External quality assessment of nucleic acid testing in provincial or municipal CDC laboratories in the COVID-19 surveillance network in China

Objective: To understand the RNA detection performance of SARS-CoV-2 Variants of Concern (VOCs) in 32 provincial or municipal CDC laboratories in COVID-19 surveillance network in China through an external quality assessment (EQA), and evaluate the clinical performance of the current SARS-CoV-2 nucleic acids detection kits used and provided by the participating laboratories.

Dynamic Zero-Covid strategy curtails mutagenesis and emergence of new variants of the SARS-CoV-2 (preprint)

Within the local outbreak period of SARS-CoV-2 Delta variant in Nanjing and Yangzhou, China, we analyzed the mutation process of the Delta variants in 520 cases, as well as the production, spread and elimination of new mutant strains under the non-pharmaceutical interventions (NPI) strategy. The investigation on distribution of COVID-19 cases and phylogenetic analysis of SARS-CoV-2 genome sequences attributed to tracking the transmission chains, transmission chains were terminated by the isolation of the COVID-19 patients and quarantine of close-contracts, suggesting the importance of NPI in prompting some mutations to disappear and stopping the transmission of new variants. Dynamic zero-Covid strategy has been implemented successfully to against the second-largest local epidemic caused by an imported COVID-19 case in China.

Antibody neutralization to SARS-CoV-2 and variants after one year in Wuhan (preprint)

Most COVID-19 patients can build effective humoral immunity against SARS-CoV-2 after recovery . However, it remains unknown how long the protection can maintain and how efficiently it can protect people from the reinfection of the emerging SARS-CoV-2 variants. Here we evaluated the sera from 248 COVID-19 convalescents around one year post-infection in Wuhan, the earliest epicenter of SARS-CoV-2. We demonstrated that the SARS-CoV-2 immunoglobulin G (IgG) maintains at a high level and potently neutralizes the infection of the original strain (WT) and the B.1.1.7 variant in most patients. However, they showed varying degrees of efficacy reduction against the other variants of concern (P.1, B.1.525, and especially B.1.351) in a patient-specific manner. Mutations in RBD including K417N, E484K, and E484Q/L452R (B.1.617) remarkably impair the neutralizing activity of the convalescents' sera. Encouragingly, we found that a small fraction of patients' sera showed broad neutralization potency to multiple variants and mutants, suggesting the existence of broadly neutralizing antibodies recognizing the epitopes beyond the mutation sites. Our results suggest that the SARS-CoV-2 vaccination effectiveness relies more on the timely re-administration of the epitope-updated vaccine than the durability of the neutralizing antibodies.

Occurrence of COVID-19 symptoms during SARS-CoV-2 infection defines waning of humoral immunity (preprint)

Approximately half of the SARS-CoV-2 infections occur without apparent symptoms, raising questions regarding long-term humoral immunity in asymptomatic individuals. Plasma levels of immunoglobulin G (IgG) and M (IgM) against the viral spike or nucleoprotein were determined for 25,091 individuals enrolled in a surveillance program in Wuhan, China. We compared 405 asymptomatic individuals with 459 symptomatic COVID-19 patients. The well-defined duration of the SARS-CoV-2 endemic in Wuhan allowed a side-by-side comparison of antibody responses following symptomatic and asymptomatic infections without subsequent antigen re-exposure. IgM responses rapidly declined in both groups. However, both the prevalence and durability of IgG responses and neutralizing capacities correlated positively with symptoms. Regardless of sex, age, and body weight, asymptomatic individuals lost their SARS-CoV-2-specific IgG antibodies more often and rapidly than symptomatic patients. These findings have important implications for immunity and favour immunization programs including individuals after asymptomatic infections. One-Sentence SummaryPrevalence and durability of SARS-CoV-2-specific IgG responses and neutralizing capacities correlate with COVID-19 symptoms.

Toll-Like Receptor Agonist R848 Protects Mice from Lethal SARS-CoV-2 Infections (preprint)

Background: An ideal animal model to study SARS-coronavirus 2 (SARS-CoV-2) pathogenesis and evaluate therapies and vaccines should reproduce SARS-CoV-2 infection and recapitulate lung disease like those seen in humans. The angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV-2, but mice are resistant to the infection because their ACE2 is incompatible with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Methods: We generated a mouse-adapted strain SARS-CoV-2 by serial passages in the lung of BALB/c mice. Complete genome deep sequencing of different generations of viruses was performed to characterize the dynamics of the adaptive mutations in SARS-CoV-2. Indirect immunofluorescence analysis and Biolayer interferometry experiments demonstrated that two mutations in RBD significantly increased its binding affinity towards mouse ACE2. Significantly, TLR7/8 agonist Resiquimod block SARS-CoV-2 in vitro and in vivo. Findings: We adapted a wild-type SARS-CoV-2 by serial passages in the lung of BALB/c mice. The mouse-adapted strain WBP-1 showed increased infectivity in BALB/c mice and led to severe interstitial pneumonia. We characterized the dynamics of the adaptive mutations in SARS-CoV-2 and demonstrated that Q493K and Q498H in RBD significantly increased its binding affinity towards mouse ACE2. Additionally, The TLR7/8 agonist Resiquimod was able to protect mice against WBP-1 challenge, demonstrating this mouse-adapted strain is a useful tool to investigate COVID-19 and develop new therapies. Interpretation: We found for the first time that the Q493K and Q498H mutations in the RBD of WBP-1 enhanced its interactive affinities with mACE2. The mouse-adapted SARS-CoV-2 provides a valuable tool for the evaluation of novel antiviral and vaccine strategies, especially in determining the immunopathological consequences of any intervention. This study also verified the antiviral activity of TLR7/8 agonist Resiquimod against SARS-CoV-2 in vitro and in vivo.Funding Statement: This research was funded by Emergency Science and Technology Project of Hubei Province（2020FCA046）and Independent Science and Technology Innovation Fund of Huazhong Agricultural University in 2020 (2662020PY002).Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: The animal experiments were approved by the Research Ethics Committee, Huazhong Agricultural University, Hubei, China (HZAUMO-2020-0007). All the animal experiments were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals from the Research Ethics Committee, Huazhong Agricultural University, Hubei, China.

A SARS-CoV-2 neutralizing antibody with exceptional spike binding coverage and optimized therapeutic potentials (preprint)

The Coronavirus Disease of 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health and economy. Therapeutic options such as monoclonal antibodies (mAbs) against SARS-CoV-2 are in urgent need. We have identified potent monoclonal antibodies binding to SARS-CoV-2 Spike protein from COVID-19 convalescent patients and one of these antibodies, P4A1, interacts directly and covers the majority of the Receptor Binding Motif (RBM) of Spike receptor-binding domain (RBD), shown by high-resolution complex structure analysis. We further demonstrated P4A1 binding and neutralizing activities against wild type and mutant spike proteins. P4A1 was subsequently engineered to reduce the potential risk for antibody-dependent enhancement (ADE) of infection and to extend its half-life. The engineered mAb exhibits optimized pharmacokinetic and safety profile, and results in complete viral clearance in a rhesus monkey model of COVID-19 following a single injection.

The analysis of the long-term impact of SARS-CoV-2 on the cellular immune system in individuals recovering from COVID-19 reveals a profound NKT cell impairment (preprint)

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affects millions of people and killed hundred-thousands of individuals. While acute and intermediate interactions between SARS-CoV-2 and the immune system have been studied extensively, long-term impacts on the cellular immune system remained to be analyzed. Here, we comprehensively characterized immunological changes in peripheral blood mononuclear cells in 49 COVID-19 convalescent individuals (CI) in comparison to 27 matched SARS-CoV-2 unexposed individuals (UI). Despite recovery from the disease for more than 2 months, CI showed significant decreases in frequencies of invariant NKT and NKT-like cells compared to UI. Concomitant with the decrease in NKT-like cells, an increase in the percentage of Annexin V and 7-AAD double positive NKT-like cells was detected, suggesting that the reduction in NKT-like cells results from cell death months after recovery. Significant increases in regulatory T cell frequencies, TIM-3 expression on CD4 and CD8 T cells, as well as PD-L1 expression on B cells were also observed in CI, while the cytotoxic potential of T cells and NKT-like cells, defined by GzmB expression, was significantly diminished. However, both CD4 and CD8 T cells of CI showed increased Ki67 expression and were fully capable to proliferate and produce effector cytokines upon TCR stimulation. Collectively, we provide the first comprehensive characterization of immune signatures in patients recovering from SARS-CoV-2 infection, suggesting that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease.

SARS-CoV-2 infection induces sustained humoral immune responses in convalescent patients following symptomatic COVID-19 (preprint)

Long-term antibody responses and neutralizing activities following SARS-CoV-2 infections have not yet been elucidated. We quantified immunoglobulin M (IgM) and G (IgG) antibodies recognizing the SARS-CoV-2 receptor-binding domain (RBD) of the spike (S) or the nucleocapsid (N) protein, and neutralizing antibodies during a period of six months following COVID-19 disease onset in 349 symptomatic COVID-19 patients, which were among the first world-wide being infected. The positivity rate and magnitude of IgM-S and IgG-N responses increased rapidly. High levels of IgM-S/N and IgG-S/N at 2-3 weeks after disease onset were associated with virus control and IgG-S titers correlated closely with the capacity to neutralize SARS-CoV-2. While specific IgM-S/N became undetectable 12 weeks after disease onset in most patients, IgG-S/N titers showed an intermediate contraction phase, but stabilized at relatively high levels over the six months observation period. At late time points the positivity rates for binding and neutralizing SARS-CoV-2-specific antibodies was still over 70%. Taken together, our data indicate sustained humoral immunity in recovered patients who suffer from symptomatic COVID-19, suggesting prolonged immunity.

Profiling B cell immune responses to identify neutralizing antibodies from convalescent COVID-19 patients (preprint)

The pandemic Coronavirus Disease 2019 (COVID-19) causes noticeable morbidity and mortality worldwide. In addition to vaccine and antiviral drug therapy, the use of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) neutralizing antibodies for treatment purposes is a viable alternative. In this study, we aimed to profile the humoral responses and identify neutralizing antibodies against SARS-CoV-2 using high-throughput single-cell sequencing that tailored to B cell receptor sequencing. From two convalescent patients with high serum titer against SARS-COV-2, we identified seven antibodies specifically binding to SARS-CoV-2. Among these, the most potent antibody, P4A1 was demonstrated to block the binding of spike protein to its receptor angiotensin-converting enzyme 2 (ACE2), and prevent the viral infection in neutralization assays with pseudovirus as well as live virus at nM to sub-nM range. Moreover, antibody P4A1 can also bind strongly to spike protein with N354D/D364Y, R408I, W436R, V367F or D614G mutations respectively, suggesting that the antibody alone or in combination with other antibodies that recognize different variations of SARS-CoV-2, may provide a broad spectrum therapeutic option for COVID-19 patients. Authors Lisu Huang, Bingqing Shen, Yu Guo, and Shu Shen contributed equally to this work.

Airborne SARS-CoV-2 and the Use of Masks for Protection against Its Spread in Wuhan, China (preprint)

The outbreak of COVID-19 has caused a global public health crisis. The spread of SARS-CoV-2 by contact is widely accepted, but the relative importance of aerosol transmission for the spread of COVID-19 is controversial. Here we characterize the distribution of SARA-CoV-2 in 123 aerosol samples, 63 masks, and 30 surface samples collected at various locations in Wuhan, China. The positive percentages of viral RNA included 21% of the aerosol samples from an intensive care unit and 39% of the masks from patients with a range of conditions. A viable virus was isolated from the surgical mask of one critically ill patient while all viral RNA positive aerosol samples were cultured negative. The SARS-CoV-2 detected in masks from patients, ambient air, and respirators from health workers compose a chain of emission, transport, and recipient of the virus. Our results indicate that masks are effective in protecting against the spread of viruses, and it is strongly recommended that people throughout the world wear masks to break the chain of virus transmission and thus protect themselves and others from SARS-CoV-2.

Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin (preprint)

Since the SARS outbreak 18 years ago, a large number of severe acute respiratory syndrome related coronaviruses (SARSr-CoV) have been discovered in their natural reservoir host, bats1-4. Previous studies indicated that some of those bat SARSr-CoVs have the potential to infect humans5-7. Here we report the identification and characterization of a novel coronavirus (nCoV-2019) which caused an epidemic of acute respiratory syndrome in humans, in Wuhan, China. The epidemic, started from December 12th, 2019, has caused 198 laboratory confirmed infections with three fatal cases by January 20th, 2020. Full-length genome sequences were obtained from five patients at the early stage of the outbreak. They are almost identical to each other and share 79.5% sequence identify to SARS-CoV. Furthermore, it was found that nCoV-2019 is 96% identical at the whole genome level to a bat coronavirus. The pairwise protein sequence analysis of seven conserved non-structural proteins show that this virus belongs to the species of SARSr-CoV. The nCoV-2019 virus was then isolated from the bronchoalveolar lavage fluid of a critically ill patient, which can be neutralized by sera from several patients. Importantly, we have confirmed that this novel CoV uses the same cell entry receptor, ACE2, as SARS-CoV.