The potential for traditional Chinese therapy in treating sleep disorders caused by COVID-19 through the cholinergic anti-inflammatory pathway.

Since the outbreak of Coronavirus disease (COVID-19) in 2019, it has spread rapidly across the globe. Sleep disorders caused by COVID-19 have become a major concern for COVID-19 patients and recovered patients. So far, there's no effective therapy on this. Traditional Chinese therapy (TCT) has a great effect on sleep disorders, with rare side effects and no obvious withdrawal symptoms. The cholinergic anti-inflammatory pathway, a neuroregulatory pathway in the central nervous system that uses cholinergic neurons and neurotransmitters to suppress inflammatory responses, has been reported to be associated with sleep disorders and psychiatric symptoms. Many studies have shown that TCT activates the cholinergic anti-inflammatory pathway (CAP), inhibits inflammation, and relieves associated symptoms. Therefore, we believe that TCT may be a potential therapeutic strategy to alleviate sleep disorders induced by COVID-19 through CAP. In this review, we analyzed the relationship between cytokine storm induced by Coronavirus and sleep disorders, explained the influence of CAP on sleep disorders, discussed the TCT's effect on CAP, and summarized the treatment effect of TCT on sleep disorders. Based on these practical researches and theoretical basis, we propose potential strategies to effectively improve the sleep disorders caused by COVID-19.

Study the Outbreak and End Characteristics of Epidemic and Social Transmission from the Perspective of Data

Method: This paper starts from the perspective of data, based on the theory of complex networks, analyses the collected data related to influenza, measles and COVID-19, and draws on the epidemic transmission theory to study the characteristics of the spread and end of the epidemic from the perspective of data, and successively explores the hysteresis loop phenomenon The estimation method of news dissemination potential in social media and the influence of susceptibility and contact matrix on the spread and control of COVID-19. According to Bayesian theorem, the time series of the number of new participants in daily topic discussion follows Poisson distribution, which is related to the distribution of generation time and effective regeneration number. According to the collected data related to COVID-19, the impact of susceptibility on the transmission of COVID-19 was studied. According to Ro=1, the threshold of infection rate can be calculated ßc.

An Engineered IgG-VHH Bispecific Antibody against SARS-CoV-2 and Its Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies are shown to be effective therapeutics for providing coronavirus disease 2019 (COVID-19) protection. However, recurrent variants arise and facilitate significant escape from current antibody therapeutics. Bispecific antibodies (bsAbs) represent a unique platform to increase antibody breadth and to reduce neutralization escape. Herein, a novel immunoglobulin G-variable domains of heavy-chain-only antibody (IgG-VHH) format bsAb derived from a potent human antibody R15-F7 and a humanized nanobody P14-F8-35 are rationally engineered. The resulting bsAb SYZJ001 efficiently neutralizes wild-type SARS-CoV-2 as well as the alpha, beta, gamma, and delta variants, with superior efficacy to its parental antibodies. Cryo-electron microscopy structural analysis reveals that R15-F7 and P14-F8-35 bind to nonoverlapping epitopes within the RBD and sterically hindered ACE2 receptor binding. Most importantly, SYZJ001 shows potent prophylactic and therapeutic efficacy against SARS-CoV-2 in three established mouse models. Collectively, the current results demonstrate that the novel bsAb format is feasible and effective, suggesting great potential as an inspiring antiviral strategy.

The potential for traditional Chinese therapy in treating sleep disorders caused by COVID-19 through the cholinergic anti-inflammatory pathway

Since the outbreak of Coronavirus disease (COVID-19) in 2019, it has spread rapidly across the globe. Sleep disorders caused by COVID-19 have become a major concern for COVID-19 patients and recovered patients. So far, there’s no effective therapy on this. Traditional Chinese therapy (TCT) has a great effect on sleep disorders, with rare side effects and no obvious withdrawal symptoms. The cholinergic anti-inflammatory pathway, a neuroregulatory pathway in the central nervous system that uses cholinergic neurons and neurotransmitters to suppress inflammatory responses, has been reported to be associated with sleep disorders and psychiatric symptoms. Many studies have shown that TCT activates the cholinergic anti-inflammatory pathway (CAP), inhibits inflammation, and relieves associated symptoms. Therefore, we believe that TCT may be a potential therapeutic strategy to alleviate sleep disorders induced by COVID-19 through CAP. In this review, we analyzed the relationship between cytokine storm induced by Coronavirus and sleep disorders, explained the influence of CAP on sleep disorders, discussed the TCT’s effect on CAP, and summarized the treatment effect of TCT on sleep disorders. Based on these practical researches and theoretical basis, we propose potential strategies to effectively improve the sleep disorders caused by COVID-19.

Research progress of mRNA technology for viral infectious diseases

Messenger RNA (mRNA, mRNA) vaccines and antibodies are a new type of vaccine and antibody technology emerging in recent years. Compared with traditional vaccines, mRNA vaccines have the advantages of high safety, good balanced immunity, short development cycle, and low production costs. mRNA antibodies exert biological effects in vivo earlier and longer than other forms of delivered antibodies. With the rapid development of mRNA modification and delivery technology, mRNA technology is rapidly maturing, showing broad application prospects in tumor treatment, prevention and treatment of viral infectious diseases, etc. In particular, the new coronavirus mRNA vaccine has been completed at a record speed The development and successful application paves the way for the promotion of mRNA technology in the future. This paper reviews the important breakthroughs in the field of mRNA technology, focusing on the major progress of mRNA vaccines and antibodies in response to viral infectious diseases, and looks forward to the future research trends of this technology in the field of anti-viral infection.

Neutralization of ARCoV-induced sera against SARS-CoV-2 variants.

ARCoV is a candidate mRNA vaccine encoding receptor-binding domain of SARS-CoV-2. Its safety, tolerability, and immunogenicity profile have been confirmed in the phase 1 clinical trial in China. A multi-regional phase 3 clinical trial is currently underway to test the efficacy of ARCoV (NCT04847102). Here, we tested the cross-neutralization against SARS-CoV-2 variants of concern (VOCs) of a panel of serum samples from participants in the phase 1 clinical trial of ARCoV by pesudo- and authentic SARS-CoV-2. Our data suggest the immunity induced by the ARCoV vaccine reduced but still has significant neutralization against the Alpha and Delta variants. Moreover, ARCoV maintained activity against the Beta variant, despite of its obvious reduction in neutralizing titers. Our findings further support the solid protective neutralization activity against VOCs induced by ARCoV vaccine.

Safety and immunogenicity of the SARS-CoV-2 ARCoV mRNA vaccine in Chinese adults: a randomised, double-blind, placebo-controlled, phase 1 trial.

BACKGROUND: Safe and effective vaccines are urgently needed to end the COVID-19 pandemic caused by SARS-CoV-2 infection. We aimed to assess the preliminary safety, tolerability, and immunogenicity of an mRNA vaccine ARCoV, which encodes the SARS-CoV-2 spike protein receptor-binding domain (RBD). METHODS: This single centre, double-blind, randomised, placebo-controlled, dose-escalation, phase 1 trial of ARCoV was conducted at Shulan (Hangzhou) hospital in Hangzhou, Zhejiang province, China. Healthy adults aged 18-59 years negative for SARS-CoV-2 infection were enrolled and randomly assigned using block randomisation to receive an intramuscular injection of vaccine or placebo. Vaccine doses were 5 µg, 10 µg, 15 µg, 20 µg, and 25 µg. The first six participants in each block were sentinels and along with the remaining 18 participants, were randomly assigned to groups (5:1). In block 1 sentinels were given the lowest vaccine dose and after a 4-day observation with confirmed safety analyses, the remaining 18 participants in the same dose group proceeded and sentinels in block 2 were given their first administration on a two-dose schedule, 28 days apart. All participants, investigators, and staff doing laboratory analyses were masked to treatment allocation. Humoral responses were assessed by measuring anti-SARS-CoV-2 RBD IgG using a standardised ELISA and neutralising antibodies using pseudovirus-based and live SARS-CoV-2 neutralisation assays. SARS-CoV-2 RBD-specific T-cell responses, including IFN-γ and IL-2 production, were assessed using an enzyme-linked immunospot (ELISpot) assay. The primary outcome for safety was incidence of adverse events or adverse reactions within 60 min, and at days 7, 14, and 28 after each vaccine dose. The secondary safety outcome was abnormal changes detected by laboratory tests at days 1, 4, 7, and 28 after each vaccine dose. For immunogenicity, the secondary outcome was humoral immune responses: titres of neutralising antibodies to live SARS-CoV-2, neutralising antibodies to pseudovirus, and RBD-specific IgG at baseline and 28 days after first vaccination and at days 7, 15, and 28 after second vaccination. The exploratory outcome was SARS-CoV-2-specific T-cell responses at 7 days after the first vaccination and at days 7 and 15 after the second vaccination. This trial is registered with www.chictr.org.cn (ChiCTR2000039212). FINDINGS: Between Oct 30 and Dec 2, 2020, 230 individuals were screened and 120 eligible participants were randomly assigned to receive five-dose levels of ARCoV or a placebo (20 per group). All participants received the first vaccination and 118 received the second dose. No serious adverse events were reported within 56 days after vaccination and the majority of adverse events were mild or moderate. Fever was the most common systemic adverse reaction (one [5%] of 20 in the 5 µg group, 13 [65%] of 20 in the 10 µg group, 17 [85%] of 20 in the 15 µg group, 19 [95%] of 20 in the 20 µg group, 16 [100%] of 16 in the 25 µg group; p<0·0001). The incidence of grade 3 systemic adverse events were none (0%) of 20 in the 5 µg group, three (15%) of 20 in the 10 µg group, six (30%) of 20 in the 15 µg group, seven (35%) of 20 in the 20 µg group, five (31%) of 16 in the 25 µg group, and none (0%) of 20 in the placebo group (p=0·0013). As expected, the majority of fever resolved in the first 2 days after vaccination for all groups. The incidence of solicited systemic adverse events was similar after administration of ARCoV as a first or second vaccination. Humoral immune responses including anti-RBD IgG and neutralising antibodies increased significantly 7 days after the second dose and peaked between 14 and 28 days thereafter. Specific T-cell response peaked between 7 and 14 days after full vaccination. 15 µg induced the highest titre of neutralising antibodies, which was about twofold more than the antibody titre of convalescent patients with COVID-19. INTERPRETATION: ARCoV was safe and well tolerated at all five doses. The acceptable safety profile, together with the induction of strong humoral and cellular immune responses, support further clinical testing of ARCoV at a large scale. FUNDING: National Key Research and Development Project of China, Academy of Medical Sciences China, National Natural Science Foundation China, and Chinese Academy of Medical Sciences.

A highly immunogenic live-attenuated vaccine candidate prevents SARS-CoV-2 infection and transmission in hamsters.

The highly pathogenic and readily transmissible SARS-CoV-2 has caused a global coronavirus pandemic, urgently requiring effective countermeasures against its rapid expansion. All available vaccine platforms are being used to generate safe and effective COVID-19 vaccines. Here, we generated a live-attenuated candidate vaccine strain by serial passaging of a SARS-CoV-2 clinical isolate in Vero cells. Deep sequencing revealed the dynamic adaptation of SARS-CoV-2 in Vero cells, resulting in a stable clone with a deletion of seven amino acids (N679SPRRAR685) at the S1/S2 junction of the S protein (named VAS5). VAS5 showed significant attenuation of replication in multiple human cell lines, human airway epithelium organoids, and hACE2 mice. Viral fitness competition assays demonstrated that VAS5 showed specific tropism to Vero cells but decreased fitness in human cells compared with the parental virus. More importantly, a single intranasal injection of VAS5 elicited a high level of neutralizing antibodies and prevented SARS-CoV-2 infection in mice as well as close-contact transmission in golden Syrian hamsters. Structural and biochemical analysis revealed a stable and locked prefusion conformation of the S trimer of VAS5, which most resembles SARS-CoV-2-3Q-2P, an advanced vaccine immunogen (NVAX-CoV2373). Further systematic antigenic profiling and immunogenicity validation confirmed that the VAS5 S trimer presents an enhanced antigenic mimic of the wild-type S trimer. Our results not only provide a potent live-attenuated vaccine candidate against COVID-19 but also clarify the molecular and structural basis for the highly attenuated and super immunogenic phenotype of VAS5.

Lipid nanoparticle-encapsulated mRNA antibody provides long-term protection against SARS-CoV-2 in mice and hamsters.

Monoclonal antibodies represent important weapons in our arsenal to against the COVID-19 pandemic. However, this potential is severely limited by the time-consuming process of developing effective antibodies and the relative high cost of manufacturing. Herein, we present a rapid and cost-effective lipid nanoparticle (LNP) encapsulated-mRNA platform for in vivo delivery of SARS-CoV-2 neutralization antibodies. Two mRNAs encoding the light and heavy chains of a potent SARS-CoV-2 neutralizing antibody HB27, which is currently being evaluated in clinical trials, were encapsulated into clinical grade LNP formulations (named as mRNA-HB27-LNP). In vivo characterization demonstrated that intravenous administration of mRNA-HB27-LNP in mice resulted in a longer circulating half-life compared with the original HB27 antibody in protein format. More importantly, a single prophylactic administration of mRNA-HB27-LNP provided protection against SARS-CoV-2 challenge in mice at 1, 7 and even 63 days post administration. In a close contact transmission model, prophylactic administration of mRNA-HB27-LNP prevented SARS-CoV-2 infection between hamsters in a dose-dependent manner. Overall, our results demonstrate a superior long-term protection against SARS-CoV-2 conferred by a single administration of this unique mRNA antibody, highlighting the potential of this universal platform for antibody-based disease prevention and therapy against COVID-19 as well as a variety of other infectious diseases.

Long-term stability and protection efficacy of the RBD-targeting COVID-19 mRNA vaccine in nonhuman primates.

Messenger RNA (mRNA) vaccine technology has shown its power in preventing the ongoing COVID-19 pandemic. Two mRNA vaccines targeting the full-length S protein of SARS-CoV-2 have been authorized for emergency use. Recently, we have developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor-binding domain (RBD) of SARS-CoV-2 (termed ARCoV), which confers complete protection in mouse model. Herein, we further characterized the protection efficacy of ARCoV in nonhuman primates and the long-term stability under normal refrigerator temperature. Intramuscular immunization of two doses of ARCoV elicited robust neutralizing antibodies as well as cellular response against SARS-CoV-2 in cynomolgus macaques. More importantly, ARCoV vaccination in macaques significantly protected animals from acute lung lesions caused by SARS-CoV-2, and viral replication in lungs and secretion in nasal swabs were completely cleared in all animals immunized with low or high doses of ARCoV. No evidence of antibody-dependent enhancement of infection was observed throughout the study. Finally, extensive stability assays showed that ARCoV can be stored at 2-8 °C for at least 6 months without decrease of immunogenicity. All these promising results strongly support the ongoing clinical trial.

Rational development of a human antibody cocktail that deploys multiple functions to confer Pan-SARS-CoVs protection.

Structural principles underlying the composition and synergistic mechanisms of protective monoclonal antibody cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic antibody cocktail against SARS-CoV-2. On the basis of our previously identified humanized cross-neutralizing antibody H014, we systematically analyzed a fully human naive antibody library and rationally identified a potent neutralizing antibody partner, P17, which confers effective protection in animal model. Cryo-EM studies dissected the nature of the P17 epitope, which is SARS-CoV-2 specific and distinctly different from that of H014. High-resolution structure of the SARS-CoV-2 spike in complex with H014 and P17, together with functional investigations revealed that in a two-antibody cocktail, synergistic neutralization was achieved by S1 shielding and conformational locking, thereby blocking receptor attachment and viral membrane fusion, conferring high potency as well as robustness against viral mutation escape. Furthermore, cluster analysis identified a hypothetical 3rd antibody partner for further reinforcing the cocktail as pan-SARS-CoVs therapeutics.

Study on the Mixed Teaching Mode of the Program Language Courses in the New Situation

With the spread of new coronavirus, new forms of online teaching are widely used in colleges and universities in China. Teachers are the main parts relying on the existing online teaching platform, which can make up for the defect of single online platform, simulate off-line teaching better, complement the advantage of off-line teaching, and improve the quality of online teaching effectively by using the new hybrid teaching mode of program language courses with other network tools software.

Humoral immune response to circulating SARS-CoV-2 variants elicited by inactivated and RBD-subunit vaccines.

SARS-CoV-2 variants could induce immune escape by mutations on the receptor-binding domain (RBD) and N-terminal domain (NTD). Here we report the humoral immune response to circulating SARS-CoV-2 variants, such as 501Y.V2 (B.1.351), of the plasma and neutralizing antibodies (NAbs) elicited by CoronaVac (inactivated vaccine), ZF2001 (RBD-subunit vaccine) and natural infection. Among 86 potent NAbs identified by high-throughput single-cell VDJ sequencing of peripheral blood mononuclear cells from vaccinees and convalescents, near half anti-RBD NAbs showed major neutralization reductions against the K417N/E484K/N501Y mutation combination, with E484K being the dominant cause. VH3-53/VH3-66 recurrent antibodies respond differently to RBD variants, and K417N compromises the majority of neutralizing activity through reduced polar contacts with complementarity determining regions. In contrast, the 242-244 deletion (242-244Δ) would abolish most neutralization activity of anti-NTD NAbs by interrupting the conformation of NTD antigenic supersite, indicating a much less diversity of anti-NTD NAbs than anti-RBD NAbs. Plasma of convalescents and CoronaVac vaccinees displayed comparable neutralization reductions against pseudo- and authentic 501Y.V2 variants, mainly caused by E484K/N501Y and 242-244Δ, with the effects being additive. Importantly, RBD-subunit vaccinees exhibit markedly higher tolerance to 501Y.V2 than convalescents, since the elicited anti-RBD NAbs display a high diversity and are unaffected by NTD mutations. Moreover, an extended gap between the third and second doses of ZF2001 leads to better neutralizing activity and tolerance to 501Y.V2 than the standard three-dose administration. Together, these results suggest that the deployment of RBD-vaccines, through a third-dose boost, may be ideal for combating SARS-CoV-2 variants when necessary, especially for those carrying mutations that disrupt the NTD supersite.

Double lock of a potent human therapeutic monoclonal antibody against SARS-CoV-2.

Receptor recognition and subsequent membrane fusion are essential for the establishment of successful infection by SARS-CoV-2. Halting these steps can cure COVID-19. Here we have identified and characterized a potent human monoclonal antibody, HB27, that blocks SARS-CoV-2 attachment to its cellular receptor at sub-nM concentrations. Remarkably, HB27 can also prevent SARS-CoV-2 membrane fusion. Consequently, a single dose of HB27 conferred effective protection against SARS-CoV-2 in two established mouse models. Rhesus macaques showed no obvious adverse events when administrated with 10 times the effective dose of HB27. Cryo-EM studies on complex of SARS-CoV-2 trimeric S with HB27 Fab reveal that three Fab fragments work synergistically to occlude SARS-CoV-2 from binding to the ACE2 receptor. Binding of the antibody also restrains any further conformational changes of the receptor binding domain, possibly interfering with progression from the prefusion to the postfusion stage. These results suggest that HB27 is a promising candidate for immuno-therapies against COVID-19.

Adaptation of SARS-CoV-2 in BALB/c mice for testing vaccine efficacy.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has prioritized the development of small-animal models for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We adapted a clinical isolate of SARS-CoV-2 by serial passaging in the respiratory tract of aged BALB/c mice. The resulting mouse-adapted strain at passage 6 (called MASCp6) showed increased infectivity in mouse lung and led to interstitial pneumonia and inflammatory responses in both young and aged mice after intranasal inoculation. Deep sequencing revealed a panel of adaptive mutations potentially associated with the increased virulence. In particular, the N501Y mutation is located at the receptor binding domain (RBD) of the spike protein. The protective efficacy of a recombinant RBD vaccine candidate was validated by using this model. Thus, this mouse-adapted strain and associated challenge model should be of value in evaluating vaccines and antivirals against SARS-CoV-2.

A Thermostable mRNA Vaccine against COVID-19.

There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.

A Mouse Model of SARS-CoV-2 Infection and Pathogenesis.

Since December 2019, a novel coronavirus SARS-CoV-2 has emerged and rapidly spread throughout the world, resulting in a global public health emergency. The lack of vaccine and antivirals has brought an urgent need for an animal model. Human angiotensin-converting enzyme II (ACE2) has been identified as a functional receptor for SARS-CoV-2. In this study, we generated a mouse model expressing human ACE2 (hACE2) by using CRISPR/Cas9 knockin technology. In comparison with wild-type C57BL/6 mice, both young and aged hACE2 mice sustained high viral loads in lung, trachea, and brain upon intranasal infection. Although fatalities were not observed, interstitial pneumonia and elevated cytokines were seen in SARS-CoV-2 infected-aged hACE2 mice. Interestingly, intragastric inoculation of SARS-CoV-2 was seen to cause productive infection and lead to pulmonary pathological changes in hACE2 mice. Overall, this animal model described here provides a useful tool for studying SARS-CoV-2 transmission and pathogenesis and evaluating COVID-19 vaccines and therapeutics.