Characterization of RBD-specific cross-neutralizing antibodies responses against SARS-CoV-2 variants from COVID-19 convalescents.

Introduction: The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been posing a severe threat to global public health. Although broadly neutralizing antibodies have been used to prevent or treat corona virus disease 2019 (COVID-19), new emerging variants have been proven resistant to these antibodies. Methods: In this study, we isolated receptor binding domain (RBD)-specific memory B cells using single-cell sorting method from two COVID-19 convalescents and expressed the antibody to test their neutralizing activity against diverse SARS-CoV-2 variants. Then, we resolved antibody-RBD complex structures of potent RBD-specific neutralizing antibodies by X-ray diffraction method. Finally, we analyzed the whole antibody repertoires of the two donors and studied the evolutionary pathway of potent neutralizing antibodies. Results and discussion: We identified three potent RBD-specific neutralizing antibodies (1D7, 3G10 and 3C11) from two COVID-19 convalescents that neutralized authentic SARS-CoV-2 WH-1 and Delta variant, and one of them, 1D7, presented broadly neutralizing activity against WH-1, Beta, Gamma, Delta and Omicron authentic viruses. The resolved antibody-RBD complex structures of two antibodies, 3G10 and 3C11, indicate that both of them interact with the external subdomain of the RBD and that they belong to the RBD-1 and RBD-4 communities, respectively. From the antibody repertoire analysis, we found that the CDR3 frequencies of the light chain, which shared high degrees of amino acid identity with these three antibodies, were higher than those of the heavy chain. This research will contribute to the development of RBD-specific antibody-based drugs and immunogens against multiple variants.

Delayed Clearance of Viral RNA in Sputum for Severity COVID-19 Patients with Initial High Viral Load.

Aim: To analyze the possible risk factors of delayed virus clearance in hospitalized patients with coronavirus disease 2019 (COVID-19). Methods: Retrospective analysis of patients with COVID-19 admitted to the isolation wards from our hospital from 19th Jan 2020 to 18th March 2020. We were collected patient's data including demographic, epidemiologic, and clinical information, as well as laboratory and radiologic findings. The possible confounding risk factors for prolonged viral RNA shedding of COVID-19 during hospitalization were explored by univariate analysis and any variables with a p value less than 0.05 after univariate analysis were included in a subsequent multivariate logistic regression model analysis. Results: The 104 patients included 30 mild patients and 74 severe or critically ill patients. The median duration of viral RNA positivity in sputum was 11 days, and the longest duration of viral RNA positivity was 49 days after admission. Multivariate analysis shown that the used with darunavir/cobicistat treatment (odds ratio [OR]: 4.25, 95% confidence interval [CI]: 1.25-14.42, p = 0.020), duration of fever (OR: 1.15, 95% CI: 1.03-1.30, p = 0.015) and time to radiological improvement (OR: 1.14, 95% CI: 1.01-1.30, p = 0.033) were associated with delayed clearance of SARS-CoV-2 in sputum from COVID-19 patients. Then adjusted in the multivariate binary logistic regression analysis model in severe COVID-19 and found that critical COVID-19 patients (OR: 13.25, 95% CI: 1.45-12.07, p = 0.022), lower virus cycle threshold (CT) values of RT-PCR (OR: 0.96, 95% CI: 0.93-0.99, p = 0.004) and used with darunavir/cobicistat treatment (OR: 8.44, 95% CI: 2.21-32.28, p = 0.022) were associated with delayed clearance of SARS-CoV-2 in sputum from COVID-19 patients. Conclude: Clearance of viral RNA in sputum was delayed in severe COVID-19 patients, especially with lower virus CT value. And antivirals with darunavir/cobicistat has little advantage in eliminating SARS-CoV-2.

Exploring Rapid and Effective Screening Methods for Anti-SARS-CoV-2 Neutralizing Antibodies in COVID-19 Convalescent Patients and Longitudinal Vaccinated Populations.

Assessing the duration of neutralizing antibodies (nAbs) following SARS-CoV-2 infection or vaccination is critical to evaluate the protective immunity and formulate public health strategies. In this study, SARS-CoV-2 Ab ELISA (enzyme-linked immunosorbent assay), chemiluminescent microparticle immunoassay (CMIA), as well as pseudovirus neutralization test (PVNT) were performed in two cohorts, convalescent patients (CP) from coronavirus disease 2019 (COVID-19) and BBIBP-CorV vaccinated population. It was found that nAbs and binding antibodies emerged at 14 days post the 1st dose of vaccination, reached peaks at 28 days after 2nd dose vaccination and then gradually declined over time. CP-6M (convalescent patients up to 6 months) from COVID-19 presented stronger nAbs or binding antibodies responses than vaccinees 90 days or 180 days after 2nd dose vaccination. CMIA or SARS-CoV-2 Ab ELISA correlated well with PVNT with high consistency in the two cohorts. It shown that nAbs and binding antibodies can keep 6 months both in CP and vaccinees. Most importantly, our data show the application of using CMIA and SARS-CoV-2 Ab ELISA as rapid screening tests for nAb titer and could be used as alternative strategies for quickly evaluating SARS-CoV-2 nAbs responses in vaccine research.

Predictors of COVID-19 Infection: A Prevalence Study of Hospitalized Patients.

AIM: To find the predictors of coronavirus disease 2019 (COVID-19) in hospitalized patients. METHODS: A prevalence study compared the characteristics of COVID-19 patients with non-COVID-19 patients from January 19, 2020, to February 18, 2020, during the COVID-19 outbreak. Laboratory test results and pulmonary chest imaging of confirmed COVID-19 and non-COVID-19 patients were collected by retrieving medical records in our center. RESULTS: 96 COVID-19 patients and 122 non-COVID-19 patients were enrolled in this study. COVID-19 patients were older (53 vs. 39; P < 0.001) and had higher body mass index (BMI) than non-COVID-19 group (24.21 ± 3.51 vs. 23.00 ± 3.27, P = 0.011); however, differences in gender were not observed between the two groups. Logistic regression analysis showed that exposure history (OR: 23.34, P < 0.001), rhinorrhea (odds radio (OR): 0.12, P = 0.006), alanine aminotransferase (ALT) (OR: 1.03, P = 0.049), lactate dehydrogenase (LDH) (OR: 1.01, P = 0.020), lymphocyte (OR: 0.27, P = 0.007), and bilateral involvement on chest CT imaging (OR: 23.01, P < 0.001) were independent risk factors for COVID-19. Moreover, bilateral involvement on chest CT imaging (AUC = 0.904, P < 0.001) had significantly higher AUC than others in predicting COVID-19. CONCLUSIONS: Exposure history, elevated ALT and LDH, absence of rhinorrhea, lymphopenia, and bilateral involvement on chest CT imaging provide robust evidence for the diagnosis of COVID-19, especially in resource-limited conditions where nucleic acid detection is not readily available.

Cross-neutralization of RBD mutant strains of SARS-CoV-2 by convalescent patient derived antibodies.

BACKGROUND: The emergence of COVID-19 pandemic resulted in an urgent need for the development of therapeutic interventions. Of which, neutralizing antibodies play a crucial role in the prevention and resolution of viral infection. METHODS: We generated antibody libraries from 18 different COVID-19 recovered patients and screened neutralizing antibodies to SARS-CoV-2 and its mutants. After 3 rounds of panning, 456 positive phage clones were obtained with high affinity to RBD (receptor binding domain). Clones were then reconstituted into whole human IgG for epitope binning assay and all 19 IgG were classified into 6 different epitope groups or Bins. RESULTS: Although all antibodies were found to bind RBD, the antibodies in Bin2 had superior inhibitory ability of the interaction between spike protein and angiotensin converting enzyme 2 receptor (ACE2). Most importantly, the antibodies from Bin2 showed stronger binding affinity or ability to mutant RBDs (N501Y, W463R, R408I, N354D, V367F, and N354D/D364Y) derived from different SARS-CoV-2 strains as well, suggesting the great potential of these antibodies in preventing infection of SARS-CoV-2 and its mutations. Furthermore, such neutralizing antibodies strongly restricted the binding of RBD to hACE2 overexpressed 293T cells. Consistently, these antibodies effectively neutralized wildtype and more transmissible mutant pseudovirus entry into hACE2 overexpressed 293T cells. In Vero-E6 cells, one of these antibodies can even block the entry of live SARS-CoV-2 into cells at 12.5 nM. CONCLUSIONS: These results indicate that the neutralizing human antibodies from the patient-derived antibody libraries have the potential to fight SARS-CoV-2 and its mutants in this global pandemic.

One-year follow-up of chest CT findings in patients after SARS-CoV-2 infection.

BACKGROUND: Knowledge about the 1-year outcome of COVID-19 is limited. The aim of this study was to follow-up and evaluate lung abnormalities on serial computed tomography (CT) scans in patients with COVID-19 after hospital discharge. METHODS: A prospective cohort study of patients with COVID-19 from the First Affiliated Hospital, Zhejiang University School of Medicine was conducted, with assessments of chest CT during hospitalization and at 2 weeks, 1 month, 3 months, 6 months, and 1 year after hospital discharge. Risk factors of residual CT opacities and the influence of residual CT abnormalities on pulmonary functions at 1 year were also evaluated. RESULTS: A total of 41 patients were followed in this study. Gradual recovery after hospital discharge was confirmed by the serial CT scores. Around 47% of the patients showed residual aberration on pulmonary CT with a median CT score of 0 (interquartile range (IQR) of 0-2) at 1 year after discharge, with ground-glass opacity (GGO) with reticular pattern as the major radiologic pattern. Patients with residual radiological abnormalities were older (p = 0.01), with higher rate in current smokers (p = 0.04), higher rate in hypertensives (p = 0.05), lower SaO2 (p = 0.004), and higher prevalence of secondary bacterial infections during acute phase (p = 0.02). Multiple logistic regression analyses indicated that age was a risk factor associated with residual radiological abnormalities (OR 1.08, 95% CI 1.01-1.15, p = 0.02). Pulmonary functions of total lung capacity (p = 0.008) and residual volume (p < 0.001) were reduced in patients with residual CT abnormalities and were negatively correlated with CT scores. CONCLUSION: During 1-year follow-up after discharge, COVID-19 survivors showed continuous improvement on chest CT. However, residual lesions could still be observed and correlated with lung volume parameters. The risk of developing residual CT opacities increases with age.

Thyroid Function Abnormalities in COVID-19 Patients.

Purpose: The novel coronavirus COVID-19, has caused a worldwide pandemic, impairing several human organs and systems. Whether COVID-19 affects human thyroid function remains unknown. Methods: Eighty-four hospitalized COVID-19 patients in the First Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China) were retrospectively enrolled in this study, among which 22 cases had complete records of thyroid hormones. In addition, 91 other patients with pneumonia and 807 healthy subjects were included as controls. Results: We found that levels of total triiodothyronine (TT3) and thyroid stimulating hormone (TSH) were lower in COVID-19 patients than healthy group (p < 0.001). Besides, TSH level in COVID-19 patients was obviously lower than non-COVID-19 patients (p < 0.001). Within the group of COVID-19, 61.9% (52/84) patients presented with thyroid function abnormalities and the proportion of thyroid dysfunction was higher in severe cases than mild/moderate cases (74.6 vs. 23.8%, p < 0.001). Patients with thyroid dysfunction tended to have longer viral nucleic acid cleaning time (14.1 ± 9.4 vs. 10.6 ± 8.3 days, p = 0.088). To note, thyroid dysfunction was also associated with decreased lymphocytes (p < 0.001) and increased CRP (p = 0.002). The correlation between TT3 and TSH level seemed to be positive rather than negative in the early stage, and gradually turned to be negatively related over time. Conclusion: Thyroid function abnormalities are common in COVID-19 patients, especially in severe cases. This might be partially explained by nonthyroidal illness syndrome.

Clinical efficacy of methylprednisolone and the combined use of lopinavir/ritonavir with arbidol in treatment of coronavirus disease 2019.

This study aims to comparatively analyze the therapeutic efficacy upon multiple medication plans over lopinavir/ritonavir (LPV/r), arbidol (ARB), and methylprednisolone on patients with coronavirus disease 2019 (COVID-19). Totally, 75 COVID-19 patients admitted to The First Affiliated Hospital, Zhejiang University School of Medicine from January 22, 2020 to February 29, 2020 were recruited and grouped based on whether or not LPV/r and ARB were jointly used and whether or not methylprednisolone was used. Indexes including body temperature, time for nucleic acid negative conversion, hospital stays, and laboratory indexes were examined and compared. For all patients, there were no significant differences in the change of body temperature, the time for negative conversion, and hospital stays whether LPV/r and ARB were jointly used or not. While for severe and critically severe patients, methylprednisolone noticeably reduced the time for negative conversion. Meanwhile, the clinical efficacy was superior on patients receiving methylprednisolone within 3 days upon admission, and the duration of hospital stays was much shorter when methylprednisolone was given at a total dose of 0-400 mg than a higher dose of >400 mg if all patients received a similar dose per day. Nonetheless, no significant changes across hepatic, renal, and myocardial function indexes were observed. LPV/r combined with ARB produced no noticeably better effect on COVID-19 patients relative to the single-agent treatment. Additionally, methylprednisolone was efficient in severe and critically severe cases, and superior efficacy could be realized upon its early, appropriate, and short-term application.

Factors Associated With Prolonged Viral RNA Shedding in Patients with Coronavirus Disease 2019 (COVID-19).

BACKGROUND: An outbreak of coronavirus disease 2019 (COVID-19) is becoming a public health emergency. Data are limited on the duration and host factors related to viral shedding. METHODS: In this retrospective study, risk factors associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA shedding were evaluated in a cohort of 113 symptomatic patients from 2 hospitals outside Wuhan. RESULTS: The median (interquartile range) duration of SARS-CoV-2 RNA detection was 17 (13-22) days as measured from illness onset. When comparing patients with early (<15 days) and late (≥15 days after illness onset) viral RNA clearance, prolonged SARS-CoV-2 RNA shedding was associated with male sex (P = .009), old age (P = .033), concomitant hypertension (P = .009), delayed admission to hospital after illness onset (P = .001), severe illness at admission (P = .049), invasive mechanical ventilation (P = .006), and corticosteroid treatment (P = .025). Patients with longer SARS-CoV-2 RNA shedding duration had slower recovery of body temperature (P < .001) and focal absorption on radiograph images (P < .001) than patients with early SARS-CoV-2 RNA clearance. Male sex (OR, 3.24; 95% CI, 1.31-8.02), delayed hospital admission (OR, 1.30; 95% CI, 1.10-1.54), and invasive mechanical ventilation (OR, 9.88; 95% CI, 1.11-88.02) were independent risk factors for prolonged SARS-CoV-2 RNA shedding. CONCLUSIONS: Male sex, delayed admission to hospital after illness onset, and invasive mechanical ventilation were associated with prolonged SARS-CoV-2 RNA shedding. Hospital admission and general treatments should be started as soon as possible in symptomatic COVID-19 patients, especially male patients.

Clinical Outcomes and Plasma Concentrations of Baloxavir Marboxil and Favipiravir in COVID-19 Patients: An Exploratory Randomized, Controlled Trial.

BACKGROUND: Effective antiviral drugs for COVID-19 are still lacking. This study aims to evaluate the clinical outcomes and plasma concentrations of baloxavir acid and favipiravir in COVID-19 patients. METHODS: Favipiravir and baloxavir acid were evaluated for their antiviral activity against SARS-CoV-2 in vitro before the trial initiation. We conducted an exploratory trial with 3 arms involving hospitalized adult patients with COVID-19. Patients were randomized assigned in a 1:1:1 ratio into baloxavir marboxil group, favipiravir group, and control group. The primary outcome was the percentage of subjects with viral negative by Day 14 and the time from randomization to clinical improvement. Virus load reduction, blood drug concentration and clinical presentation were also observed. The trial was registered with Chinese Clinical Trial Registry (ChiCTR 2000029544). RESULTS: Baloxavir acid showed antiviral activity in vitro with the half-maximal effective concentration (EC50) of 5.48 µM comparable to arbidol and lopinavir, but favipiravir didn't demonstrate significant antiviral activity up to 100 µM. Thirty patients were enrolled. The percentage of patients who turned viral negative after 14-day treatment was 70%, 77%, and 100% in the baloxavir marboxil, favipiravir, and control group respectively, with the medians of time from randomization to clinical improvement was 14, 14 and 15 days, respectively. One reason for the lack of virological effect and clinical benefits may be due to insufficient concentrations of these drugs relative to their antiviral activities. One of the limitations of this study is the time from symptom onset to randomization, especially in the baloxavir marboxil and control groups, which is higher than the favipiravir group. CONCLUSIONS: Our findings could not prove a benefit of addition of either baloxavir marboxil or favipiravir under the trial dosages to the existing standard treatment.

The Clinical and Radiological Manifestations in Coronavirus Disease 2019 With Negative Nucleic Acid Results.

BACKGROUND: Coronavirus disease 2019 (COVID-19) was a new emerging disease with high infectiousness. Its diagnosis primarily depended on real-time polymerase chain reaction (RT-PCR) results. This study investigated epidemiological, clinical, and radiological characteristics of COVID-19 with negative RT-PCR results before confirmation. METHODS: Patients with COVID-19 were enrolled and divided into 2 groups: a negative group with negative RT-PCR results before confirmation and a positive group with positive results at the first detection. Epidemiological and clinical features were compared. Dynamic chest computerized tomography (CT) images of the negative group were evaluated. RESULTS: Ninety-nine laboratory-confirmed patients with COVID-19 including 8 patients (8%) with negative RT-PCR results were included. Patients from the negative group had similar epidemiological features: the average age (50.25 ±â€…13.27 years in the negative group and 53.70 ±â€…16.64 years in the positive group) and gender distribution (males made up 50% of the negative group and 62.6% of the positive group) were comparable. No significant differences were observed in clinical symptoms between the 2 groups. We found that fever was the most common symptom for both groups, followed by cough, expectoration, chest distress, fatigue, and gastroenterological symptoms. Moreover, ground-glass opacities and consolidations were the main manifestation in chest CT of patients with COVID-19 with or without confirmed RT-PCR results. CONCLUSIONS: Regardless of initial RT-PCR results, patients with COVID-19 had similar epidemiological, clinical, and chest CT features. Our study suggests value from early chest CT scans in COVID-19 screening and dynamic significance of radiology in disease monitoring should guide clinical decisions.

A Deep Learning System to Screen Novel Coronavirus Disease 2019 Pneumonia.

The real-time reverse transcription-polymerase chain reaction (RT-PCR) detection of viral RNA from sputum or nasopharyngeal swab had a relatively low positive rate in the early stage of coronavirus disease 2019 (COVID-19). Meanwhile, the manifestations of COVID-19 as seen through computed tomography (CT) imaging show individual characteristics that differ from those of other types of viral pneumonia such as influenza-A viral pneumonia (IAVP). This study aimed to establish an early screening model to distinguish COVID-19 from IAVP and healthy cases through pulmonary CT images using deep learning techniques. A total of 618 CT samples were collected: 219 samples from 110 patients with COVID-19 (mean age 50 years; 63 (57.3%) male patients); 224 samples from 224 patients with IAVP (mean age 61 years; 156 (69.6%) male patients); and 175 samples from 175 healthy cases (mean age 39 years; 97 (55.4%) male patients). All CT samples were contributed from three COVID-19-designated hospitals in Zhejiang Province, China. First, the candidate infection regions were segmented out from the pulmonary CT image set using a 3D deep learning model. These separated images were then categorized into the COVID-19, IAVP, and irrelevant to infection (ITI) groups, together with the corresponding confidence scores, using a location-attention classification model. Finally, the infection type and overall confidence score for each CT case were calculated using the Noisy-OR Bayesian function. The experimental result of the benchmark dataset showed that the overall accuracy rate was 86.7% in terms of all the CT cases taken together. The deep learning models established in this study were effective for the early screening of COVID-19 patients and were demonstrated to be a promising supplementary diagnostic method for frontline clinical doctors.

CAR T-cell treatment during the COVID-19 pandemic: Management strategies and challenges.

The pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is spreading rapidly across the world. Currently, the COVID-19 pandemic is affecting the continuity of essential routine healthcare services and procedures, including chimeric antigen receptor T-cell (CAR-T) therapy, a life-saving option for patients with relapsed/refractory (R/R) hematologic malignancies. Due to the rapid disease progression of hematological malignancies, there is an urgent need to manufacture and utilize CAR T-cells. However, CAR-T treatment has become extraordinarily challenging during this COVID-19 pandemic. Thus, many medical and technical factors must now be taken into consideration before, during, and after CAR-T therapy. The purpose of this review is to provide brief suggestions for rational decision-making strategies in evaluating and selecting CAR T-cell treatment and appropriate CAR T-cell products, and protective strategies for medical staff and patients to prevent infection in the midst of the current COVID-19 pandemic.

Erratum to: Transmission risk of patients with COVID-19 meeting discharge criteria should be interpreted with caution.

Erratum to: J Zhejiang Univ-Sci B (Biomed & Biotechnol) 2020 21(5):408-410. https://doi.org/10.1631/jzus.B2000117. The original version of this article unfortunately contained a mistake. For Fig. 1a in p.409, the citation of a reference, as well as the permission to reprint this picture, was missing. The correct version and the corresponding reference are given below: (a) Chest computed tomography (CT) image of Patient 1 on admission presents multiple ground-glass opacities distributed in the periphery of inferior lobe of both lungs. Reprinted from Zhang et al. (2020), with kind permission from Springer Nature.

Epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (COVID-19) with gastrointestinal symptoms.

OBJECTIVE: The SARS-CoV-2-infected disease (COVID-19) outbreak is a major threat to human beings. Previous studies mainly focused on Wuhan and typical symptoms. We analysed 74 confirmed COVID-19 cases with GI symptoms in the Zhejiang province to determine epidemiological, clinical and virological characteristics. DESIGN: COVID-19 hospital patients were admitted in the Zhejiang province from 17 January 2020 to 8 February 2020. Epidemiological, demographic, clinical, laboratory, management and outcome data of patients with GI symptoms were analysed using multivariate analysis for risk of severe/critical type. Bioinformatics were used to analyse features of SARS-CoV-2 from Zhejiang province. RESULTS: Among enrolled 651 patients, 74 (11.4%) presented with at least one GI symptom (nausea, vomiting or diarrhoea), average age of 46.14 years, 4-day incubation period and 10.8% had pre-existing liver disease. Of patients with COVID-19 with GI symptoms, 17 (22.97%) and 23 (31.08%) had severe/critical types and family clustering, respectively, significantly higher than those without GI symptoms, 47 (8.14%) and 118 (20.45%). Of patients with COVID-19 with GI symptoms, 29 (39.19%), 23 (31.08%), 8 (10.81%) and 16 (21.62%) had significantly higher rates of fever >38.5°C, fatigue, shortness of breath and headache, respectively. Low-dose glucocorticoids and antibiotics were administered to 14.86% and 41.89% of patients, respectively. Sputum production and increased lactate dehydrogenase/glucose levels were risk factors for severe/critical type. Bioinformatics showed sequence mutation of SARS-CoV-2 with m6A methylation and changed binding capacity with ACE2. CONCLUSION: We report COVID-19 cases with GI symptoms with novel features outside Wuhan. Attention to patients with COVID-19 with non-classic symptoms should increase to protect health providers.