Novel pectin from crude polysaccharide of Syzygium aromaticum against SARS-CoV-2 activities by targeting 3CLpro (preprint)

To date, COVID-19 is still a severe threat to public health, hence specific effective therapeutic drugs development against SARS-CoV-2 is urgent needed. 3CLpro and PLpro and RdRp are the enzymes required for the SARS-CoV-2 RNA synthesis. Therefore, binding to the enzyme may interfere the enzyme function. Before, we found that sulfated polysaccharide binding to 3CLpro might block the virus replication. Hence, we hypothesize that negative charged pectin glycan may also impede the virus replication. Here we show that 922 crude polysaccharide from Syzygium aromaticum may near completely block SARS-CoV-2 replication. The inhibition rate was 99.9% (EC50 : 0.90 muM). Interestingly, 922 can associates with 3CLpro, PLpro and RdRp. We further show that the homogeneous glycan 922211 from 922 may specifically attenuate 3CL protease activity. The IC50s of 922 and 922211 against 3CLpro are 4.73 plusmn 1.05 muM and 0.18 plusmn 0.01 muM, respectively. Monosaccharide composition analysis reveals that 922211 with molecular weight of 78.7 kDa is composed of rhamnose, galacturonic acid, galactose and arabinose in the molar ratio of 8.21 : 37.81 : 3.58 : 4.49. The structure characterization demonstrated that 922211 is a homogalacturonan linked to RG-I pectin polysaccharide. The linear homogalacturonan part in the backbone may be partly methyl esterified while RG-I type part bearing 1, 4-linked alpha-GalpA, 1, 4-linked alpha-GalpAOMe and 1, 2, 4-linked alpha-Rhap. There are four branches attached to C-1 or C4 position of Rhamnose glycosyl residues on the backbone. The branches are composed of 1, 3-linked beta-Galp, terminal (T)-linked beta-Galp, 1, 5-linked alpha-Araf, T-linked alpha-Araf, 4-linked alpha-GalpA and/or 4-linked beta-GalpA. The above results suggest that 922 and 922211 might be a potential novel leading compound for anti-SARS-CoV-2 new drug development.

Treatment of Six COVID-19 Patients with Convalescent Plasma (preprint)

Objective The global mortality toll of coronavirus disease 2019 (COVID-19) is rapidly increasing. Current antiviral therapy is insufficient, so it is crucial to develop new treatments. We assessed the efficacy of convalescent plasma transfusion for COVID-19 patients.Methods This retrospective, single-center study was conducted on six COVID-19 patients treated with convalescent plasma at Guizhou Provincial Jiangjunshan Hospital in China, from January 29 to April 30, 2020; the final follow-up data was collected on May 20, 2020. The efficacy of convalescent plasma was evaluated symptom relief and improvements in laboratory indicators and chest imaging abnormalities. Results Following treatment with convalescent plasma, the laboratory indicators and chest imaging examination of patients 1–3 changed from abnormal to normal. Regarding the relapsed patients (patients 4–6), two obtained negative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) real-time polymerase chain reaction (RT-PCR) test results on consecutive throat swabs after receiving convalescent plasma therapy (on days 24 and 3 for patients 4 and 6, respectively). Patient 5 received only one round of convalescent plasma transfusion; this patient’s laryngeal swab test results for SARS-CoV-2 have remained consistently positive so far.Conclusions The clinical conditions of six patients with COVID-19 improved following treatment with antiviral drugs and systemic corticosteroids combined with appropriate rounds of convalescent plasma therapy, indicating that infusion with convalescent plasma may be beneficial for patients with COVID-19.This study was registered in Chinese Clinical Trial Registry Center. (CCTR number: ChiCTR 2000033056, registered 19 May 2020)

Structural characterization of cocktail-like targeting polysaccharides from Ecklonia kurome Okam and their anti-SARS-CoV-2 activities in vitro (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the worldwide coronavirus disease 2019 (COVID-19) outbreak. Investigation has confirmed that polysaccharide heparan sulfate can bind to the spike protein and block SARS-CoV-2 infection. Theoretically, similar structure of nature polysaccharides may also have the impact on the virus. Indeed, some marine polysaccharide has been reported to inhibit SARS-Cov-2 infection in vitro, however the convinced targets and mechanism are still vague. By high throughput screening to target 3CLpro enzyme, a key enzyme that plays a pivotal role in the viral replication and transcription using nature polysaccharides library, we discover the mixture polysaccharide 375 from seaweed Ecklonia kurome Okam completely block 3Clpro enzymatic activity (IC50, 0.48 {micro}M). Further, the homogeneous polysaccharide 37502 from the 375 may bind to 3CLpro molecule well (kD value : 4.23 x 10-6). Very interestingly, 37502 also can potently disturb spike protein binding to ACE2 receptor (EC50, 2.01 {micro}M). Importantly, polysaccharide 375 shows good anti-SARS-CoV-2 infection activity in cell culture with EC50 values of 27 nM (99.9% inhibiting rate at the concentration of 20 {micro}g/mL), low toxicity (LD50: 136 mg/Kg on mice). By DEAE ion-exchange chromatography, 37501, 37502 and 37503 polysaccharides are purified from native 375. Bioactivity test show that 37501 and 37503 may impede SARS-Cov-2 infection and virus replication, however their individual impact on the virus is significantly less that of 375. Surprisingly, polysaccharide 37502 has no inhibition effect on SARS-Cov-2. The structure study based on monosaccharide composition, methylation, NMR spectrum analysis suggest that 375 contains guluronic acid, mannuronic acid, mannose, rhamnose, glucouronic acid, galacturonic acid, glucose, galactose, xylose and fucose with ratio of 1.86 : 9.56 : 6.81 : 1.69 : 1.00 : 1.75 : 1.19 : 11.06 : 4.31 : 23.06. However, polysaccharide 37502 is an aginate which composed of mannuronic acid (89.3 %) and guluronic acid (10.7 %), with the molecular weight (Mw) of 27.9 kDa. These results imply that mixture polysaccharides 375 works better than the individual polysaccharide on SARS-Cov-2 may be the cocktail-like polysaccharide synergistic function through targeting multiple key molecules implicated in the virus infection and replication. The results also suggest that 375 may be a potential drug candidate against SARS-CoV-2.

CAR-NK Cells Effectively Target the D614 and G614 SARS-CoV-2-infected Cells (preprint)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly contagious presenting a significant public health issue. Current therapies used to treat coronavirus disease 2019 (COVID-19) include monoclonal antibody cocktail, convalescent plasma, antivirals, immunomodulators, and anticoagulants, though the current therapeutic options remain limited and expensive. The vaccines from Pfizer and Moderna have recently been authorized for emergency use, which are invaluable for the prevention of SARS-CoV-2 infection. However, their long-term side effects are not yet to be documented, and populations with immunocompromised conditions (e.g., organ-transplantation and immunodeficient patients) may not be able to mount an effective immune response. In addition, there are concerns that wide-scale immunity to SARS-CoV-2 may introduce immune pressure that could select for escape mutants to the existing vaccines and monoclonal antibody therapies. Emerging evidence has shown that chimeric antigen receptor (CAR)- natural killer (NK) immunotherapy has potent antitumor response in hematologic cancers with minimal adverse effects in recent studies, however, the potentials of CAR-NK cells in preventing and treating severe cases of COVID-19 has not yet been fully exploited. Here, we improve upon a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2 and its D614G mutant. CAR-NK cells were generated using the scFv domain of S309 (henceforward, S309-CAR-NK), a SARS-CoV and SARS-CoV-2 neutralizing antibody that targets the highly conserved region of SARS-CoV-2 spike (S) glycoprotein, therefore would be more likely to recognize different variants of SARS-CoV-2 isolates. S309-CAR-NK cells can specifically bind to pseudotyped SARS-CoV-2 virus and its D614G mutant. Furthermore, S309-CAR-NK cells can specifically kill target cells expressing SARS-CoV-2 S protein in vitro and show superior killing activity and cytokine production, compared to that of the recently published CR3022-CAR-NK cells. Thus, these results pave the way for generating off-the-shelf S309-CAR-NK cells for treatment in high-risk individuals as well as provide an alternative strategy for patients unresponsive to current vaccines.

Enhanced Cholesterol-dependent Hemifusion by Internal Fusion Peptide 1 of SARS Coronavirus-2 Compared to its N-terminal Counterpart (preprint)

Membrane fusion is an important step for the entry of the lipid-sheathed viruses into the host cells. The fusion process is being carried out by fusion proteins present in the viral envelope. The class I viruses contains a 20-25 amino acid sequence at its N-terminal of the fusion domain, which is instrumental in fusion, and is termed as fusion peptide. However, Severe Acute Respiratory Syndrome Coronavirus (SARS) coronaviruses contain more than one fusion peptide sequences. We have shown that the internal fusion peptide 1 (IFP1) of SARS-CoV is far more efficient than its N-terminal counterpart (FP) to induce hemifusion between small unilamellar vesicles. Moreover, the ability of IFP1 to induce hemifusion formation increases dramatically with growing cholesterol content in the membrane. Interestingly, IFP1 is capable of inducing hemifusion, but fails to open pore.

Erythrocyte Sedimentation Rate in COVID-19 Infections (preprint)

Objectives To compare the clinical characteristics between the rapid cohort and the normal cohort of erythrocyte sedimentation rate (ESR) in COVID-19 infections, analyze the variables with significant differences, and explore the influencing factors of rapid ESR. Methods Selected a total of 80 patients with ESR detection during hospitalization were measured in 146 patients who received medical observation in concentrated isolation hospital in Guizhou province in China , collected and compared demographic information, epidemiological data, clinical symptoms, laboratory test data and CT image data during the observation between rapid cohort and normal group of ESR. Results By comparison, the proportion of male in the rapid cohort was higher than female. The average age was more than 35 years old, with a large age gap. The proportion of severe and critical patients was more than 26.53% (13/49). However, in the normal cohort the proportion of female was more than male, and the average age was about 8 years lower than the rapid cohort, and the age gap was smaller. The proportion of severe and critical patients was 12.90%, which was less than half of the rapid group. In the two groups, the proportion of clustered cases accounted for more than 50%, and the average number of patients in one family was more than 3. The most common clinical symptoms were cough, sputum, fever, sore throat and weakness of limbs. There were significant differences in ALT, {gamma}-GT and C-reactive protein between the rapid and normal cohort (P<0.05), but no statistically significant in other indicators. Hemoglobin and C-reactive protein have a significant effect on erythrocyte sedimentation rate. Conclusions In this study, we found that ESR is related to Hemoglobin and C-reactive protein.

Treatment of 6 COVID-19 Patients with Convalescent Plasma (preprint)

Objective To describe the efficacy of convalescent plasma transfusion for COVID-19 patients. Methods This is a retrospective study of 6 COVID-19 patients with convalescent plasma at Guizhou Provincial Jiangjunshan Hospital [boxh] a tertial hospital, in Guiyang, Guizhou, China, from January 29, to April 30, 2020; final data of follow-up was May 12, 2020. Through the review of the electronic medical records of Guizhou Jiangjunshan Hospital, clinical data of 6 patients were obtained. Three patients with worsening symptoms after empirical treatment with antivirals were transfused convalescent plasma therapy for the first treatment, while the other three severe or critical COVID-19 patients with rapid progression were transfused. The efficacy of convalescent plasma depends on the relief of symptoms, changes in laboratory indicators and chest imaging abnormalities. Results The PaO2 / FiO2 and lymphocyte count of patients 1, 2 and 3 treated with convalescent plasma treatment for the first treatment period were changed from abnormal to normal. The levels of inflammation markers CRP and IL-6 of the patients decreased significantly. Chest imaging examination showed that the lung lesions gradually subsided. The relapsed patients (No. 4 and No. 6), after using convalescent plasma therapy, turned negative on two consecutive throat swab tests on Day 24 and Day 3, respectively. Conclusions Convalescent plasma treatment of COVID-19 is beneficial for those patients with be difficult to turn to negative or re-positive RT-PCR.