Hybridoma-derived neutralizing monoclonal antibodies against Beta and Delta variants of SARS-CoV-2 in vivo.

Neutralizing monoclonal antibodies (mAb) are a major therapeutic strategy for the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The continuous emergence of new SARS-CoV-2 variants worldwide has increased the urgency for the development of new mAbs. In this study, we immunized mice with the receptor-binding domain (RBD) of the SARS-CoV-2 prototypic strain (WIV04) and screened 35 RBD-specific mAbs using hybridoma technology. Results of the plaque reduction neutralization test showed that 25 of the mAbs neutralized authentic WIV04 strain infection. The 25 mAbs were divided into three categories based on the competitive enzyme-linked immunosorbent assay results. A representative mAb was selected from each category (RD4, RD10, and RD14) to determine the binding kinetics and median inhibitory concentration (IC50) of WIV04 and two variants of concern (VOC): B.1.351 (Beta) and B.1.617.2 (Delta). RD4 neutralized the B.1.617.2 variant with an IC50 of 2.67 â€‹ng/mL; however, it completely lost neutralizing activity against the B.1.351 variant. RD10 neutralized both variants with an IC50 exceeding 100 â€‹ng/mL; whereas RD14 neutralized two variants with a higher IC50 (>1 â€‹mg/mL). Animal experiments were performed to evaluate the protective effects of RD4 and RD10 against various VOC infections. RD4 could protect Adv-hACE2 transduced mice from B.1.617.2 infection at an antibody concentration of 25 â€‹mg/kg, while RD10 could protect mice from B.1.351 infection at an antibody concentration of 75 â€‹mg/kg. These results highlight the potential for future modifications of the mAbs for practical use.

Exploiting an early warning Nomogram for predicting the risk of ICU admission in patients with COVID-19: a multi-center study in China.

BACKGROUND: Novel coronavirus disease 2019 (COVID-19) is a global public health emergency. Here, we developed and validated a practical model based on the data from a multi-center cohort in China for early identification and prediction of which patients will be admitted to the intensive care unit (ICU). METHODS: Data of 1087 patients with laboratory-confirmed COVID-19 were collected from 49 sites between January 2 and February 28, 2020, in Sichuan and Wuhan. Patients were randomly categorized into the training and validation cohorts (7:3). The least absolute shrinkage and selection operator and logistic regression analyzes were used to develop the nomogram. The performance of the nomogram was evaluated for the C-index, calibration, discrimination, and clinical usefulness. Further, the nomogram was externally validated in a different cohort. RESULTS: The individualized prediction nomogram included 6 predictors: age, respiratory rate, systolic blood pressure, smoking status, fever, and chronic kidney disease. The model demonstrated a high discriminative ability in the training cohort (C-index = 0.829), which was confirmed in the external validation cohort (C-index = 0.776). In addition, the calibration plots confirmed good concordance for predicting the risk of ICU admission. Decision curve analysis revealed that the prediction nomogram was clinically useful. CONCLUSION: We established an early prediction model incorporating clinical characteristics that could be quickly obtained on hospital admission, even in community health centers. This model can be conveniently used to predict the individual risk for ICU admission of patients with COVID-19 and optimize the use of limited resources.

Contribution of Trp63CreERT2-labeled cells to alveolar regeneration is independent of tuft cells.

Viral infection often causes severe damage to the lungs, leading to the appearance of ectopic basal cells (EBCs) and tuft cells in the lung parenchyma. Thus far, the roles of these ectopic epithelial cells in alveolar regeneration remain controversial. Here, we confirm that the ectopic tuft cells are originated from EBCs in mouse models and COVID-19 lungs. The differentiation of tuft cells from EBCs is promoted by Wnt inhibition while suppressed by Notch inhibition. Although progenitor functions have been suggested in other organs, pulmonary tuft cells don't proliferate or give rise to other cell lineages. Consistent with previous reports, Trp63CreERT2 and KRT5-CreERT2-labeled ectopic EBCs do not exhibit alveolar regeneration potential. Intriguingly, when tamoxifen was administrated post-viral infection, Trp63CreERT2 but not KRT5-CreERT2 labels islands of alveolar epithelial cells that are negative for EBC biomarkers. Furthermore, germline deletion of Trpm5 significantly increases the contribution of Trp63CreERT2-labeled cells to the alveolar epithelium. Although Trpm5 is known to regulate tuft cell development, complete ablation of tuft cell production fails to improve alveolar regeneration in Pou2f3-/- mice, implying that Trpm5 promotes alveolar epithelial regeneration through a mechanism independent of tuft cells.

Establishment of Human Pluripotent Stem Cell-Derived Skin Organoids Enabled Pathophysiological Model of SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19) patients with impact on skin and hair loss are reported. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is detected in the skin of some patients; however, the detailed pathological features of skin tissues from patients infected with SARS-CoV-2 at a molecular level are limited. Especially, the ability of SARS-CoV-2 to infect skin cells and impact their function is not well understood. A proteome map of COVID-19 skin is established here and the susceptibility of human-induced pluripotent stem cell (hiPSC)-derived skin organoids with hair follicles and nervous system is investigated, to SARS-CoV-2 infection. It is shown that KRT17+ hair follicles can be infected by SARS-CoV-2 and are associated with the impaired development of hair follicles and epidermis. Different types of nervous system cells are also found to be infected, which can lead to neuron death. Findings from the present work provide evidence for the association between COVID-19 and hair loss. hiPSC-derived skin organoids are also presented as an experimental model which can be used to investigate the susceptibility of skin cells to SARS-CoV-2 infection and can help identify various pathological mechanisms and drug screening strategies.

A human antibody of potent efficacy against SARS-CoV-2 in rhesus macaques showed strong blocking activity to B.1.351.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease-2019 (COVID-19), interacts with the host cell receptor angiotensin-converting enzyme 2 (hACE2) via its spike 1 protein during infection. After the virus sequence was published, we identified two potent antibodies against the SARS-CoV-2 receptor binding domain (RBD) from antibody libraries using a phage-to-yeast (PtY) display platform in only 10 days. Our lead antibody JMB2002, now in a Phase 1 clinical trial (ChiCTR2100042150), showed broad-spectrum in vitro blocking activity against hACE2 binding to the RBD of multiple SARS-CoV-2 variants, including B.1.351 that was reportedly much more resistant to neutralization by convalescent plasma, vaccine sera and some clinical-stage neutralizing antibodies. Furthermore, JMB2002 has demonstrated complete prophylactic and potent therapeutic efficacy in a rhesus macaque disease model. Prophylactic and therapeutic countermeasure intervention of SARS-CoV-2 using JMB2002 would likely slow down the transmission of currently emerged SARS-CoV-2 variants and result in more efficient control of the COVID-19 pandemic.

Protective Efficacy of Inactivated Vaccine against SARS-CoV-2 Infection in Mice and Non-Human Primates.

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused more than 96 million infections and over 2 million deaths worldwide so far. However, there is no approved vaccine available for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the disease causative agent. Vaccine is the most effective approach to eradicate a pathogen. The tests of safety and efficacy in animals are pivotal for developing a vaccine and before the vaccine is applied to human populations. Here we evaluated the safety, immunogenicity, and efficacy of an inactivated vaccine based on the whole viral particles in human ACE2 transgenic mouse and in non-human primates. Our data showed that the inactivated vaccine successfully induced SARS-CoV-2-specific neutralizing antibodies in mice and non-human primates, and subsequently provided partial (in low dose) or full (in high dose) protection of challenge in the tested animals. In addition, passive serum transferred from vaccine-immunized mice could also provide full protection from SARS-CoV-2 infection in mice. These results warranted positive outcomes in future clinical trials in humans.

Correlation of autopsy pathological findings and imaging features from 9 fatal cases of COVID-19 pneumonia.

ABSTRACT: We aimed to investigate the relationship of radiological features and the corresponding pulmonary pathology of patients with Coronavirus Disease (COVID-19) pneumonia.In this multicenter study, serial chest CT and radiographic images from 9 patients (51-85 years old, 56% male) were reviewed and analyzed. Postmortem lungs were sampled and studied from these autopsies, with a special focus on several corresponding sites based on imaging features.The predominant pattern of pulmonary injury in these 9 cases was diffuse alveolar damage (DAD) and interstitial inflammation. Moreover, acute fibrinous exudates, organization, inflammatory cell infiltration, hyaline membranes, pulmonary edema, pneumocyte hyperplasia, and fibrosis were all observed. The histopathology features varied according to the site and severity of each lesion. In most of the 9 cases, opacities started from a subpleural area and peripheral structures were more severely damaged based on gross views and pathological examinations. Fibrosis could occur in early stages of infection and this was supported by radiological and pathological findings. The radiological features of COVID-19 pneumonia, at the critically ill stage, were diffuse ground-glass opacities with consolidation, interstitial thickening, and fibrous stripes, which was based in the fibrous tissue proliferation in the alveolar and interlobular septa, and filled alveoli with organizing exudation. Fungal and bacterial co-infections were also observed in 6 cases.Typical imaging features can be correlated with underlying pathological findings. Combining assessments of imaging features with pathological findings therefore can enhance our understanding of the histopathological mechanism of COVID-19 pneumonia, and facilitate early radiological diagnosis and prognosis estimation of COVID-19 pneumonia, which has important implications for the development of clinical targeted treatments and research related to COVID-19 pneumonia.

Pathological changes in the lungs and lymphatic organs of 12 COVID-19 autopsy cases.

Systematic autopsy and comprehensive pathological analyses of COVID-19 decedents should provide insights into the disease characteristics and facilitate the development of novel therapeutics. In this study, we report the autopsy findings from the lungs and lymphatic organs of 12 COVID-19 decedents-findings that evaluated histopathological changes, immune cell signature and inflammatory factor expression in the lungs, spleen and lymph nodes. Here we show that the major pulmonary alterations included diffuse alveolar damage, interstitial fibrosis and exudative inflammation featured with extensive serous and fibrin exudates, macrophage infiltration and abundant production of inflammatory factors (IL-6, IP-10, TNFα and IL-1ß). The spleen and hilar lymph nodes contained lesions with tissue structure disruption and immune cell dysregulation, including lymphopenia and macrophage accumulation. These findings provide pathological evidence that links injuries of the lungs and lymphatic organs with the fatal systematic respiratory and immune malfunction in critically ill COVID-19 patients.

Clinicopathologic Features of COVID-19: A Case Report and Value of Forensic Autopsy in Studying SARS-CoV-2 Infection.

ABSTRACT: As of August 23, 2020, the 2019 novel coronavirus disease (COVID-19) has infected more than 23,518,340 people and caused more than 810,492 deaths worldwide including 4,717 deaths in China. We present a case of a 53-year-old woman who was admitted to the hospital because of dry coughs and high fever on January 26, 2020, in Wuhan, China. She was not tested for SARS-CoV-2 RNA until on hospital day 11 (illness day 21) because of a significant shortage of test kits at the local hospital. Then, her test was positive for COVID-19 on hospital day 20. Despite intensive medical treatments, she developed respiratory failure with secondary bacterial infection and expired on hospital day 23 (3 days after she was tested positive for SARS-CoV-2 RNA). A systemic autopsy examination, including immunohistochemistry and ultrastructural studies, demonstrates that SARS-CoV-2 can infect multiple organs with profound adverse effect on the immune system, and the lung pathology is characterized by diffuse alveolar damage. Extrapulmonary SARS-CoV-2 RNA was detected in several organs postmortem. The detailed pathological features are described. In addition, this report highlights the value of forensic autopsy in studying SARS-CoV-2 infection and the importance of clinicopathological correlation in better understanding the pathogenesis of COVID-19.

SARS-CoV-2 triggers inflammatory responses and cell death through caspase-8 activation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can lead to respiratory illness and multi-organ failure in critically ill patients. Although the virus-induced lung damage and inflammatory cytokine storm are believed to be directly associated with coronavirus disease 2019 (COVID-19) clinical manifestations, the underlying mechanisms of virus-triggered inflammatory responses are currently unknown. Here we report that SARS-CoV-2 infection activates caspase-8 to trigger cell apoptosis and inflammatory cytokine processing in the lung epithelial cells. The processed inflammatory cytokines are released through the virus-induced necroptosis pathway. Virus-induced apoptosis, necroptosis, and inflammation activation were also observed in the lung sections of SARS-CoV-2-infected HFH4-hACE2 transgenic mouse model, a valid model for studying SARS-CoV-2 pathogenesis. Furthermore, analysis of the postmortem lung sections of fatal COVID-19 patients revealed not only apoptosis and necroptosis but also massive inflammatory cell infiltration, necrotic cell debris, and pulmonary interstitial fibrosis, typical of immune pathogenesis in the lung. The SARS-CoV-2 infection triggered a dual mode of cell death pathways and caspase-8-dependent inflammatory responses may lead to the lung damage in the COVID-19 patients. These discoveries might assist the development of therapeutic strategies to treat COVID-19.

An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques.

The rapid spread of coronavirus SARS-CoV-2 greatly threatens global public health but no prophylactic vaccine is available. Here, we report the generation of a replication-incompetent recombinant serotype 5 adenovirus, Ad5-S-nb2, carrying a codon-optimized gene encoding Spike protein (S). In mice and rhesus macaques, intramuscular injection with Ad5-S-nb2 elicits systemic S-specific antibody and cell-mediated immune (CMI) responses. Intranasal inoculation elicits both systemic and pulmonary antibody responses but weaker CMI response. At 30 days after a single vaccination with Ad5-S-nb2 either intramuscularly or intranasally, macaques are protected against SARS-CoV-2 challenge. A subsequent challenge reveals that macaques vaccinated with a 10-fold lower vaccine dosage (1 × 1010 viral particles) are also protected, demonstrating the effectiveness of Ad5-S-nb2 and the possibility of offering more vaccine dosages within a shorter timeframe. Thus, Ad5-S-nb2 is a promising candidate vaccine and warrants further clinical evaluation.

Deficiency of Tfh Cells and Germinal Center in Deceased COVID-19 Patients.

The COVID-19 pandemic caused by SARS-CoV2 is characterized by a remarkable variation in clinical severity ranging from a mild illness to a fatal multi-organ disease. Understanding the dysregulated human immune responses in the fatal subjects is critical for management of COVID-19 patients and the pandemic. In this study, we examined the immune cell compositions in the lung tissues and hilar lymph nodes using immunohistochemistry on 6 deceased COVID-19 patients and 4 focal organizing pneumonia (FOP) patients who underwent lung surgery and served as controls. We found a dominant presence of macrophages and a general deficiency of T cells and B cells in the lung tissues from deceased COVID-19 patients. In contrast to the FOP patients, Tfh cells and germinal center formation were largely absent in the draining hilar lymph nodes in the deceased COVID-19 patients. This was correlated with reduced IgM and IgG levels compared to convalescent COVID-19 patients. In summary, our data highlight a defect of germinal center structure in deceased COVID-19 patients leading to an impaired humoral immunity. Understanding the mechanisms of this deficiency will be one of the key points for the management of this epidemic.

Infection with novel coronavirus (SARS-CoV-2) causes pneumonia in Rhesus macaques.

The 2019 novel coronavirus (SARS-CoV-2) outbreak is a major challenge for public health. SARS-CoV-2 infection in human has a broad clinical spectrum ranging from mild to severe cases, with a mortality rate of ~6.4% worldwide (based on World Health Organization daily situation report). However, the dynamics of viral infection, replication and shedding are poorly understood. Here, we show that Rhesus macaques are susceptible to the infection by SARS-CoV-2. After intratracheal inoculation, the first peak of viral RNA was observed in oropharyngeal swabs one day post infection (1 d.p.i.), mainly from the input of the inoculation, while the second peak occurred at 5 d.p.i., which reflected on-site replication in the respiratory tract. Histopathological observation shows that SARS-CoV-2 infection can cause interstitial pneumonia in animals, characterized by hyperemia and edema, and infiltration of monocytes and lymphocytes in alveoli. We also identified SARS-CoV-2 RNA in respiratory tract tissues, including trachea, bronchus and lung; and viruses were also re-isolated from oropharyngeal swabs, bronchus and lung, respectively. Furthermore, we demonstrated that neutralizing antibodies generated from the primary infection could protect the Rhesus macaques from a second-round challenge by SARS-CoV-2. The non-human primate model that we established here provides a valuable platform to study SARS-CoV-2 pathogenesis and to evaluate candidate vaccines and therapeutics.