ACE2 Can Act as the Secondary Receptor in the FcγR-Dependent ADE of SARS-CoV-2 (preprint)

Currently, it is not clear whether antibody-mediated enhancement (ADE) is involved in the pathogenesis of COVID-19, and the occurrence condition for ADE needs to be elucidated. We demonstrated that different from the reported RBD-targeting neutralizing antibody XG005 which elicits an ACE2-independent ADE on Raji cells, neutralizing antibody CB6, mouse anti-S1 serum and convalescent plasma could not elicit ADE on Raji cells; instead, they could elicit ADE on cells with FcγRIIA/CD32A expression and small amount of endogenous ACE2. ADE occurred at sub-neutralizing antibody concentrations, and the maximum induction concentration of ADE was correlated with IC50, implying that part of unneutralized S protein is essential for ADE. The enhanced infectivity of 614G variant was higher than that of 614D wildtype in the presence of antibodies, further indicating that ADE may be influenced by the virus strains with different ACE2 binding affinity and infectivity. ADE was proved to be FcγRII-dependent, and knockdown of ACE2 or the application of fusion-inhibition peptide EK1C4 significantly impaired ADE. In conclusion, our results identified a novel ADE mechanism for neutralizing antibodies against SARS-CoV-2. In certain circumstance, ACE2 may act as the secondary receptor in the antibody and FcγR-mediated enhanced entry of SARS-CoV-2.Funding: This work is supported by National Key R&D Program of China (2020YFA0707600), Natural Science Foundation of China (82170015), Medical and Health Science Technology Innovation Project of Chinese Academy of Medical Sciences (2020-I2M-2-014), Project of BRC-BC (Biomedical Translational Engineering Research Center of BUCT-CJFH) (XK2020-09), Natural Science Foundation of China (82041011/H0104 & 81800002/H0101); Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (CIFMS 2018-I2M-1-003 & 2020-I2M-CoV19-005).Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: The plasma from convalescent patients were collected 6 months after their discharge, with informed consent signed by every patient. The procedures were approved by the Ethics Committee in China- Japan Friendship Hospital (Beijing, China), and complied with all relevant ethical regulations regarding human research.

Dynamics of the Upper Respiratory Tract Microbiota and Its Association with Fatality in COVID-19 Patients (preprint)

Background: The pandemic of Coronavirus disease 2019 (COVID-19) is ongoing globally, which is a big challenge for public health. Alteration of human microbiota had been observed in COVID-19. However, it is unknown how the microbiota is associated with the fatality in COVID-19.Methods: We conducted metatranscriptome sequencing on 588 longitudinal oropharyngeal swab specimens collected from 192 COVID-19 patients recruited in the LOTUS clinical trial (Registration number: ChiCTR2000029308) (including 39 deceased patients), and 95 healthy controls from the same geographic area.Findings: The upper respiratory tract (URT) microbiota in COVID-19 patients differed from that in healthy controls, while deceased patients possessed a more distinct microbiota. Streptococcus was enriched in recovered patients, whereas potential pathogens, including Candida and Enterococcus, were more abundant in deceased patients. Moreover, the microbiota dominated by Streptococcus was more stable than that dominated by other species. In contrast, the URT microbiota in deceased patients showed a more significant alteration and became more deviated from the norm after admission. The abundance of Streptococcus on admission, particularly that of S. parasanguis, was identified as a strong predictor of fatality by Cox and L1 regularized logistic regression analysis, thus could be used as a potential prognostic biomarker of COVID-19.Interpretation Alteration of the URT microbiota was observed in COVID-19 patients and was associated with the fatality rate. A higher abundance of Streptococcus, especially S. parasanguis, on admission in oropharyngeal swabs predicts a better outcome. The generalization of the results in other populations and underlying mechanisms need further investigations.Trial Registration: Participants were enrolled in ChiCTR2000029308.Funding: This study was funded in part by the National Major Science & Technology Project for Control and Prevention of Major Infectious Diseases in China (2017ZX10103004, 2018ZX10301401), the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (2019-I2M-2-XX, 2016-I2M-1-014, 2018-I2M-1-003), The Non-profit Central Research Institute Fund of CAMS (2020HY320001, 2019PT310029), Beijing Advanced Innovation Center for Genomics (ICG), and Beijing Advanced Innovation Center for Structural Biology (ICSB).Declaration of Interests: All authors declare no competing interests.Ethics Approval Statement: The study was approved by the Institutional Review Board of Jin Yin-Tan Hospital (KY2020-02.01). Written informed consent was obtained from all patients or their legal representatives if they were too unwell to provide consent.

Dynamics of the upper respiratory tract microbiota and its association with fatality in COVID-19 patients (preprint)

The pandemic of Coronavirus disease 2019 (COVID-19) is ongoing globally, which is a big challenge for public health. Alteration of human microbiota had been observed in COVID-19. However, it is unknown how the microbiota is associated with the fatality in COVID-19. We conducted metatranscriptome sequencing on 588 longitudinal oropharyngeal swab specimens collected from 192 COVID-19 patients recruited in the LOTUS clinical trial (Registration number: ChiCTR2000029308) (including 39 deceased patients), and 95 healthy controls from the same geographic area. The upper respiratory tract (URT) microbiota in COVID-19 patients differed from that in healthy controls, while deceased patients possessed a more distinct microbiota. Streptococcus was enriched in recovered patients, whereas potential pathogens, including Candida and Enterococcus, were more abundant in deceased patients. Moreover, the microbiota dominated by Streptococcus was more stable than that dominated by other species. In contrast, the URT microbiota in deceased patients showed a more significant alteration and became more deviated from the norm after admission. The abundance of Streptococcus on admission, particularly that of S. parasanguinis, was identified as a strong predictor of fatality by Cox and L1 regularized logistic regression analysis, thus could be used as a potential prognostic biomarker of COVID-19. The generalization of the results in other populations and underlying mechanisms needs further investigations.

Cross-Reactive Antibody Against Human Coronavirus OC43 Spike Protein Correlates with Disease Severity in COVID-19 Patients: A Retrospective Study (preprint)

Background: Seasonal human coronaviruses (HCoVs) including HCoV-229E, -OC43, -NL63 and -HKU1 are widely spreading in global human populations. However, the relevance of humoral response against seasonal HCoVs to COVID-19 pathogenesis is elusive.Methods: We profiled the temporal changes of IgG antibodies against spike (S; S-IgG) proteins of SARS-CoV-2 and seasonal HCoVs in 838 plasma samples collected from 344 COVID-19 patients. We tested the antigenic cross-reactivity of S protein between SARS-CoV-2 and seasonal HCoVs and evaluated the correlations between HCoV-OC43 S-IgG antibody and disease severity in COVID-19 patients.Findings: SARS-CoV-2 S-IgG titers mounted until days 22–28, whereas HCoV-OC43 antibody titers increased until days 15–21 and then plateaued until day 46. However, IgG antibody titers against HCoV-NL63, -229E, and -HKU1 showed no significant increasing. A two-way cross-reactivity was identified between SARS-CoV-2 and HCoV-OC43. Neutralizing antibodies against SARS-CoV-2 were not detected in healthy controls who were positive for HCoV-OC43 S-IgG. HCoV-OC43 S-IgG titers were significantly higher in patients with severe disease than those in mild/moderate patients at days 1–21 post symptom onset (PSO). Higher levels of HCoV-OC43 S-IgG were also observed in patients requiring mechanical ventilation and the elderly. At days 1–10 PSO, HCoV-OC43 S-IgG titers correlated to disease severity in all age groups, and to fatality in over 60-year group.Interpretation: Our data indicate that there exist a humoral cross-reactive response between HCoV-OC43 and SARS-CoV-2. The cross-reactive HCoV-OC43 S-IgG antibody is not protective against SARS-CoV-2, but may be a risk factor for the severity and adverse outcome of COVID-19.Funding Statement: This study was funded in part by the National Major Science & Technology Project for Control and Prevention of Major Infectious Diseases in China (2017ZX10204401, 2018ZX10734404), Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (2016-I2M-1–014, 2018-I2M-1-003, 2020-I2M-1-001, 2020-I2M-CoV19-005), Natural Science Foundation of China (82041011/H0104), and National Key R&D Program of China (2020YFA0707600). Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: This study was approved by the Ethical Review Board of Wuhan Jinyintan Hospital, Infectious Disease Hospital of Heilongjiang Province (Harbin), and Institute of Pathogen Biology, Chinese Academy of Medical Sciences. Written informed consent was obtained from each healthy volunteer and COVID-19 patients in cohort 4. Written informed consents from the remaining patients were waived in light of the emerging infectious disease of high public health relevance.

Augmentation of anti-MDA5 antibody implies severe disease in COVID-19 patients (preprint)

Recent studies have provided insights into the autoinflammation triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection, which is associated with high mortality of coronavirus disease 2019 (COVID-19). Striking similarities has been noted between COVID-19 and anti-melanoma differentiation-associated gene 5 (MDA5) antibody (Ab)-related dermatomyositis (DM), implying a shared autoinflammatory aberrance. However, it is unclear whether anti-MDA5 Ab is present in COVID-19 and correlates with the severity and adverse outcome of COVID-19 patients. Here, we found that the positive rate of anti-MDA5 Ab in patients with COVID-19 was 48.2% and the anti-MDA5 Ab positive patients tended to develop severe disease (88.6% vs 66.9%, P<0.0001). In particular, the titer of anti-MDA5 Ab was increased in the non-survivals (5.95{+/-}5.16 vs 8.22{+/-}6.64, P=0.030) and the positive rate was also higher than that in the survivals (23.5% vs 12.0%, P=0.012). Regarding to severe COVID-19 patients, we found that high titer of anti-MDA5 Ab ([≥]10.0 U/mL) was more prevalent in the non-survivals (31.2% vs 14.0%, P=0.006). Moreover, early profiling of anti-MDA5 Ab could distinguish severe patients from those with non-severe ones. Overall, our data reveal that anti-MDA5 Ab is prevalent in the COVID-19 patients and high titer of this antibody is correlated with severe disease and unfavorable outcomes.

Endpoints for randomized controlled clinical trials for COVID-19 treatments (preprint)

Introduction: Endpoint choice for randomized controlled trials of treatments for COVID-19 is complex. A new disease brings many uncertainties, but trials must start rapidly. COVID-19 is heterogeneous, ranging from mild disease that improves within days to critical disease that can last weeks and can end in death. While improvement in mortality would provide unquestionable evidence about clinical significance of a treatment, sample sizes for a study evaluating mortality are large and may be impractical. Furthermore, patient states in between "cure" and "death" represent meaningful distinctions. Clinical severity scores have been proposed as an alternative. However, the appropriate summary measure for severity scores has been the subject of debate, particularly in relating to the uncertainty about the time-course of COVID-19. Outcomes measured at fixed time-points may risk missing the time of clinical benefit. An endpoint such as time-to-improvement (or recovery), avoids the timing problem. However, some have argued that power losses will result from reducing the ordinal scale to a binary state of "recovered" vs "not recovered." Methods: We evaluate statistical power for possible trial endpoints for COVID-19 treatment trials using simulation models and data from two recent COVID-19 treatment trials. Results: Power for fixed-time point methods depends heavily on the time selected for evaluation. Time-to-improvement (or recovery) analyses do not specify a time-point. Time-to-event approaches have reasonable statistical power, even when compared to a fixed time-point method evaluated at the optimal time. Discussion: Time-to-event analyses methods have advantages in the COVID-19 setting, unless the optimal time for evaluating treatment effect is known in advance. Even when the optimal time is known, a time-to-event approach may increase power for interim analyses.

Viral load in upper respiratory tract of COVID-19 patients detected by digital PCR (preprint)

Background: Upper respiratory tract specimens are widely applicable for the diagnosis of COVID-19. To date, no study has analyzed the actual viral loads in upper respiratory tract and its relationship with the severity of lung lesions, Ct value of RT-PCR and transmission capacity in COVID-19 patients.Methods: We retrospectively enrolled nine COVID-19 patients. Clinical data and close contacts of these patients were investigated. Respiratory samples were tested for SARS-CoV-2 with both normal RT-PCR and droplet digital PCR.Results: All the COVID-19 patients complicated with pneumonia. Viral loads in nasopharyngeal swabs were accurately quantified, and they had no direct correspondence with the severity of lung lesions. The Cycle Threshold (Ct) value of RT-PCR was approximately consistent with the absolute quantification of digital PCR. The spearman correlation coefficient between them was -0.952 with P value < 0.001. Close contacts of patients with very low viral load or no detected virus were not infected.Conclusions: Viral loads in nasopharyngeal swabs, could not predict the severity of lung lesions revealed by CT in COVID-19 patients. The infectious capacity of patients with low or absent viral load in upper respiratory tract was relatively weak, and wearing mask might be helpful for lower its spread.

Household transmission of COVID-19, Shenzhen, January-February 2020 (preprint)

Coronavirus disease 2019 has led to more than three million cases globally. Since the first family cluster of COVID-19 cases identified in Shenzhen in early January, most of the local transmission occurred within household contacts. Identifying the factors associated with household transmission is of great importance to guide preventive measures.

Evaluation of the Efficacy and Safety of Intravenous Remdesivir in Adult Patients with Severe Pneumonia caused by COVID-19 virus Infection: study protocol for a Phase 3 Randomized, Double-blind, Placebo-controlled, Multicentre trial (preprint)

Background: A novel coronavirus emerged in Wuhan, Hubei Province, China towards the end of 2019 (SARS-CoV-2 or COVID-19 virus). Large scale spread within China and internationally led the World Health Organisation to declare a Public Health Emergency of International Concern on 30th January 2020. The clinical manifestations of COVID-19 virus infection include asymptomatic infection, mild upper respiratory symptoms, severe viral pneumonia with respiratory failure and even death. There are no antivirals of proven clinical efficacy in coronavirus infections. Remdesivir (GS-5734), a nucleoside analogue, has inhibitory effects on animal and human highly pathogenic coronaviruses, including MERS-CoV and SARS-CoV, in in-vitro and in-vivo experiments. It is also inhibitory against the COVID-19 virus in-vitro. The aim of this study is to assess the efficacy and safety of remdesivir in adult patients with severe pneumonia caused by COVID-19 virus infection.Methods: The protocol is prepared in accordance with the SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) guidelines. This is a phase 3, randomized, double-blind, placebo-controlled, multicentre trial. Adults (≥18 years) with laboratory confirmed COVID-19 virus infection, and severe pneumonia signs or symptoms, and radiologically confirmed severe pneumonia are randomly assigned in a 2:1 ratio to intravenous remdesivir or placebo for 10 days. The primary endpoint is time to clinical improvement (censored at Day 28), defined as the time (in days) from randomization of study treatment (remdesivir or placebo) until a decline of two categories on a six-category ordinal scale of clinical status (1 ꞊ discharged; 6 ꞊ death) or live discharge from hospital. One interim analysis for efficacy and futility will be conducted once half of the total number of events required had been observed.Discussion: This is the first randomized, placebo-controlled trial in 2019-nCoV. Enrolment began in sites in Wuhan, Hubei Province, China on 6th February 2020.Trial registration: ClinicalTrials.gov, NCT04257656, 6th February 2020.