Development of neutralizing antibodies against SARS-CoV-2, using a high-throughput single-B-cell cloning method.

Background: Rapid and efficient strategies are needed to discover neutralizing antibodies (nAbs) from B cells derived from virus-infected patients. Methods: Here, we report a high-throughput single-B-cell cloning method for high-throughput isolation of nAbs targeting diverse epitopes on the SARS-CoV-2-RBD (receptor binding domain) from convalescent COVID-19 patients. This method is simple, fast and highly efficient in generating SARS-CoV-2-neutralizing antibodies from COVID-19 patients' B cells. Results: Using this method, we have developed multiple nAbs against distinct SARS-CoV-2-RBD epitopes. CryoEM and crystallography revealed precisely how they bind RBD. In live virus assay, these nAbs are effective in blocking viral entry to the host cells. Conclusion: This simple and efficient method may be useful in developing human therapeutic antibodies for other diseases and next pandemic.

The pre-analytical process management status and influencing factors of laboratory test before prescribing antimicrobial in developing country.

INTRODUCTION: The results of laboratory testing are crucial basis for clinicians to prescribe antimicrobial. Laboratory testing is a highly complex process, and increasing evidence suggests that errors and obstacles in the pre-analytical process (PP) will affect reasonable antimicrobial use. However, PP was an easily neglected link in hospital infection management and the current situation of it and the influencing factors of management are not clear. METHODS: A cross-sectional survey was conducted in the department of clinical, specimen collection, transportation, and inspection in 109 secondary and tertiary hospitals in Central China. The rate of antimicrobial susceptibility test request (AST) and related indexes of above departments were calculated to describe the situation. Management characteristics (frequency of training etc.) were described as proportions and fractional probit regression analysis was used to determine the influencing factors. RESULTS: The average rate of non restricted-use antimicrobial was 63%, the restricted-use was 86%, the special-use was 95%. The zero obstacle rate of specimen collection was 27.3%, of specimen transportation was 19.4% and of inspection feedback was 61.7%. There was a difference between the secondary and tertiary hospitals on non restricted-use (X2 = 22.968, P < 0.001); restricted-use (X2 = 29.466, P < 0.001); special-use (X2 = 27.317, P < 0.001). Taking non restricted-use as an example, training (OR = 0.312, 95%CI: 0.148,0.429), low-frequency appraisal (OR = 0.153, 95%CI: 0.082,0.224), guidance (OR = 0.32, 95%CI: 0.237,0.403) and information technology (OR = 0.104, 95%CI: 0.009,0.199) were positive factors. CONCLUSIONS: There were substantial differences in the rate of AST request in clinical department between secondary and tertiary hospitals. The zero obstacle rate in collection, transportation and inspection department were still low. In most departments, training and performance appraisal were positive factors, guidance and information technology were positive supporting factors.

Characterization of cross-reactive monoclonal antibodies against SARS-CoV-1 and SARS-CoV-2: Implication for rational design and development of pan-sarbecovirus vaccines and neutralizing antibodies.

Emergence of various circulating SARS-CoV-2 variants of concern (VOCs) promotes the identification of pan-sarbecovirus vaccines and broadly neutralizing antibodies (bNAbs). Here, to characterize monoclonal antibodies cross-reactive against both SARS-CoV-1 and SARS-CoV-2 and to search the criterion for bNAbs against all emerging SARS-CoV-2, we isolated several SARS-CoV-1-cross-reactive monoclonal antibodies (mAbs) from a wildtype SARS-CoV-2 convalescent donor. These antibodies showed broad binding capacity and cross-neutralizing potency against various SARS-CoV-2 VOCs, including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta), but failed to efficiently neutralize Omicron variant and its sublineages. Structural analysis revealed how Omicron sublineages, but not other VOCs, efficiently evade an antibody family cross-reactive against SARS-CoV-1 through their escape mutations. Further evaluation of a series of SARS-CoV-1/2-cross-reactive bNAbs showed a negative correlation between the neutralizing activities against SARS-CoV-1 and SARS-CoV-2 Omicron variant. Together, these results suggest the necessity of using cross-neutralization against SARS-CoV-1 and SARS-CoV-2 Omicron as criteria for rational design and development of potent pan-sarbecovirus vaccines and bNAbs.

Eugenol Alleviates TGEV-Induced Intestinal Injury via Suppressing ROS/NLRP3/GSDMD-Dependent Pyroptosis.

Transmissible gastroenteritis virus (TGEV), a coronavirus, is one of the main causative agents of diarrhea in piglets and significantly impacts the global swine industry. Pyroptosis is involved in the pathogenesis of coronavirus, but its role in TGEV-induced intestinal injury has yet to be fully elucidated. Eugenol, an essential plant oil, plays a vital role in antiviral innate immune responses. We demonstrate the preventive effect of eugenol on TGEV infection. Eugenol alleviates TGEV-induced intestinal epithelial cell pyroptosis and reduces intestinal injury in TGEV-infected piglets. Mechanistically, eugenol reduces the activation of NLRP3 inflammasome, thereby inhibiting TGEV-induced intestinal epithelial cell pyroptosis. In addition, eugenol scavenges TGEV-induced reactive oxygen species (ROS) increase, which in turn prevents TGEV-induced NLRP3 inflammasome activation and pyroptosis. Overall, eugenol protects the intestine by reducing TGEV-induced pyroptosis through inhibition of NLRP3 inflammasome activation, which may be mediated through intracellular ROS levels. These findings propose that eugenol may be an effective strategy to prevent TGEV infection.

Eugenol Attenuates Transmissible Gastroenteritis Virus-Induced Oxidative Stress and Apoptosis Via ROS-NRF2-ARE Signaling.

Transmissible gastroenteritis virus (TGEV), a coronavirus that causes severe diarrhea due to oxidative stress in the piglet intestine, is a major cause of economic loss in the livestock industry. However, limited interventions have been shown to be effective in the treatment of TGEV. Here, we demonstrate the therapeutic activity of eugenol in TGEV-induced intestinal oxidative stress and apoptosis. Our data show that eugenol supplementation protects intestine and IPEC-J2 cells from TGEV-induced damage. Mechanistically, eugenol reduces TGEV-induced oxidative stress in intestinal epithelial cells by reducing reactive oxygen species levels. Interestingly, eugenol also inhibits TGEV-induced intestinal cell apoptosis in vitro and in vivo. In conclusion, our data suggest that eugenol prevents TGEV-induced intestinal oxidative stress by reducing ROS-mediated damage to antioxidant signaling pathways. Therefore, eugenol may be a promising therapeutic strategy for TGEV infection.

Eugenol alleviates transmissible gastroenteritis virus-induced intestinal epithelial injury by regulating NF-κB signaling pathway.

Increasing evidence supports the ability of eugenol to maintain intestinal barrier integrity and anti-inflammatory in vitro and in vivo; however, whether eugenol alleviates virus-mediated intestinal barrier damage and inflammation remains a mystery. Transmissible gastroenteritis virus (TGEV), a coronavirus, is one of the main causative agents of diarrhea in piglets and significantly impacts the global swine industry. Here, we found that eugenol could alleviate TGEV-induced intestinal functional impairment and inflammatory responses in piglets. Our results indicated that eugenol improved feed efficiency in TGEV-infected piglets. Eugenol not only increased serum immunoglobulin concentration (IgG) but also significantly decreased serum inflammatory cytokine concentration (TNF-α) in TGEV-infected piglets. In addition, eugenol also significantly decreased the expression of NF-κB mRNA and the phosphorylation level of NF-κB P65 protein in the jejunum mucosa of TGEV-infected piglets. Eugenol increased villus height and the ratio of villus height to crypt depth in the jejunum and ileum, and decreased serum D-lactic acid levels. Importantly, eugenol increased tight junction protein (ZO-1) and mRNA expression levels of nutrient transporter-related genes (GluT-2 and CaT-1) in the jejunum mucosa of TGEV-infected piglets. Meanwhile, compared with TGEV-infected IPEC-J2 cells, treatment with eugenol reduced the cell cytopathic effect, attenuated the inflammatory response. Interestingly, eugenol did not increase the expression of ZO-1 and Occludin in IPEC-J2 cells. However, western blot and immunofluorescence results showed that eugenol restored TGEV-induced down-regulation of ZO-1 and Occludin, while BAY11-7082 (The NF-κB specific inhibitor) enhanced the regulatory ability of eugenol. Our findings demonstrated that eugenol attenuated TGEV-induced intestinal injury by increasing the expression of ZO-1 and Occludin, which may be related to the inhibition of NF-κB signaling pathway. Eugenol may offer some therapeutic opportunities for coronavirus-related diseases.

Memory B cell repertoire from triple vaccinees against diverse SARS-CoV-2 variants.

Omicron (B.1.1.529), the most heavily mutated SARS-CoV-2 variant so far, is highly resistant to neutralizing antibodies, raising concerns about the effectiveness of antibody therapies and vaccines1,2. Here we examined whether sera from individuals who received two or three doses of inactivated SARS-CoV-2 vaccine could neutralize authentic Omicron. The seroconversion rates of neutralizing antibodies were 3.3% (2 out of 60) and 95% (57 out of 60) for individuals who had received 2 and 3 doses of vaccine, respectively. For recipients of three vaccine doses, the geometric mean neutralization antibody titre for Omicron was 16.5-fold lower than for the ancestral virus (254). We isolated 323 human monoclonal antibodies derived from memory B cells in triple vaccinees, half of which recognized the receptor-binding domain, and showed that a subset (24 out of 163) potently neutralized all SARS-CoV-2 variants of concern, including Omicron. Therapeutic treatments with representative broadly neutralizing monoclonal antibodies were highly protective against infection of mice with SARS-CoV-2 Beta (B.1.351) and Omicron. Atomic structures of the Omicron spike protein in complex with three classes of antibodies that were active against all five variants of concern defined the binding and neutralizing determinants and revealed a key antibody escape site, G446S, that confers greater resistance to a class of antibodies that bind on the right shoulder of the receptor-binding domain by altering local conformation at the binding interface. Our results rationalize the use of three-dose immunization regimens and suggest that the fundamental epitopes revealed by these broadly ultrapotent antibodies are rational targets for a universal sarbecovirus vaccine.

Structural and functional characterizations of infectivity and immune evasion of SARS-CoV-2 Omicron.

The SARS-CoV-2 Omicron variant with increased fitness is spreading rapidly worldwide. Analysis of cryo-EM structures of the spike (S) from Omicron reveals amino acid substitutions forging interactions that stably maintain an active conformation for receptor recognition. The relatively more compact domain organization confers improved stability and enhances attachment but compromises the efficiency of the viral fusion step. Alterations in local conformation, charge, and hydrophobic microenvironments underpin the modulation of the epitopes such that they are not recognized by most NTD- and RBD-antibodies, facilitating viral immune escape. Structure of the Omicron S bound with human ACE2, together with the analysis of sequence conservation in ACE2 binding region of 25 sarbecovirus members, as well as heatmaps of the immunogenic sites and their corresponding mutational frequencies, sheds light on conserved and structurally restrained regions that can be used for the development of broad-spectrum vaccines and therapeutics.

An ultrapotent pan-ß-coronavirus lineage B (ß-CoV-B) neutralizing antibody locks the receptor-binding domain in closed conformation by targeting its conserved epitope.

New threats posed by the emerging circulating variants of SARS-CoV-2 highlight the need to find conserved neutralizing epitopes for therapeutic antibodies and efficient vaccine design. Here, we identified a receptor-binding domain (RBD)-binding antibody, XG014, which potently neutralizes ß-coronavirus lineage B (ß-CoV-B), including SARS-CoV-2, its circulating variants, SARS-CoV and bat SARSr-CoV WIV1. Interestingly, antibody family members competing with XG014 binding show reduced levels of cross-reactivity and induce antibody-dependent SARS-CoV-2 spike (S) protein-mediated cell-cell fusion, suggesting a unique mode of recognition by XG014. Structural analyses reveal that XG014 recognizes a conserved epitope outside the ACE2 binding site and completely locks RBD in the non-functional "down" conformation, while its family member XG005 directly competes with ACE2 binding and position the RBD "up". Single administration of XG014 is effective in protection against and therapy of SARS-CoV-2 infection in vivo. Our findings suggest the potential to develop XG014 as pan-ß-CoV-B therapeutics and the importance of the XG014 conserved antigenic epitope for designing broadly protective vaccines against ß-CoV-B and newly emerging SARS-CoV-2 variants of concern.

Correlation of Seizure Increase and COVID-19 Outbreak in Adult Patients with Epilepsy: Findings and Suggestions from a Nationwide Multi-centre Survey in China.

OBJECTIVES: To investigate the impact of the COVID-19 outbreak on the behaviours, mental health and seizure control of adult patients with epilepsy (PWE) and to identify the correlation of seizure increase and the COVID-19 outbreak to guide the medical care of individuals with epilepsy during a public health crisis. METHODS: This study was conducted at 28 centres from February 2020 to April 2020. Participants filled out a 62-item online survey including sociodemographic, COVID-19-related, epilepsy-related and psychological variables and were divided into two groups based on whether their seizure frequency increased during the COVID-19 pandemic. Chi-square tests and t-tests were used to test differences in significant characteristics. Multiple logistic regression analyses were used to identify risk factors for seizure worsening. RESULTS: A total of 1,237 adult PWE were enrolled for analysis. Of this sample, 31 (8.33%) patients experienced an increase in seizures during the pandemic. Multivariate logistic regression suggested that feeling nervous about the pandemic (P < 0.05), poor quality of life (P = 0.001), drug reduction/withdrawal (P = 0.032), moderate anxiety during the COVID-19 outbreak (P = 0.046) and non-seizure free before the COVID-19 outbreak (P < 0.05) were independently related to seizure increase during the pandemic. CONCLUSIONS: During the COVID-19 pandemic, PWE with poor quality of life and mental status, as well as AED reduction/withdrawal, were more likely to experience seizure increase. This observation highlights the importance of early identification of the population at high risk of seizure worsening and implementation of preventive strategies during the pandemic.

A retrospective view of pediatric cases infected with SARS-CoV-2 of a middle-sized city in mainland China.

ABSTRACT: The coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 had resulted in a global pandemic. A comprehensive analysis of pediatric COVID-19 cases is essential to decipher the natural features of children under the risk of this disease.In the epidemic period, all the children infected with SARS-CoV-2 in Wuxi, a city with a stable medical system during the COVID-19 outbreak in China, were enrolled for comprehensive data documenting their clinical, prognosis, follow-up, treatment and various tests results. Combing their family cluster characteristics, the epidemiological, hospitalization, and transmission features of children with SARS-CoV-2 were analyzed and discussed.A total of 7 children were enrolled, including 4 mild cases, 1 moderate case, and 2 asymptomatic cases. The common symptoms were fever and dry cough. The length of viral nucleic acid duration in nasopharynx varied and was irrelevant to the severity of the symptom, whether symptomatic or asymptomatic. Two cases showed viral nucleic acid positive recurrence after discharge from the hospital. A child with type 1 diabetes was also focused, for the elevated blood sugar during hospitalization. All these children had close contacts with their family members, some of those were confirmed COVID-19 cases.We provided a holistic and detailed portrayal of the pediatric COVID-19 cases in a typical city of timely response to the epidemic. While the family cluster exhibits the major transmission mode, attention should be paid for the potential risk since the expanded social space of children in future.

Pediatric massage therapy for restoring pediatric lung function from COVID-19: A protocol for systematic review and meta-analysis.

BACKGROUND: The novel coronavirus disease 2019 (COVID-19) has caused an international outbreak of a respiratory illness and grown to be a global public health emergency since patients were first detected in Wuhan, China. Given the rapidly growing pandemic and the overwhelmed medical system, there is an urgent need of alternative medicine to help children relieve symptoms during self-quarantine, and possibly to help increase their chances of survival and recovery from COVID-19. By using various manual techniques at specified locations on the surface of the body, pediatric massage manipulation can unblock meridians, promote the circulation of qi and blood and strengthen resistance to pathogens. METHODS: We will search the following electronic databases: Wanfang and Pubmed Database, CNKI, CENTRAL, CINAHL, EMBASE and MEDLINE. Each database will be searched from inception to June 2020. The entire process will include study selection, data extraction, risk of bias assessment and meta-analyses. RESULTS: This systematic review will evaluate the existing evidence of pediatric massage therapy for restoring pediatric lung function from COVID-19. The outcomes will include the improvement of pulmonary function and adverse effect. CONCLUSION: This proposed systematic review will evaluate the existing evidence and explore the potential role of pediatric massage therapy on the effectiveness and safety in pulmonary function of COVID-19 convalescent children. PROSPERO REGISTRATION NUMBER: CRD42020193396.