Nucleolin mediates SARS-CoV-2 replication and viral-induced apoptosis of host cells.

Host-oriented antiviral therapeutics are promising treatment options to combat COVID-19 and its emerging variants. However, relatively little is known about the cellular proteins hijacked by SARS-CoV-2 for its replication. Here we show that SARS-CoV-2 induces expression and cytoplasmic translocation of the nucleolar protein, nucleolin (NCL). NCL interacts with SARS-CoV-2 viral proteins and co-localizes with N-protein in the nucleolus and in stress granules. Knockdown of NCL decreases the stress granule component G3BP1, viral replication and improved survival of infected host cells. NCL mediates viral-induced apoptosis and stress response via p53. SARS-CoV-2 increases NCL expression and nucleolar size and number in lungs of infected hamsters. Inhibition of NCL with the aptamer AS-1411 decreases viral replication and apoptosis of infected cells. These results suggest nucleolin as a suitable target for anti-COVID therapies.

The SARS-CoV-2 spike protein binds and modulates estrogen receptors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 as its primary infection mechanism. Interactions between S and endogenous proteins occur after infection but are not well understood. We profiled binding of S against >9000 human proteins and found an interaction between S and human estrogen receptor α (ERα). Using bioinformatics, supercomputing, and experimental assays, we identified a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects. Non-invasive imaging in SARS-CoV-2-infected hamsters localized lung pathology with increased ERα lung levels. Postmortem lung experiments from infected hamsters and humans confirmed an increase in cytoplasmic ERα and its colocalization with S in alveolar macrophages. These findings describe the discovery of a S-ERα interaction, imply a role for S as an NRC, and advance knowledge of SARS-CoV-2 biology and coronavirus disease 2019 pathology.

Sulforaphane exhibits antiviral activity against pandemic SARS-CoV-2 and seasonal HCoV-OC43 coronaviruses in vitro and in mice.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has incited a global health crisis. Currently, there are limited therapeutic options for the prevention and treatment of SARS-CoV-2 infections. We evaluated the antiviral activity of sulforaphane (SFN), the principal biologically active phytochemical derived from glucoraphanin, the naturally occurring precursor present in high concentrations in cruciferous vegetables. SFN inhibited in vitro replication of six strains of SARS-CoV-2, including Delta and Omicron, as well as that of the seasonal coronavirus HCoV-OC43. Further, SFN and remdesivir interacted synergistically to inhibit coronavirus infection in vitro. Prophylactic administration of SFN to K18-hACE2 mice prior to intranasal SARS-CoV-2 infection significantly decreased the viral load in the lungs and upper respiratory tract and reduced lung injury and pulmonary pathology compared to untreated infected mice. SFN treatment diminished immune cell activation in the lungs, including significantly lower recruitment of myeloid cells and a reduction in T cell activation and cytokine production. Our results suggest that SFN should be explored as a potential agent for the prevention or treatment of coronavirus infections.

Progression and Resolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection in Golden Syrian Hamsters.

To catalyze severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research, including development of novel interventive and preventive strategies, the progression of disease was characterized in a robust coronavirus disease 2019 (COVID-19) animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at 2, 4, 7, 14, and 28 days after inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal time points, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 after inoculation, corresponding with widespread necrosis and inflammation. At day 7, when the presence of infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 after inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

Pharmacokinetics of high-titer anti-SARS-CoV-2 human convalescent plasma in high-risk children.

BACKGROUNDWhile most children who contract COVID-19 experience mild disease, high-risk children with underlying conditions may develop severe disease, requiring interventions. Kinetics of antibodies transferred via COVID-19 convalescent plasma early in disease have not been characterized.METHODSIn this study, high-risk children were prospectively enrolled to receive high-titer COVID-19 convalescent plasma (>1:320 anti-spike IgG; Euroimmun). Passive transfer of antibodies and endogenous antibody production were serially evaluated for up to 2 months after transfusion. Commercial and research ELISA assays, virus neutralization assays, high-throughput phage-display assay utilizing a coronavirus epitope library, and pharmacokinetic analyses were performed.RESULTSFourteen high-risk children (median age, 7.5 years) received high-titer COVID-19 convalescent plasma, 9 children within 5 days (range, 2-7 days) of symptom onset and 5 children within 4 days (range, 3-5 days) after exposure to SARS-CoV-2. There were no serious adverse events related to transfusion. Antibodies against SARS-CoV-2 were transferred from the donor to the recipient, but antibody titers declined by 14-21 days, with a 15.1-day half-life for spike protein IgG. Donor plasma had significant neutralization capacity, which was transferred to the recipient. However, as early as 30 minutes after transfusion, recipient plasma neutralization titers were 6.2% (range, 5.9%-6.7%) of donor titers.CONCLUSIONConvalescent plasma transfused to high-risk children appears to be safe, with expected antibody kinetics, regardless of weight or age. However, current use of convalescent plasma in high-risk children achieves neutralizing capacity, which may protect against severe disease but is unlikely to provide lasting protection.Trial registrationClinicalTrials.gov NCT04377672.FundingThe state of Maryland, Bloomberg Philanthropies, and the NIH (grants R01-AI153349, R01-AI145435-A1, K08-AI139371-A1, and T32-AI052071).

Hamsters as a Model of Severe Acute Respiratory Syndrome Coronavirus-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), rapidly spread across the world in late 2019, leading to a pandemic. While SARS-CoV-2 infections predominately affect the respiratory system, severe infections can lead to renal and cardiac injury and even death. Due to its highly transmissible nature and severe health implications, animal models of SARS-CoV-2 are critical to developing novel therapeutics and preventatives. Syrian hamsters (Mesocricetus auratus) are an ideal animal model of SARS-CoV-2 infections because they recapitulate many aspects of human infections. After inoculation with SARS-CoV-2, hamsters become moribund, lose weight, and show varying degrees of respiratory disease, lethargy, and ruffled fur. Histopathologically, their pulmonary lesions are consistent with human infections including interstitial to broncho-interstitial pneumonia, alveolar hemorrhage and edema, and granulocyte infiltration. Similar to humans, the duration of clinical signs and pulmonary pathology are short lived with rapid recovery by 14 d after infection. Immunocompromised hamsters develop more severe infections and mortality. Preclinical studies in hamsters have shown efficacy of therapeutics, including convalescent serum treatment, and preventatives, including vaccination, in limiting or preventing clinical disease. Although hamster studies have contributed greatly to our understanding of the pathogenesis and progression of disease after SARS-CoV-2 infection, additional studies are required to better characterize the effects of age, sex, and virus variants on clinical outcomes in hamsters. This review aims to describe key findings from studies of hamsters infected with SARS-CoV-2 and to highlight areas that need further investigation.

124I-Iodo-DPA-713 Positron Emission Tomography in a Hamster Model of SARS-CoV-2 Infection.

PURPOSE: Molecular imaging has provided unparalleled opportunities to monitor disease processes, although tools for evaluating infection remain limited. Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by lung injury that we sought to model. Activated macrophages/phagocytes have an important role in lung injury, which is responsible for subsequent respiratory failure and death. We performed pulmonary PET/CT with 124I-iodo-DPA-713, a low-molecular-weight pyrazolopyrimidine ligand selectively trapped by activated macrophages cells, to evaluate the local immune response in a hamster model of SARS-CoV-2 infection. PROCEDURES: Pulmonary 124I-iodo-DPA-713 PET/CT was performed in SARS-CoV-2-infected golden Syrian hamsters. CT images were quantified using a custom-built lung segmentation tool. Studies with DPA-713-IRDye680LT and a fluorescent analog of DPA-713 as well as histopathology and flow cytometry were performed on post-mortem tissues. RESULTS: Infected hamsters were imaged at the peak of inflammatory lung disease (7 days post-infection). Quantitative CT analysis was successful for all scans and demonstrated worse pulmonary disease in male versus female animals (P < 0.01). Increased 124I-iodo-DPA-713 PET activity co-localized with the pneumonic lesions. Additionally, higher pulmonary 124I-iodo-DPA-713 PET activity was noted in male versus female hamsters (P = 0.02). DPA-713-IRDye680LT also localized to the pneumonic lesions. Flow cytometry demonstrated a higher percentage of myeloid and CD11b + cells (macrophages, phagocytes) in male versus female lung tissues (P = 0.02). CONCLUSION: 124I-Iodo-DPA-713 accumulates within pneumonic lesions in a hamster model of SARS-CoV-2 infection. As a novel molecular imaging tool, 124I-Iodo-DPA-713 PET could serve as a noninvasive, clinically translatable approach to monitor SARS-CoV-2-associated pulmonary inflammation and expedite the development of novel therapeutics for COVID-19.

Sex Differences in Lung Imaging and SARS-CoV-2 Antibody Responses in a COVID-19 Golden Syrian Hamster Model.

In the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males than in females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8 to 10 weeks of age) were inoculated intranasally with 105 50% tissue culture infective dose (TCID50) of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developed more extensive pneumonia as noted on chest computed tomography, and recovered more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including interferon-ß (IFN-ß) and tumor necrosis factor-α (TNF-α), were comparable between the sexes. However, during the recovery phase of infection, females mounted 2-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole-inactivated SARS-CoV-2 and mutant S-RBDs as well as virus-neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2-associated sex differences seen in the human population. IMPORTANCE Men experience more severe outcomes from coronavirus disease 2019 (COVID-19) than women. Golden Syrian hamsters were used to explore sex differences in the pathogenesis of a human isolate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). After inoculation, male hamsters experienced greater sickness, developed more severe lung pathology, and recovered more slowly than females. Sex differences in disease could not be reversed by estradiol treatment in males and were not explained by either virus replication kinetics or the concentrations of inflammatory cytokines in the lungs. During the recovery period, antiviral antibody responses in the respiratory tract and plasma, including to newly emerging SARS-CoV-2 variants, were greater in female than in male hamsters. Greater lung pathology during the acute phase combined with lower antiviral antibody responses during the recovery phase of infection in males than in females illustrate the utility of golden Syrian hamsters as a model to explore sex differences in the pathogenesis of SARS-CoV-2 and vaccine-induced immunity and protection.

Rapid detection of SARS-CoV-2 using a radiolabeled antibody.

PURPOSE: Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus 2019 disease (COVID-19), poses a serious risk to humanity and represents a huge challenge for healthcare systems worldwide. Since the early days of the COVID-19 pandemic, it has been evident that adequate testing is an essential step in limiting and controlling the spread of SARS-CoV-2. Here, we present an accurate, inexpensive, scalable, portable, and rapid detection kit to directly detect SARS-CoV-2 in biological samples that could even be translated for population testing. We have demonstrated that our method can reliably identify viral load and could be used to reach those fractions of the population with limited access to more sophisticated and expensive tests. PROCEDURES: The proposed SARS-CoV-2 detection kit is based on the combination of a SARS-CoV-2-targeted antibody (CR3022) that targets spike protein S1 domain on the viral surface. This antibody was radiolabeled with a long-lived isotope (Iodine-125) to allow us to detect bound antibody in samples with SARS-CoV-2. We used a series of in vitro assays to determine sensitivity and specificity and facilitate automation of the testing kit. Bound antibody was extracted from saliva samples via a centrifugation step and a semi-permeable membrane. Our kit was further validated using SARS-CoV-2 virions. RESULTS: We were able to accomplish radiosynthesis of [125I]I-CR3022 reliably without loss of binding. The SARS-CoV-2-sensing antibody was shown to maintain its spike S1 affinity and to bind to as low as 2.5-5 ng of spike protein. We then used beads-bound spike S1 to develop a separation kit which proved to be both easy to use and inexpensive. The kit made it possible to extract bound antibody from the saliva-like sample. We were able to validate the separation kit using intact SARS-CoV-2 virions and showed that our kit can detect a viral concentration as low as 19,700 PFU/mL (~ 9.22%TBF) and as high as 1,970,000 PFU/mL (45.04%TBF). CONCLUSION: Here we report the development and validation of a SARS-CoV-2 detection system based on the combination of a specific radiolabeled antibody and a separation membrane. We demonstrate our system to be comparable to other SARS-CoV-2 detection kits already approved by the FDA and believe this technology could be easily deployed to countries with limited resources for the diagnosis of COVID-19. Furthermore, workflows could be easily adapted to target other antigens and therefore other types of diseases.

Imaging Enterobacterales infections in patients using pathogen-specific positron emission tomography.

Enterobacterales represent the largest group of bacterial pathogens in humans and are responsible for severe, deep-seated infections, often resulting in sepsis or death. They are also a prominent cause of multidrug-resistant (MDR) infections, and some species are recognized as biothreat pathogens. Tools for noninvasive, whole-body analysis that can localize a pathogen with specificity are needed, but no such technology currently exists. We previously demonstrated that positron emission tomography (PET) with 2-deoxy-2-[18F]fluoro-d-sorbitol (18F-FDS) can selectively detect Enterobacterales infections in murine models. Here, we demonstrate that uptake of 18F-FDS by bacteria occurs via a metabolically conserved sorbitol-specific pathway with rapid in vitro 18F-FDS uptake noted in clinical strains, including MDR isolates. Whole-body 18F-FDS PET/computerized tomography (CT) in 26 prospectively enrolled patients with either microbiologically confirmed Enterobacterales infection or other pathologies demonstrated that 18F-FDS PET/CT was safe, could rapidly detect and localize Enterobacterales infections due to drug-susceptible or MDR strains, and differentiated them from sterile inflammation or cancerous lesions. Repeat imaging in the same patients monitored antibiotic efficacy with decreases in PET signal correlating with clinical improvement. To facilitate the use of 18F-FDS, we developed a self-contained, solid-phase cartridge to rapidly (<10 min) formulate ready-to-use 18F-FDS from commercially available 2-deoxy-2-[18F]fluoro-d-glucose (18F-FDG) at room temperature. In a hamster model, 18F-FDS PET/CT also differentiated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia from secondary Klebsiella pneumoniae pneumonia-a leading cause of complications in hospitalized patients with COVID-19. These data support 18F-FDS as an innovative and readily available, pathogen-specific PET technology with clinical applications.