Nanobodies against SARS-CoV-2 RBD from a Two-step Phage Screening of Universal and Focused Synthetic Libraries (preprint)

Coronavirus disease 2019 (COVID-19) is an evolving global pandemic, and nanobody (Nb) is recognized as a potential diagnostic and therapeutic tool for infectious disease. Here, we designed and synthesized a humanized and highly diverse phage Nbs library hsNb-U (Humanized synthetic Nbs Library - Universal). We expressed and purified the SARS-CoV-2 receptor-binding domain (RBD), and screened this univeral library against the RBD protein target. Then, the CDR1 and CDR2 sequences of five leads obtained from the hsNb-U phage panning were combined with randomly mutated CDR3 to construct a targeted (focused) phage display library, hsNb-RBD, for subsequent phage panning and screening. From the obtained sequences, we expressed 45 unique anti-RBD candidate Nbs. Among the selected Nbs, eight were found to be highly expressed, and five of these show high-affinity to RBD (EC50 less than 100nM). Finally, we found that Nb39 can compete with angiotensin converting enzyme 2 (ACE2) for binding to RBD. Overall, this two-step strategy of synthetic phage display libraries enables rapid selection of SARS-CoV-2 RBD nanobodies with potential therapeutic activity, and this two-step strategy can potentially be used for rapid discovery of Nbs against other targets.

An alternative way of SARS-COV-2 to induce cell stress and elevated DNA damage risk in cardiomyocytes without direct infection.

BACKGROUND: The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) in 2020 has led to millions of deaths worldwide. Case reports suggested that infection of SARS-CoV-2 is potentially associated with occurrences of cardiovascular pathology. However, the mode of action and mechanisms of SARS-CoV-2 influencing cardiomyocytes still remain largely unclear. AIMS: To explore the mechanisms underlying cardiomyocytes damage induced by SARS-CoV-2 infection. MATERIALS & METHODS: the serum markers of cardiovascular injury were analyzed by ELISA. The isolated SARS-CoV-2 virus were co-cultured with human cardiomyocytes (AC16) and immunofluorescence assay was used evaluate the invasion of virus. Moreover, serum obtained from acute stage of SARS-CoV-2 infected patients and healthy controls were used to incubate with AC16 cells, then indicators associated with cell stress and DNA damage were analyzed by Western-blot. RESULTS: we found that high-sensitivity troponin T (hsTnT), an indicator of cardiovascular disease, was higher in the acute stage of COVID-19. Additionally, in vitro coculture of SARS-CoV-2 and AC16 cells showed almost no infectious ability of SARS-CoV-2 to directly infect AC16 cells. Results of serum treatment suggested that serum from infected subjects induced cell stress (upregulation of p53 and HSP70) and elevation of DNA damage risk (increased γH2Ax and H3K79me2) in AC16. DISCUSSION: our observations indicated a hard way for SARS-CoV-2 to infect cardiomyocytes directly. However, infection-induced immune storm in serum could bring stress and elevated DNA damage risks to cardiovascular system. CONCLUSION: These findings indicated the possibilities of SARS-CoV-2 inducing stress and elevating DNA damage risk to cardiomyocytes without direct infection.

Myocardial Infarction and Azygos Vein Thrombosis After ChAdOx1 nCoV-19 Vaccination in a Hemodialysis Patient.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication after vaccination of Oxford-AstraZeneca coronavirus disease 2019 (COVID-19) vaccine (AZD1222) or Janssen COVID-19 vaccine. It makes a rare complication of thrombosis at common and/or uncommon organs with thrombocytopenia after COVID-19 vaccination four to 28 days later and most patients were younger than 60 years of age. We reported the case of a 75-year-old female with end-stage renal disease who received regular hemodialysis. She received Oxford-AstraZeneca COVID-19 vaccination eight days ago and then she suffered from intermittent chest tightness and epigastric pain with tarry stool passage for two days. Severe thrombocytopenia with elevated D-dimer value was noted and computed tomography of the chest showed azygos vein thrombosis. Elevated cardiac enzyme with ST-T change in 12-lead electrocardiogram was also noted. For positive anti-platelet factor 4 antibodies, VITT with myocardial infarction and azygos vein thrombosis was diagnosed.

The impact of COVID-19 on cardio-oncology care in Taiwan.

COVID-19 has not only affected the respiratory but the cardiovascular system. Taiwan has encountered a less severe COVID-19 pandemic. We reported the current situation in Taiwan. In this study, we retrospectively analyzed the data from our cardio-oncology program since October of 2019 to April of 2020 (the initial months of COVID-19 pandemic). In our cardio-oncology program, newly diagnosed breast cancer patients preparing for epirubicin therapy were included. Echocardiography, 6-min walking distance and major adverse cardiovascular events (MACEs) were recorded. To evaluate whether the social atmosphere affects cardio-oncology care, we analyzed the objective (physical) and subjective (emotional) parameters before and after January 21, 2020, when the first case of COVID-19 was confirmed in Taiwan. There was no significant decrease in patients' return ratio and LVEFs. However, there was a trend of subjective shortness of breath reported by the patients but no decline in 6 MWT. Notably, none of the enrolled patients reported MACEs during the COVID pandemic. We observed an impact of anxiety on patients receiving epirubicin but it did not influence their return ratio.

The Pivotal Role of SOD1 for Prediction of COVID-19 Progression Revealed by Proteomic and Bioinformatic Strategies (preprint)

Background: Severe acute respiratory distress syndrome-associated coronavirus-2 (SARS-CoV-2) is still threatening the whole human population worldwide. Patients infected with SARS-CoV-2 present diverse symptoms regarding to the severity of the disease. Determining the proteome changes associated with diverse symptoms and in different infection stages is beneficial for clinical diagnosis and management. Methods: We performed a TMT labeling proteomic study on the plasma of healthy controls and COVID-19 patients, including those with asymptomatic infection (NS), mild syndrome (MS), and severe syndrome in the early phase (SSEP) and later phase (SSLP). Bioinformatic and ELISA were used for the data analysis and identification. Findings: Hundreds of proteins were dysregulated in the plasma of COVID-19 patients including all the clinical symptoms. Bioinformatics analysis of the dysregulated proteins revealed that oxidative stress, complement activation and glycolysis-related proteins were affected after infection with SARS-CoV-2. ELISA analysis confirmed that SOD1, PRDX2 and LDHA levels were increased along with severe symptoms and did not change after recovery compared with those in healthy controls. Both GSEA and ROC analysis indicated that SOD1 could be a pivotal player in the progression of COVID-19. Interpretation: Our results indicated that plasma proteome changes differed based on symptoms and disease stages and SOD1 could be an important protein for indicating COVID-19 progression. These results may also provide new understanding for COVID-19 diagnosis and treatment. Funding: This project is supported by Shenzhen Bay Laboratory Opening Fund, Shenzhen Key Medical Discipline Construction Fund, Guangdong Natural Science Foundation, Sanming Project of Medicine in Shenzhen.Declaration of Interests: All the authors declared there are no conflicts of interest exist.Ethics Approval Statement: This research was approved by the ethics committee of the Shenzhen Center for Disease Control and Prevention [Approval number: 2020-025A].

SARS-CoV-2 manipulates the SR-B1-mediated HDL uptake pathway for its entry (preprint)

The recently emerged pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly, leading to a global COVID-19 pandemic. Binding of the viral spike protein (SARS-2-S) to cell surface receptor angiotensin-converting enzyme 2 (ACE2) mediates host cell infection. In the present study, we demonstrate that in addition to ACE2, the S1 subunit of SARS-2-S binds to HDL and that SARS-CoV-2 hijacks the SR-B1-mediated HDL uptake pathway to facilitate its entry. SR-B1 facilitates SARS-CoV-2 entry into permissive cells by augmenting virus attachment. MAb (monoclonal antibody)-mediated blocking of SARS-2-S-HDL binding and SR-B1 antagonists strongly inhibit HDL-enhanced SARS-CoV-2 infection. Notably, SR-B1 is co-expressed with ACE2 in human pulmonary and extrapulmonary tissues. These findings revealed a novel mechanism for SARS-CoV-2 entry and could provide a new target to treat SARS-CoV-2 infection.

Cholesterol Metabolism--Impact for SARS-CoV-2 Infection Prognosis, Entry, and Antiviral Therapies (preprint)

Abstract The recently emerged pathogenic SARS-coronavirus 2 (SARS-CoV-2) has spread rapidly, leading to a global pandemic. In this study, we show that SARS-CoV-2 infection was associated with clinically significant lower level of HDL cholesterol (HDL-C), which can be used as indicators of disease severity and poor prognosis. Importantly, we found the spike protein of SARS-CoV-2 (SARS-2-S) bound to HDL. Antagonists of HDL receptor-Scavenger receptor class B type I (SR-B1), strongly inhibited SARS-CoV-2 infection. Notably, the lipids transfer function of SR-B1 was indispensable for this inhibition, offering explanations for the reduced serum HDL level observed in COVID-19 patients. Basing on findings here, we speculate that SR-B1-mediated pulmonary HDL-vitamin E uptake could participate in mediating SARS-CoV-2 infection of lung cells, and the unique expression profile of SR-B1 may also affect SARS-CoV-2 cell and tissue tropism. These findings might help to provide further insights into viral transmission, pathological characteristics and reveal therapeutic targets.