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Principal Investigator: Sonja Best
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2023
Award: $482,755
Funding agency: National Institute of Allergy and Infectious Diseases
The SARS-CoV-2 global pandemic has a continuing demand for greater understanding of the mechanisms of disease and the development of therapeutics to complement success in vaccination. Several mouse models have emerged that can be used, albeit with limitations, as models of severe SARS-Cov2 infection. Therefore we have initiated two major efforts to employ mouse models that can be utilized in the efforts to understand dysregulation of innate and adaptive immunity associated with severe viral pneumonia. The ultimate goal is to define these processes as they relate to SARS-CoV-2 to identify points of intervention that can be targeted therapeutically.
The first major initiative is to develop mouse models of SARS-CoV-2 infection that model human disease. To achieve this, we have partnered with Jackson Laboratories to genetically engineer mice that express a humanized ACE2 gene to enable virus replication in tissues. We have tested 4 separate strategies to humanize ACE2 at the endogenous locus, or as a transgene. We have also tested mouse backgrounds for susceptibility. The ultimate goal will be to fully characterize the host responses as they relate to pathology in these models, and then utilize the models for testing biologics that block various events in inflammatory cascades.
During this year, we finalized establishing 12 models of SARS-CoV-2 infection in 10 strains of mice. In humans, the range of disease phenotypes observed is extreme, from asymptomatic to critical, and is dependent on age, sex, genetics and metabolic status. The 10 strains of mice used included the 8 founder strains of the Collaborative Cross (B6, A/J, 129SJ, NZO, NOD, PWK, CAST and WSB) with additional strains Balb/c and DSB. Together, these strains represent over 90% of the genetic diversity within Mus musculus and have enabled us to model distinct disease phenotypes including a) sensitive mice with high sustained virus replication in lung and CNS (B6,A/J), b) resistant mouse strains associated with lower peak virus titer and earlier control of replication in the lung with no or low dissemination to other organs (PWK, NZO), and c) sex bias where resistance is independent of virus titer in the lung suggesting a sex-differences in host response (CAST, NOD, WSB). Cytokine analysis in the BAL revealed that resistance to disease in males was associated with high IFNb expression at 3dpi. Cytokine profiles generally modeled human responses with lethality associated with sustained high IP-10, as well as increasing MCP3, TNFa, IL-10, RANTES, IFNg and IL1b. Taken together, these mouse models represent a powerful tool to understand mechanisms of immune-mediated control and pathology following SARS-CoV-2 infection. The work was published (PMID: 37491352) and additional follow up data is being analysed including RNAseq data from lungs and brain of all models, as well as additional spatial transcriptomics of lungs.
We have developed a protocol for safe fixation and subsequent multiplex imaging of the lungs of SAR-COv2 infected animals. Remarkably, in the K18-hACE2 transgenic mouse model of SARS-CoV-2 lethality, lungs on day two after infection showed almost no inflammatory infiltrate and no evidence of type 1 interferon signaling, whereas the influenza-infected animals showed robust innate immune cell infiltrates and interferon signaling at this time point. This reinforces existing evidence that coronaviruses potently suppress type 1 interferon responses and markedly change the inflammatory process. We are pursuing these observations across a more complete time course, using more markers to identify cell types and cell states, to better understand how these changes in innate immunity affect later adaptive responses and if the discoordination of viral spread and innate immunity plays a special role in pathogenesis. However, because cerebral infection plays a major role in lethality in this model, we are seeking alternatives as a substrate for testing therapeutics, and have recently obtained a mouse adapted version of SARS-CoV-2 that causes pulmonary dysfunction. This strain is presently being titrated to establish the proper model for transferring interventions from the influenza model described below.
The second major initiative is the employment of a lethal influenza infection as a model for severe viral pneumonia. Ongoing studies involve (i) tests of interventions in the lethal influenza model that might have clinical utility and (ii) molecular, cell, and tissue level studies aimed at better understanding the underlying mechanism(s) of tissue damage and why interventions that constrain viral replication or innate immunity often fail after an early point in infection but well before death of the host. Using a severe influenza infection model that bypasses early nasopharyngeal replication and leads to rapid deep lung infection, we found that only very early treatment with the anti-viral oseltamivir phosphate (Tamiflu) could prevent death. Among 50 single or combined treatments covering many of the agents tested or used clinically for COVID-19 treatment (anti-IL-6, PANAM-G3, PMX205, inosine Pranobex, anti-PSGL1, ruxolitinib, inbrutinib, acalabrutinib, dypridamole, baricitinib, colchicine, silvelestat, AZD5059, anti-IL-6R, anti-CCL2, and Zileuton among others), none reduced weight loss or led to survival of any of the infected animals, and several worsened disease. These findings argue that either (i) multiple damaging activities are involved and blunting only one is insufficient for a clinical effect, and/or (ii) that irreversible tissue damage occurs early and once this occurs, interfering independently with viral replication or host immunity does not play a major role in preventing eventual death.
Imaging of whole lung lobes using our IBEX method for multiplex staining showed that in this influenza infection model, there was early infiltration by neutrophils, extensive spread of the virus, loss of pro-surfactant and associated type 2 pneumocytes, alveolar disruption, and myeloid cell bronchiolar plugging, followed by later arrival of T cells in concert with marked loss of viable lung tissue. While some treatments modified the balance and extent of cell infiltrates, none prevented the damage and parenchymal loss. From these data, we developed the hypothesis that the infected animals rapidly pass a tipping point with respect to residual functional pulmonary capacity and that after this point, interference with inflammatory processes alone is insufficient to rescue the animals. This led to a change in strategy based on combining arrest of further damage and promoting recovery of functional lung structures by enabling more effective repair of damaged pulmonary epithelium. Results from other laboratories indicated that type 1 interferons can inhibit pneumocyte proliferation, while other studies suggest that late arrival of cytotoxic T cells in the lung can cause loss of residual functional epithelial cells. We therefore combined low dose Tamiflu administered late in the course of infection in combination with one of two additional treatments that either promote pneumocyte replication (blocking of interferon signaling) or limit further immune destruction (depletion of CD8+ T cells) can rescue mice from death. Remarkably, these combinations prevented death in more than 50% of the infected animals and we are combining Paxlovid in place of Tamiflu with each of these two immune interventions to examine if this combination is effective in the mouse adapted SARS-CoV-2 model.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><ACE2><Active Follow-up><Acute><Affect><Age><Alveolar><Animals><Attention><B cell differentiation factor><B cell stimulating factor 2><B-Cell Differentiation Factor><B-Cell Differentiation Factor-2><B-Cell Stimulatory Factor-2><BALB C Mouse><BALB/c><BCDF><BSF-2><BSF2><Biological Testing><Body Tissues><Body Weight decreased><Brain><Brain Nervous System><Bypass><CCL2><CCL2 gene><CCL5><CCL7><CCL7 gene><CD183><CD8 Cell><CD8 T cells><CD8 lymphocyte><CD8+ T cell><CD8+ T-Lymphocyte><CD8-Positive Lymphocytes><CD8-Positive T-Lymphocytes><CKR-L2><CMKAR3><COVID associated ARDS><COVID associated acute respiratory distress syndrome><COVID crisis><COVID epidemic><COVID induced ARDS><COVID induced acute respiratory distress syndrome><COVID pandemic><COVID related ARDS><COVID related acute respiratory distress syndrome><COVID-19><COVID-19 associated ARDS><COVID-19 associated acute respiratory distress syndrome><COVID-19 crisis><COVID-19 epidemic><COVID-19 global health crisis><COVID-19 global pandemic><COVID-19 health crisis><COVID-19 induced ARDS><COVID-19 induced acute respiratory distress syndrome><COVID-19 infection><COVID-19 pandemic><COVID-19 public health crisis><COVID-19 related ARDS><COVID-19 related acute respiratory distress syndrome><COVID-19 therapy><COVID-19 treatment><COVID-19 virus><COVID-19 virus infection><COVID-19/ARDS><COVID-19/acute respiratory distress syndrome><COVID/ARDS><COVID/acute respiratory distress syndrome><COVID19><COVID19 associated ARDS><COVID19 associated acute respiratory distress syndrome><COVID19 crisis><COVID19 epidemic><COVID19 global health crisis><COVID19 global pandemic><COVID19 health crisis><COVID19 induced ARDS><COVID19 induced acute respiratory distress syndrome><COVID19 infection><COVID19 pandemic><COVID19 public health crisis><COVID19 related ARDS><COVID19 related acute respiratory distress syndrome><COVID19 therapy><COVID19 treatment><COVID19 virus><COVID19/ARDS><COVID19/acute respiratory distress syndrome><CRG-2><CSIF><CSIF-10><CV-19><CV19><CXCL10><CXCL10 gene><CXCR3><CXCR3 gene><Cell Body><Cell Communication and Signaling><Cell Signaling><Cell-Mediated Lympholytic Cells><Cells><Cerebrum><Cessation of life><Chemokine (C-C Motif) Ligand 5><Chemokine (C-X-C Motif) Receptor 3><Chemokine, CC Motif, Ligand 2><Clinical><CoV-2><CoV2><Colchicine><Combined Modality Therapy><Complement><Complement Proteins><Coronaviridae><Coronavirus><Cytokine Synthesis Inhibitory Factor><Cytolytic T-Cell><Cytotoxic T Cell><Cytotoxic T-Lymphocytes><D17S136E><Data><Death><Differences between sexes><Differs between sexes><Disease><Disease Resistance><Disorder><Dose><Dysfunction><Early treatment><Employment><Encephalon><Endogenous Interferon Beta><Epithelial Cells><Epithelium><Equilibrium><Event><Fibroblast Interferon><Fixation><Functional disorder><G Protein-Coupled Receptor 9><GEM model><GEMM model><GPR9><Gender Bias><Genes><Genetic><Genetic Diversity><Genetic Variation><Genetically Engineered Mouse><Goals><Grippe><HPGF><Hepatocyte-Stimulating Factor><Host Defense Mechanism><House mice><Human><Hybridoma Growth Factor><IFI10><IFN><IFN-Beta><IFN-beta 2><IFN-β><IFNB2><IFNb><IL-10><IL-6><IL10><IL10A><IL6 Protein><INP10><IP-10><IP10><IP10 Receptor><IP10-Mig receptor><IP10-R><Image><Immune><Immune destruction><Immune infiltrates><Immune mediated destruction><Immune response><Immunes><Immunity><Immunological response><Inbred BALB C Mice><Infection><Infiltration><Inflammatory><Inflammatory Infiltrate><Inflammatory Response><Influenza><Innate Immune Response><Innate Immunity><Inosine><Interferon-beta><Interferon-β><Interferons><Interleukin 10 Precursor><Interleukin-10><Interleukin-6><Intervention><Intervention Strategies><Intracellular Communication and Signaling><K-18><K-18 conjugate><K18><K18 combination><Laboratories><Lung><Lung Parenchyma><Lung Respiratory System><Lung Tissue><Lung damage><Lung infections><MARC><MCAF><MCP-1><MCP-3><MCP1><MCP3><MGC17164><MGI-2><MOB-1><Mediating><Metabolic><Methods><Mice><Mice Mammals><Mig Receptor><Mig-R><MigR><Modeling><Modern Man><Molecular><Monocyte Chemoattractant Protein-1><Monocyte Chemotactic Protein-1><Monocyte Chemotactic and Activating Factor><Monocyte Chemotactic and Activating Protein><Monocyte Chemotactive and Activating Factor><Monocyte Secretory Protein JE><Morbidity><Morbidity - disease rate><Mouse Strains><Multimodal Therapy><Multimodal Treatment><Murine><Mus><Mus musculus><Myeloid Cells><Myeloid Differentiation-Inducing Protein><NC28><Nasopharynx><Native Immunity><Natural Immunity><Natural Interferon Beta><Natural human interferon beta><Neutrophil Infiltration><Neutrophil Recruitment><Neutrophilic Infiltrate><Non-Specific Immunity><Nonspecific Immunity><Organ><Oseltamivir><Pathogenesis><Pathologic><Pathology><Paxlovid><Phosphates><Physiopathology><Plasmacytoma Growth Factor><Play><Predisposition><Process><Proliferating><Protocol><Protocols documentation><Publishing><Pulmonary imaging><RANTES><RNA Seq><RNA sequencing><RNAseq><Recovery of Function><Residual><Residual state><Resistance><Rhinopharynx><Role><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 associated ARDS><SARS-CoV-2 associated acute respiratory distress syndrome><SARS-CoV-2 epidemic><SARS-CoV-2 global health crisis><SARS-CoV-2 global pandemic><SARS-CoV-2 induced ARDS><SARS-CoV-2 induced acute respiratory distress syndrome><SARS-CoV-2 infection><SARS-CoV-2 pandemic><SARS-CoV-2 related ARDS><SARS-CoV-2 related acute respiratory distress syndrome><SARS-CoV-2 therapy><SARS-CoV-2 treatment><SARS-CoV-2/ARDS><SARS-CoV-2/acute respiratory distress syndrome><SARS-CoV2><SARS-CoV2 epidemic><SARS-CoV2 infection><SARS-CoV2 pandemic><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-coronavirus-2 epidemic><SARS-coronavirus-2 pandemic><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><SCYA2><SCYA5><SCYA7><SCYB10><SIS delta><SIS-delta><SISd><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome CoV 2 epidemic><Severe Acute Respiratory Syndrome CoV 2 pandemic><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 epidemic><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome coronavirus 2 pandemic><Severe acute respiratory syndrome related corona virus 2><Sex Bias><Sex Differences><Sexual differences><Signal Transduction><Signal Transduction Systems><Signaling><Small Inducible Cytokine A2><Small Inducible Cytokine A5><Staining method><Stains><Structure><Structure of parenchyma of lung><Susceptibility><T-Cell RANTES Protein><T-Cell Specific Protein p288><T-Cells><T-Lymphocyte><T8 Cells><T8 Lymphocytes><TCP228><Tamiflu><Testing><Thrombus><Time><Tissues><Titrations><Transgenes><Transgenic Mice><Vaccination><Viral><Viral Pneumonia><Virus><Virus Replication><Weight Loss><Weight Reduction><Work><Wuhan coronavirus><Zileuton><active followup><adaptive immune response><adaptive immunity><after COVID-19 infection><after SARS-CoV-2 infection><after SARS-CoV2 infection><after infection by SARS-CoV-2><after severe acute respiratory distress syndrome CoV-2 infection><ages><angiotensin converting enzyme 2><angiotensin converting enzyme II><balance><balance function><biological signal transduction><body weight loss><cell type><cerebral><clinical effect><combination therapy><combined modality 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coronavirus 2><lung imaging><lung injury><lung lobe><lung scanning><male><model of human><mortality><mouse model><multi-modal therapy><multi-modal treatment><multiplexed imaging><murine model><nCoV2><nasopharnygeal><pathophysiology><pneumocyte><post SARS-CoV-2 infection><preservation><prevent><preventing><previous COVID-19 infection><previous SARS-CoV-2 infection><previous SARS-CoV2 infection><previous severe acute respiratory distress syndrome CoV-2 infection><prior COVID-19 infection><prior SARS-CoV-2 infection><prior SARS-CoV2 infection><prior severe acute respiratory distress syndrome CoV-2 infection><pulmonary><pulmonary damage><pulmonary infections><pulmonary injury><pulmonary tissue damage><pulmonary tissue injury><repair><repaired><resistance to disease><resistant><resistant disease><resistant to disease><response><sample fixation><serious COVID><serious COVID-19><serious SARS-CoV-2><serious coronavirus disease><serious coronavirus disease 2019><serious severe acute respiratory 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disease 19><severe coronavirus disease 2019><severe severe acute respiratory syndrome coronavirus 2><sex><sex-dependent differences><sex-related differences><sex-specific differences><social role><success><surfactant><targeted drug therapy><targeted drug treatments><targeted therapeutic><targeted therapeutic agents><targeted therapy><targeted treatment><therapeutic agent development><therapeutic development><therapeutic evaluation><therapeutic testing><thymus derived lymphocyte><tool><transcriptome sequencing><transcriptomic sequencing><transcriptomics><transgene><treat COVID-19><treat COVID19><treat SARS-CoV-2><treat coronavirus disease 2019><treat severe acute respiratory syndrome coronavirus 2><viral multiplication><viral replication><virus multiplication><wt-loss>