Preclinical evaluation of RQ3013, a broad-spectrum mRNA vaccine against SARS-CoV-2 variants (preprint)

The global emergence of SARS-CoV-2 variants has led to increasing breakthrough infections in vaccinated populations, calling for an urgent need to develop more effective and broad-spectrum vaccines to combat COVID-19. Here we report the preclinical development of RQ3013, an mRNA vaccine candidate intended to bring broad protection against SARS-CoV-2 variants of concern (VOCs). RQ3013, which contains pseudouridine-modified mRNAs formulated in lipid nanoparticles, encodes the spike(S) protein harboring a combination of mutations responsible for immune evasion of VOCs. Here we characterized the expressed S immunogen and evaluated the immunogenicity, efficacy, and safety of RQ3013 in various animal models. RQ3013 elicited robust immune responses in mice, hamsters, and nonhuman primates (NHP). It can induce high titers of antibodies with broad cross-neutralizing ability against the Wild-type, B.1.1.7, B.1.351, B.1.617.2, and the omicron B.1.1.529 variants. In mice and NHP, two doses of RQ3013 protected the upper and lower respiratory tract against infection by SARS-CoV-2 and its variants. We also proved the safety of RQ3013 in NHP models. Our results provided key support for the evaluation of RQ3013 in clinical trials.

Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid and Potential Utility for SARS-CoV-2 Detection (preprint)

Enzymatic beacons, or E-beacons, are 1:1 bioconjugates of the nanoluciferase enzyme linked covalently at its C-terminus to hairpin forming DNA oligonucleotides equipped with a dark quencher. We prepared E-beacons biocatalytically using the promiscuous "hedgehog" protein-cholesterol ligase, HhC. Instead of cholesterol, HhC attached nanoluciferase site-specifically to mono-sterylated hairpin DNA, prepared in high yield by solid phase synthesis. We tested three potential E-beacon dark quenchers: Iowa Black, Onyx-A, and dabcyl. Prototype E-beacon carrying each of those quenchers provided sequence-specific nucleic acid sensing through turn-on bioluminescence. For practical application, we prepared dabcyl-quenched E-beacons for potential use in detecting the COVID-19 virus, SARS-CoV-2. Targeting the E484 codon of the SARS-CoV-2 Spike protein, E-beacons (80 x 10-12 M) reported wild-type SARS-CoV-2 nucleic acid at [≥]1 x 10-9 M with increased bioluminescence of 8-fold. E-beacon prepared for the E484K variant of SARS-CoV-2 functioned with similar sensitivity. These E-beacons could discriminate their complementary target from nucleic acid encoding the E484Q mutation of the SARS-CoV-2 Kappa variant. Along with specificity, detection sensitivity with E-beacons is two to three orders of magnitude better than synthetic molecular beacons, rivaling the most sensitive nucleic acid detection agents reported to date.

Stock market reactions to the COVID-19 pandemic: The moderating role of corporate big data strategies based on Word2Vec

By developing a machine learning-based measure of corporate big data strategies, this study empirically explores how stock markets respond to the COVID-19 pandemic and whether corporate big data strategies make firms immune to the pandemic effect. We find that except for information technology and health care sectors, firms in most sectors in China are negatively affected by the COVID-19 outbreak. Among these firms, an increase in the number of daily new confirmed cases in the city of a firm's headquarters is associated with a decrease in its stock prices, however, such a decline is attenuated for firms with a high emphasis on big data strategies. Our results are robust when we use COVID-19 cases at the whole country level.

Analysis of the Effect of Proton Pump Inhibitors on the Course of Common COVID-19 (preprint)

Background/aims: This study is to evaluate the effect of proton pump inhibitors on the course of common COVID-19.Methods: Clinical data of common COVID-19 patients admitted to the Shanghai Public Health Clinical Center for treatment from January 20, 2020 to March 16, 2020 were collected. A retrospective study was conducted and the patients were divided into two groups according to whether they used proton pump inhibitors or not. The differences in SARS-CoV-2 clearance and hospital stay between the two groups were compared by univariate and multivariate analyses.Results: A total of 154 COVID-19 common cases were included in this study, including 80 males (51.9%), 35 patients (22.7%) in the proton pump inhibitors group, and 119 patients (77.3%) in the control group. In the proton pump inhibitors group and the control group, the duration of the SARS-CoV-2 clearance were 7(6-9) and 7(6-11) days, and the duration of the hospital stay was 21(16-25) and 20(15-26) days, respectively. There was no significant difference between the two groups in the cumulative incidence of the SARS-CoV-2 clearance and the discharge, all P > 0.05. Multivariate analysis showed that chronic gastropathy prolonged the duration of SARS-CoV-2 clearance, the HR was 20.924(3.547-123.447). Hypertension, chronic obstructive pulmonary disease, chronic liver disease and malignant tumor all increased the duration of hospital stay for COVID-19, and the HR were 1.820 (1.073-3.085), 4.370 (1.205-15.844), 9.011 (2.681-30.290) and 5.270 (1.237-22.456), respectively; the duration of the hospital stay in COVID-19 patients was shortened by the SARS-CoV-2 clearance, and the HR was 0.907 (0.869-0.947); all P < 0.05.Conclusion:  Proton pump inhibitors use has no effect on the prolonging or shortening of the course of adults hospitalized with COVID-19.

Distinct mechanisms for TMPRSS2 expression explain organ-specific inhibition of SARS-CoV-2 infection by enzalutamide (preprint)

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly become a global public health threat due to the lack of effective drugs or vaccines against SARS-CoV-2. The efficacy of several repurposed drugs has been evaluated in clinical trials. Among these drugs, a relatively new antiandrogen agent, enzalutamide, was proposed because it reduces the expression of transmembrane serine protease 2 (TMPRSS2), a key component mediating SARS-CoV-2-driven entry into host cells, in prostate cancer cells. However, definitive evidence for the therapeutic efficacy of enzalutamide in COVID-19 is lacking. Here, we evaluated the antiviral efficacy of enzalutamide in prostate cancer cells, lung cancer cells, human lung organoids and SARS-CoV-2-infected Ad-ACE2-transduced Tmprss2 knockout (Tmprss2-KO) and wild-type (WT) mice. TMPRSS2 knockout significantly inhibited SARS-CoV-2 infection in vivo. Enzalutamide effectively inhibited SARS-CoV-2 infection in human prostate cancer cells (LNCaP) but not in human lung cancer cells or patient-derived lung organoids. Although Tmprss2 knockout effectively blocked SARS-CoV-2 infection in ACE2-transduced mice, enzalutamide showed no antiviral activity due to the AR independence of TMPRSS2 expression in mouse and human lung epithelial cells. Moreover, we observed distinct AR binding patterns between prostate cells and lung cells and a lack of direct binding of AR to TMPRSS2 in human lung cells. Thus, our findings do not support the postulated protective role of enzalutamide in treating COVID-19.

S494 O-glycosylation site on the SARS-COV-2 RBD Affects the Virus Affinity to ACE2 and its Infectivity; A Molecular Dynamics Study (preprint)

SARS-COV-2 is a strain of Coronavirus family which caused the extensive pandemic of COVID-19, which is still going on. Several studies showed that the glycosylation of virus spike (S) protein and the Angiotensin-Converting Enzyme 2 (ACE2) receptor on the host cell is critical for the virus infectivity. Molecular Dynamics (MD) simulations were used to explore the role of a novel mutated O-glycosylation site (D494S) on the Receptor Binding Domain (RBD) of S protein. This site was suggested as a key mediator of virus-host interaction. We showed that the decoration of S494 with core and elongated O-glycans results in stabilized interactions on the direct RBD-ACE2 interface with more favorable binding free energies for longer oligosaccharides. Hence, the further drug design attempts should take this crucial factor into account, while suggesting any novel therapeutic candidate.

A bacterial artificial chromosome (BAC)-vectored noninfectious replicon of SARS-CoV-2 (preprint)

Vaccines and antiviral agents are in urgent need to stop the COVID-19 pandemic. To facilitate antiviral screening against SARS-CoV-2 without requirement for high biosafety level facility, we developed a bacterial artificial chromosome (BAC)-vectored replicon of SARS-CoV-2, nCoV-SH01 strain, in which secreted Gaussia luciferase (sGluc) was encoded in viral subgenomic mRNA as a reporter gene. The replicon was devoid of structural genes spike (S), membrane (M), and envelope (E). Upon transfection, the replicon RNA replicated in various cell lines, and was sensitive to interferon alpha (IFN-), remdesivir, but was resistant to hepatitis C virus inhibitors daclatasvir and sofosbuvir. Replication of the replicon was also sensitive overexpression of zinc-finger antiviral protein (ZAP). We also constructed a four-plasmid in-vitro ligation system that is compatible with the BAC system, which makes it easy to introduce desired mutations into the assembly plasmids for in-vitro ligation. This replicon system would be helpful for performing antiviral screening and dissecting virus-host interactions.

Determinants of SARS-CoV-2 receptor gene expression in upper and lower airways (preprint)

The recent outbreak of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has led to a worldwide pandemic. One week after initial symptoms develop, a subset of patients progresses to severe disease, with high mortality and limited treatment options. To design novel interventions aimed at preventing spread of the virus and reducing progression to severe disease, detailed knowledge of the cell types and regulating factors driving cellular entry is urgently needed. Here we assess the expression patterns in genes required for COVID-19 entry into cells and replication, and their regulation by genetic, epigenetic and environmental factors, throughout the respiratory tract using samples collected from the upper (nasal) and lower airways (bronchi). Matched samples from the upper and lower airways show a clear increased expression of these genes in the nose compared to the bronchi and parenchyma. Cellular deconvolution indicates a clear association of these genes with the proportion of secretory epithelial cells. Smoking status was found to increase the majority of COVID-19 related genes including ACE2 and TMPRSS2 but only in the lower airways, which was associated with a significant increase in the predicted proportion of goblet cells in bronchial samples of current smokers. Both acute and second hand smoke were found to increase ACE2 expression in the bronchus. Inhaled corticosteroids decrease ACE2 expression in the lower airways. No significant effect of genetics on ACE2 expression was observed, but a strong association of DNA- methylation with ACE2 and TMPRSS2- mRNA expression was identified in the bronchus.

Prime-boost protein subunit vaccines against SARS-CoV-2 are highly immunogenic in mice and macaques (preprint)

SARS-CoV-2 vaccines are advancing into human clinical trials, with emphasis on eliciting high titres of neutralising antibodies against the viral spike (S). However, the merits of broadly targeting S versus focusing antibody onto the smaller receptor binding domain (RBD) are unclear. Here we assessed prototypic S and RBD subunit vaccines in homologous or heterologous prime-boost regimens in mice and non-human primates. We find S is highly immunogenic in mice, while the comparatively poor immunogenicity of RBD was associated with limiting germinal centre and T follicular helper cell activity. Boosting S-primed mice with either S or RBD significantly augmented neutralising titres, with RBD-focussing driving moderate improvement in serum neutralisation. In contrast, both S and RBD vaccines were comparably immunogenic in macaques, eliciting serological neutralising activity that generally exceed levels in convalescent humans. These studies confirm recombinant S proteins as promising vaccine candidates and highlight multiple pathways to achieving potent serological neutralisation.

Causal associations between COVID-19 and Atrial Fibrillation: A bidirectional Mendelian randomization study (preprint)

Abstract Background Observational studies showed that coronavirus disease 2019 (COVID-19) attacks universally and its most menacing progression uniquely endangers the elderly with cardiovascular disease (CVD). Whether COVID-19 is causally related to increasing susceptibility and severity of atrial fibrillation (AF), the main form of CVD, remains still unknown. Methods The study aims to investigate the bidirectional causal relations of COVID-19 with AF using two-sample Mendelian randomization (MR) analysis. Results MR evidence suggested genetically predicted severe COVID-19 was significantly associated with higher risk of AF (odds ratio [OR], 1.041; 95% confidence interval (CI), 1.007-1.076; P = 0.017), while genetically predicted AF was not causally associated with severe COVID-19 (OR, 0.831; 95% CI, 1.619-1.115; P=0.217). There was limited evidence to support association of genetically proxied COVID-19 with risk of AF (OR, 1.051; 95% CI, 0.991-1.114; P=0.097), and vice versa (OR, 0.163; 95% CI, 0.004-6.790; P=0.341). MR-Egger indicated no evidence of pleiotropic bias. Conclusion Overall, severe COVID-19 may causally affect AF through independent biological pathway. Survivors from severe COVID-19 might be of high risk of AF in the future. Key words Coronavirus disease 2019; Atrial Fibrillation; Bidirectional mendelian randomization

Association between Alzheimer's disease and COVID-19: A bidirectional Mendelian randomization (preprint)

Background In observational studies, Alzheimer's disease (AD) has been associated with an increased risk of Coronavirus disease 2019 (COVID-19), and the prognosis of COVID-19 can affect nervous systems. However, the causality between these conditions remains to be determined. Methods This study sought to investigate the bidirectional causal relations of AD with COVID-19 using two-sample Mendelian randomization (MR) analysis. Results We found that genetically predicted AD was significantly associated with higher risk of severe COVID-19 (odds ratio [OR], 3.329; 95% confidence interval [CI], 1.139-9.725; P=0.028). It's interesting that genetically predicted severe COVID-19 was also significantly associated with higher risk of AD (OR, 1.004; 95% CI, 1.001-1.007; P=0.018). In addition, the two strong genetic variants associated with severe COVID-19 was associated with higher AD risk (OR, 1.018; 95% CI, 1.003-1.034; P=0.018). There is no evidence to support that genetically predicted AD was significantly associated with COVID-19 susceptibility, and vice versa. No obvious pleiotropy bias and heterogeneity were observed. Conclusion Overall, AD may causally affect severe COVID-19, and vice versa, performing bidirectional regulation through independent biological pathways.

Core Outcome Set for Traditional Chinese and Western Medicine Clinical Trials of COVID-19 (preprint)

Background: Development of a core outcome set (COS) for clinical trials for COVID-19 is urgent because of the pandemic wreaking havoc worldwide and the heterogeneity of outcomes in clinical trials. Methods: A preliminary list of outcomes were developed after a systematic review of protocols of clinical trials for COVID-19. Then, two rounds of the Delphi survey were conducted. Stakeholders were traditional Chinese medicine (TCM) experts, Western medicine (WM) experts, nurses and the public. Patients with confirmed COVID-19 were also invited to participate in a questionnaire written in understandable language. Frontline clinicians, as well as nurse, methodologist, evidence based-medicine researcher, and staff from the Chinese Clinical Trials Registry participated by video conference to vote. Results: Ninety-seven eligible study protocols were identified from 160 clinical trials. Seventy-six outcomes were identified from TCM clinical trials and 126 outcomes were identified from WM clinical trials. Finally, 145 outcomes were included in the first round of the Delphi survey. Then, a COS for clinical trials of TCM and WM was developed. The COS include clinical outcomes (recovery/improvement/progression/death), etiology (SARS-CoV-2 nucleic-acid tests, viral load), inflammatory factor (C-reactive protein), vital signs (temperature, respiration), blood and lymphatic-system parameters (lymphocytes, virus antibody), respiratory outcomes (Pulmonary imaging, blood oxygen saturation, PaO2/FiO2 ratio, arterial blood gas analysis, mechanical ventilation, oxygen intake, pneumonia severity index), clinical efficacy (prevalence of preventing patients with mild-to-moderate disease progressing to severe disease), symptoms (clinical symptom score). Outcomes were recommended according to different types of disease. Outcome measurement instrument/definition were also recommended. Conclusion: A COS for COVID-19 may improve consistency of outcome reporting in clinical trials.

Clinical Characteristics of SARS-CoV-2 Infections Involving 325 Hospitalized Patients outside Wuhan (preprint)

Background/Objective To date, the clinical features of SARS-CoV-2 infections were reported mainly based on cases in Wuhan. We aimed to report the clinical features of SARS-CoV-2 infections outside Wuhan. Methods We analyzed 325 SARS-COV-2 infection patients hospitalized in Shanghai Public Health Clinical Center. The epidemiological, demographic, and clinical data were compared between severe and non-severe cases. Results Of 325 patients, the median age was 51 years, 167 (51.4%) were men, and 107 (32.9%) had underlying diseases. 159 (48.9%) visited Wuhan or had contacted with people from Wuhan, but 57 (17.5%) had no clear epidemiological history. Compared with non-severe patients (n=299, 92%), severe patients (n=26, 8%) were older, had more common underlying disorder, more common lymphopenia, and higher D-dimer, creatine kinase, lactate dehydrogenase, aspartate aminotransferase, total bilirubin, blood urea nitrogen, creatinine, procalcitonin, C-reactive-protein, and troponin I level. The common complications included secondary infection (12.3%), acute cardiac injury (9.2%), ARDS (5.5%), acute kidney injury (5.8%), and shock (4.9%). To Mar 12, 311 (95.7%) patients were discharged, 3 (0.9%) died, and 11 (3.4%) still hospitalized. Conclusions The severity rate and fatality rate were low if the measures (early isolation, early diagnosis and early management) were undertaken at the early time of the outbreak.

A simple algorithm helps early identification of SARS-CoV-2 infection patients with severe progression tendency (preprint)

Objectives We aimed to develop a simple algorithm helps early identification of SARS-CoV-2 infection patients with severe progression tendency. Methods 322 SARS-COV-2 infection patients were respectively enrolled. The univariable and multivariable analysis were computed to identify the independent predictors of severe progression, and the prediction model was established based on independent predictors. The areas under the ROC curves (AUROCs) were used to evaluate the diagnostic performances. Results Of 322 confirmed SARS-COV-2 infection patients, 11 were diagnosed as severe cases on admission, 15 developed to severe cases after admission, and 296 were non-severe cases. The multivariable analysis identified age (OR=1.061, p=0.028), lactate dehydrogenase (LDH) (OR=1.006, p=0.037), and CD4 count (OR=0.993, p=0.006) as the independent predictors of severe progression. Consequently, the age-LDH-CD4 algorithm was derived as (age×LDH)/CD4. The AUROC of the age-LDH-CD4 model was significantly higher than that of single CD4 count, LDH, or age (0.92, 0.85, 0.80, and 0.75, respectively). The age-LDH-CD4 model ≥ 82 has high sensitive (81%) and specific (93%) for the early identification of patients with severe progression tendency following SARS-CoV-2 infection. Conclusions The age-LDH-CD4 model is a simple algorithm for early identifying cases with severe progression tendency in SARS-CoV-2 infection patients, and warrants further validation.