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1.
Curr Opin Neurobiol ; 76: 102608, 2022 Jun 29.
Article in English | MEDLINE | ID: covidwho-1906922

ABSTRACT

Coronavirus disease 2019 (COVID-19) has caused a historic pandemic of respiratory disease. COVID-19 also causes acute and post-acute neurological symptoms, which range from mild, such as headaches, to severe, including hemorrhages. Current evidence suggests that there is no widespread infection of the central nervous system (CNS) by SARS-CoV-2, thus what is causing COVID-19 neurological disease? Here, we review potential immunological mechanisms driving neurological disease in COVID-19 patients. We begin by discussing the implications of imbalanced peripheral immunity on CNS function. Next, we examine the evidence for dysregulation of the blood-brain barrier during SARS-CoV-2 infection. Last, we discuss the role myeloid cells may play in promoting COVID-19 neurological disease. Combined, we highlight the role of innate immunity in COVID-19 neuroinflammation and suggest areas for future research.

2.
NPJ Vaccines ; 7(1): 55, 2022 May 23.
Article in English | MEDLINE | ID: covidwho-1860374

ABSTRACT

Adjuvants enhance the magnitude and the durability of the immune response to vaccines. However, there is a paucity of comparative studies on the nature of the immune responses stimulated by leading adjuvant candidates. In this study, we compared five clinically relevant adjuvants in mice-alum, AS03 (a squalene-based adjuvant supplemented with α-tocopherol), AS37 (a TLR7 ligand emulsified in alum), CpG1018 (a TLR9 ligand emulsified in alum), O/W 1849101 (a squalene-based adjuvant)-for their capacity to stimulate immune responses when combined with a subunit vaccine under clinical development. We found that all four of the adjuvant candidates surpassed alum with respect to their capacity to induce enhanced and durable antigen-specific antibody responses. The TLR-agonist-based adjuvants CpG1018 (TLR9) and AS37 (TLR7) induced Th1-skewed CD4+ T cell responses, while alum, O/W, and AS03 induced a balanced Th1/Th2 response. Consistent with this, adjuvants induced distinct patterns of early innate responses. Finally, vaccines adjuvanted with AS03, AS37, and CpG1018/alum-induced durable neutralizing-antibody responses and significant protection against the B.1.351 variant 7 months following immunization. These results, together with our recent results from an identical study in non-human primates (NHPs), provide a comparative benchmarking of five clinically relevant vaccine adjuvants for their capacity to stimulate immunity to a subunit vaccine, demonstrating the capacity of adjuvanted SARS-CoV-2 subunit vaccines to provide durable protection against the B.1.351 variant. Furthermore, these results reveal differences between the widely-used C57BL/6 mouse strain and NHP animal models, highlighting the importance of species selection for future vaccine and adjuvant studies.

4.
mBio ; 12(6): e0274921, 2021 12 21.
Article in English | MEDLINE | ID: covidwho-1506962

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a historic pandemic of respiratory disease (coronavirus disease 2019 [COVID-19]), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2 signaling restricts the viral burden in the lung. We find that a recently developed mouse-adapted SARS-CoV-2 (MA-SARS-CoV-2) strain as well as the emerging B.1.351 variant trigger an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using intravital antibody labeling, we demonstrate that MA-SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Single-cell RNA sequencing (scRNA-Seq) analysis of lung homogenates identified a hyperinflammatory monocyte profile. We utilize this model to demonstrate that mechanistically, CCR2 signaling promotes the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified a potential CCR2-monocyte axis that is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 has caused a historic pandemic of respiratory disease (COVID-19), and current evidence suggests that severe disease is associated with dysregulated immunity within the respiratory tract. However, the innate immune mechanisms that mediate protection during COVID-19 are not well defined. Here, we characterize a mouse model of SARS-CoV-2 infection and find that early CCR2-dependent infiltration of monocytes restricts the viral burden in the lung. We find that SARS-CoV-2 triggers an inflammatory response in the lung characterized by the expression of proinflammatory cytokines and interferon-stimulated genes. Using RNA sequencing and flow cytometry approaches, we demonstrate that SARS-CoV-2 infection leads to increases in circulating monocytes and an influx of CD45+ cells into the lung parenchyma that is dominated by monocyte-derived cells. Mechanistically, CCR2 signaling promoted the infiltration of classical monocytes into the lung and the expansion of monocyte-derived cells. Parenchymal monocyte-derived cells appear to play a protective role against MA-SARS-CoV-2, as mice lacking CCR2 showed higher viral loads in the lungs, increased lung viral dissemination, and elevated inflammatory cytokine responses. These studies have identified that the CCR2 pathway is critical for promoting viral control and restricting inflammation within the respiratory tract during SARS-CoV-2 infection.


Subject(s)
Lung/immunology , Pneumonia, Viral/prevention & control , Receptors, CCR2/immunology , SARS-CoV-2/immunology , Signal Transduction/immunology , Animals , COVID-19 , Cytokines/immunology , Disease Models, Animal , Female , Immunity, Innate , Inflammation , Lung/cytology , Lung/virology , Mice , Mice, Inbred C57BL , Monocytes/immunology , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , Receptors, CCR2/genetics , Receptors, CCR2/metabolism , SARS-CoV-2/genetics , Viral Load , Virus Replication/immunology
5.
PLoS Biol ; 19(11): e3001284, 2021 11.
Article in English | MEDLINE | ID: covidwho-1502046

ABSTRACT

The emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a pandemic causing significant damage to public health and the economy. Efforts to understand the mechanisms of Coronavirus Disease 2019 (COVID-19) have been hampered by the lack of robust mouse models. To overcome this barrier, we used a reverse genetic system to generate a mouse-adapted strain of SARS-CoV-2. Incorporating key mutations found in SARS-CoV-2 variants, this model recapitulates critical elements of human infection including viral replication in the lung, immune cell infiltration, and significant in vivo disease. Importantly, mouse adaptation of SARS-CoV-2 does not impair replication in human airway cells and maintains antigenicity similar to human SARS-CoV-2 strains. Coupled with the incorporation of mutations found in variants of concern, CMA3p20 offers several advantages over other mouse-adapted SARS-CoV-2 strains. Using this model, we demonstrate that SARS-CoV-2-infected mice are protected from lethal challenge with the original Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), suggesting immunity from heterologous Coronavirus (CoV) strains. Together, the results highlight the use of this mouse model for further study of SARS-CoV-2 infection and disease.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Animals , COVID-19/pathology , COVID-19 Vaccines/therapeutic use , Cell Line , Disease Models, Animal , Female , Humans , Lung/pathology , Mice , Mice, Inbred BALB C , Reverse Genetics , Serial Passage , Virus Replication
6.
Cell Rep ; 36(2): 109353, 2021 07 13.
Article in English | MEDLINE | ID: covidwho-1275191

ABSTRACT

SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier and caused widespread disease in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterize 198 antibodies isolated from four COVID-19+ subjects and identify 14 SARS-CoV-2 neutralizing antibodies. One targets the N-terminal domain (NTD), one recognizes an epitope in S2, and 11 bind the receptor-binding domain (RBD). Three anti-RBD neutralizing antibodies cross-neutralize SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency and antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. All four cross-neutralizing antibodies neutralize the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , COVID-19/immunology , COVID-19/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Binding Sites , Cell Line , Cross Reactions , Epitopes/immunology , Female , HEK293 Cells , Humans , Mice , Neutralization Tests , Protein Binding/immunology , Protein Domains , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry
7.
Nature ; 591(7849): 293-299, 2021 03.
Article in English | MEDLINE | ID: covidwho-1046014

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic1-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses2. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.


Subject(s)
COVID-19/virology , Furin/metabolism , Mutation , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Animals , Antibodies, Neutralizing/immunology , COVID-19/pathology , COVID-19/physiopathology , Cell Line , Chlorocebus aethiops , Cricetinae , Female , Humans , Lung Diseases/pathology , Lung Diseases/physiopathology , Lung Diseases/virology , Male , Mice , Mice, Transgenic , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Proteolysis , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Replication/genetics
8.
Nat Commun ; 11(1): 6013, 2020 11 26.
Article in English | MEDLINE | ID: covidwho-947533

ABSTRACT

SARS-CoV-2 variants with spike (S)-protein D614G mutations now predominate globally. We therefore compare the properties of the mutated S protein (SG614) with the original (SD614). We report here pseudoviruses carrying SG614 enter ACE2-expressing cells more efficiently than those with SD614. This increased entry correlates with less S1-domain shedding and higher S-protein incorporation into the virion. Similar results are obtained with virus-like particles produced with SARS-CoV-2 M, N, E, and S proteins. However, D614G does not alter S-protein binding to ACE2 or neutralization sensitivity of pseudoviruses. Thus, D614G may increase infectivity by assembling more functional S protein into the virion.


Subject(s)
COVID-19/virology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Virion/metabolism , Virus Assembly/genetics , Virus Internalization , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/epidemiology , HEK293 Cells , Humans , Mutation , Pandemics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/metabolism
9.
Curr Protoc Immunol ; 131(1): e116, 2020 12.
Article in English | MEDLINE | ID: covidwho-935033

ABSTRACT

SARS-CoV-2 is a recently emerged human coronavirus that has escalated to a pandemic. There are currently no approved vaccines for SARS-CoV-2, which causes severe respiratory illness or death. Defining the antibody response to SARS-CoV-2 will be essential for understanding disease progression, long-term immunity, and vaccine efficacy. Here we describe two methods for evaluating the neutralization capacity of SARS-CoV-2 antibodies. The basic protocol is a focus reduction neutralization test (FRNT), which involves immunostaining infected cells with a chromogen deposit readout. The alternate protocol is a modification of the FRNT that uses an infectious clone-derived SARS-CoV-2 virus expressing a fluorescent reporter. These protocols are adapted for use in a high-throughput setting, and are compatible with large-scale vaccine studies or clinical testing. © 2020 Wiley Periodicals LLC Basic Protocol: Focus reduction neutralization test Alternate Protocol: mNeonGreen-based focus reduction neutralization test (FRNT-mNG).


Subject(s)
Antibodies, Neutralizing/blood , COVID-19/immunology , Neutralization Tests/methods , SARS-CoV-2/immunology , Animals , COVID-19/epidemiology , COVID-19/prevention & control , Chlorocebus aethiops , Humans , Pandemics , Vero Cells
10.
J Virol ; 94(19)2020 09 15.
Article in English | MEDLINE | ID: covidwho-852551

ABSTRACT

The newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a pandemic of respiratory illness. Current evidence suggests that severe cases of SARS-CoV-2 are associated with a dysregulated immune response. However, little is known about how the innate immune system responds to SARS-CoV-2. In this study, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral, surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by proinflammatory cytokines and chemokine induction, including interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and CXCL8, and identified NF-κB and ATF-4 as key drivers of this proinflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III interferon (IFN) response to SARS-CoV-2 infection. However, pretreatment and posttreatment with type I and III IFNs significantly reduced virus replication in pHAE cultures that correlated with upregulation of antiviral effector genes. Combined, our findings demonstrate that SARS-CoV-2 does not trigger an IFN response but is sensitive to the effects of type I and III IFNs. Our studies demonstrate the utility of pHAE cultures to model SARS-CoV-2 infection and that both type I and III IFNs can serve as therapeutic options to treat COVID-19 patients.IMPORTANCE The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. Our research identifies an excellent system to model SARS-CoV-2 infection of the human airways that can be used to test various treatments. Analysis of infection in this model system found that human airway epithelial cell cultures induce a strong proinflammatory cytokine response yet block the production of type I and III IFNs to SARS-CoV-2. However, treatment of airway cultures with the immune molecules type I or type III interferon (IFN) was able to inhibit SARS-CoV-2 infection. Thus, our model system identified type I or type III IFN as potential antiviral treatments for COVID-19 patients.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/immunology , Epithelial Cells/immunology , Interferon Type I/immunology , Interferons/immunology , Pneumonia, Viral/immunology , Animals , Betacoronavirus/physiology , Bronchi/cytology , Bronchi/immunology , Bronchi/virology , COVID-19 , Cell Line , Cells, Cultured , Chemokines/immunology , Chlorocebus aethiops , Coronavirus Infections/virology , Cytokines/immunology , Dogs , Epithelial Cells/virology , Humans , Lung/cytology , Lung/immunology , Lung/virology , Madin Darby Canine Kidney Cells , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Vero Cells , Virus Replication
11.
Cell Rep Med ; 1(3): 100040, 2020 06 23.
Article in English | MEDLINE | ID: covidwho-549041

ABSTRACT

SARS-CoV-2, the virus responsible for COVID-19, is causing a devastating worldwide pandemic, and there is a pressing need to understand the development, specificity, and neutralizing potency of humoral immune responses during acute infection. We report a cross-sectional study of antibody responses to the receptor-binding domain (RBD) of the spike protein and virus neutralization activity in a cohort of 44 hospitalized COVID-19 patients. RBD-specific IgG responses are detectable in all patients 6 days after PCR confirmation. Isotype switching to IgG occurs rapidly, primarily to IgG1 and IgG3. Using a clinical SARS-CoV-2 isolate, neutralizing antibody titers are detectable in all patients by 6 days after PCR confirmation and correlate with RBD-specific binding IgG titers. The RBD-specific binding data were further validated in a clinical setting with 231 PCR-confirmed COVID-19 patient samples. These findings have implications for understanding protective immunity against SARS-CoV-2, therapeutic use of immune plasma, and development of much-needed vaccines.

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