Liver/injuries , Liver/pathology , Male , Mice , Mice, Inbred C57BL , Obesity/pathology , Severity of Illness Index , West Nile Fever/immunology , West Nile Fever/pathology
7.
Vaccines (Basel) ; 9(8)2021 Aug 09.
Article in English | MEDLINE | ID: covidwho-1348705

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4+ and CD8+ T cells with a Th1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.

8.
PLoS Pathog ; 16(12): e1009128, 2020 12.
Article in English | MEDLINE | ID: covidwho-992722

ABSTRACT

Cytokine storm is suggested as one of the major pathological characteristics of SARS-CoV-2 infection, although the mechanism for initiation of a hyper-inflammatory response, and multi-organ damage from viral infection is poorly understood. In this virus-cell interaction study, we observed that SARS-CoV-2 infection or viral spike protein expression alone inhibited angiotensin converting enzyme-2 (ACE2) receptor protein expression. The spike protein promoted an angiotensin II type 1 receptor (AT1) mediated signaling cascade, induced the transcriptional regulatory molecules NF-κB and AP-1/c-Fos via MAPK activation, and increased IL-6 release. SARS-CoV-2 infected patient sera contained elevated levels of IL-6 and soluble IL-6R. Up-regulated AT1 receptor signaling also influenced the release of extracellular soluble IL-6R by the induction of the ADAM-17 protease. Use of the AT1 receptor antagonist, Candesartan cilexetil, resulted in down-regulation of IL-6/soluble IL-6R release in spike expressing cells. Phosphorylation of STAT3 at the Tyr705 residue plays an important role as a transcriptional inducer for SOCS3 and MCP-1 expression. Further study indicated that inhibition of STAT3 Tyr705 phosphorylation in SARS-CoV-2 infected and viral spike protein expressing epithelial cells did not induce SOCS3 and MCP-1 expression. Introduction of culture supernatant from SARS-CoV-2 spike expressing cells on a model human liver endothelial Cell line (TMNK-1), where transmembrane IL-6R is poorly expressed, resulted in the induction of STAT3 Tyr705 phosphorylation as well as MCP-1 expression. In conclusion, our results indicated that the presence of SARS-CoV-2 spike protein in epithelial cells promotes IL-6 trans-signaling by activation of the AT1 axis to initiate coordination of a hyper-inflammatory response.


Subject(s)
COVID-19/immunology , Interleukin-6/immunology , Receptors, Angiotensin/metabolism , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , COVID-19/metabolism , Cytokine Release Syndrome/immunology , Cytokine Release Syndrome/metabolism , Cytokine Release Syndrome/virology , Epithelial Cells/immunology , Epithelial Cells/metabolism , Epithelial Cells/virology , Humans , Interleukin-6/metabolism , Respiratory Mucosa/immunology , Respiratory Mucosa/metabolism , Respiratory Mucosa/virology , SARS-CoV-2/metabolism , Signal Transduction/physiology , Transcriptional Activation
9.
PLoS Pathog ; 16(12): e1009163, 2020 12.
Article in English | MEDLINE | ID: covidwho-978951

ABSTRACT

The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic. Critical to the rapid evaluation of vaccines and antivirals against SARS-CoV-2 is the development of tractable animal models to understand the adaptive immune response to the virus. To this end, the use of common laboratory strains of mice is hindered by significant divergence of the angiotensin-converting enzyme 2 (ACE2), which is the receptor required for entry of SARS-CoV-2. In the current study, we designed and utilized an mRNA-based transfection system to induce expression of the hACE2 receptor in order to confer entry of SARS-CoV-2 in otherwise non-permissive cells. By employing this expression system in an in vivo setting, we were able to interrogate the adaptive immune response to SARS-CoV-2 in type 1 interferon receptor deficient mice. In doing so, we showed that the T cell response to SARS-CoV-2 is enhanced when hACE2 is expressed during infection. Moreover, we demonstrated that these responses are preserved in memory and are boosted upon secondary infection. Importantly, using this system, we functionally identified the CD4+ and CD8+ structural peptide epitopes targeted during SARS-CoV-2 infection in H2b restricted mice and confirmed their existence in an established model of SARS-CoV-2 pathogenesis. We demonstrated that, identical to what has been seen in humans, the antigen-specific CD8+ T cells in mice primarily target peptides of the spike and membrane proteins, while the antigen-specific CD4+ T cells target peptides of the nucleocapsid, membrane, and spike proteins. As the focus of the immune response in mice is highly similar to that of the humans, the identification of functional murine SARS-CoV-2-specific T cell epitopes provided in this study will be critical for evaluation of vaccine efficacy in murine models of SARS-CoV-2 infection.


Subject(s)
Adaptive Immunity/immunology , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/immunology , RNA, Messenger/metabolism , SARS-CoV-2/immunology , T-Lymphocytes/immunology , Virus Replication , Angiotensin-Converting Enzyme 2/genetics , Animals , COVID-19/metabolism , COVID-19/virology , Chlorocebus aethiops , Epitopes, T-Lymphocyte/immunology , Humans , Mice , Mice, Knockout , Mice, Transgenic , RNA, Messenger/genetics , Receptor, Interferon alpha-beta/physiology , T-Lymphocytes/virology , Vero Cells
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