Progress in the role of ubiquitin-proteasome system in hepatitis B virus X protein-mediated HBV infection and hepatocarcinogenesis / 中华微生物学和免疫学杂志

Chronic hepatitis B virus (HBV) infection is one of the leading causes of hepatocellular carcinoma (HCC). Ubiquitin-proteasome system (UPS) mediates the post-translational modification and degradation of protein in eukaryotic cells. Recent studies have revealed that UPS plays an important role in HBV infection and hepatocarcinogenesis. Targeting HBx to intervene in the progression of HBV infection or HBV-related HCC has become a research hotspot both domestically and internationally in recent years. This article reviewed the progress in HBx promoting HBV infection and hepatocarcinogenesis through UPS.

A cocktail containing two synergetic antibodies broadly neutralizes SARS-CoV-2 and its variants including Omicron BA.1 and BA.2

Neutralizing antibodies (NAbs) can prevent and treat infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, continuously emerging variants, such as Omicron, have significantly reduced the potency of most known NAbs. The selection of NAbs with broad neutralizing activities and the identification of conserved critical epitopes are still urgently needed. Here, we identified an extremely potent antibody (55A8) by single B-cell sorting from convalescent SARS-CoV-2-infected patients that recognized the receptor-binding domain (RBD) in the SARS-CoV-2 spike (S) protein. 55A8 could bind to wild-type SARS-CoV-2, Omicron BA.1 and Omicron BA.2 simultaneously with 58G6, a NAb previously identified by our group. Importantly, an antibody cocktail containing 55A8 and 58G6 (2-cocktail) showed synergetic neutralizing activity with a half-maximal inhibitory concentration (IC50) in the picomolar range in vitro and prophylactic efficacy in hamsters challenged with Omicron (BA.1) through intranasal delivery at an extraordinarily low dosage (25 g of each antibody daily) at 3 days post-infection. Structural analysis by cryo-electron microscopy (cryo-EM) revealed that 55A8 is a Class III NAb that recognizes a highly conserved epitope. It could block angiotensin-converting enzyme 2 (ACE2) binding to the RBD in the S protein trimer via steric hindrance. The epitopes in the RBD recognized by 55A8 and 58G6 were found to be different and complementary, which could explain the synergetic mechanism of these two NAbs. Our findings not only provide a potential antibody cocktail for clinical use against infection with current SARS-CoV-2 strains and future variants but also identify critical epitope information for the development of better antiviral agents.

Extensive neutralization against SARS-CoV-2 variants elicited by Omicron-specific subunit vaccine booster

The currently dominant variant of SARS-CoV-2 Omicron, carrying a great number of mutations, has been verified its strong capacity of immune escape in COVID-19 convalescents and vaccinated individuals. An increased risk of SARS-CoV-2 reinfection or breakthrough infection should be concerned. Here we reported higher humoral immune response elicited by Delta and Omicron variants after breaking through previous infection and cross-neutralization against VOCs, compared to the ancestral wild-type (WT) virus infection. To overcome the immune escape of Omicron, Omicron-specific vaccine was considered as a novel and potential strategy. Mouse models were used to verify whether Omicron-specific RBD subunit boost immune response by immunizing Omicron-RBD recombinant proteins. Three doses of Omicron-RBD immunization elicit comparable neutralizing antibody (NAb) titers with three doses of WT-RBD immunization, but the neutralizing activity was not cross-active. By contrast, two doses of WT-RBD with an Omicron-RBD booster increased the NAb geometric mean titers against Omicron by 9 folds. Moreover, an additional boost vaccination with Omicron-RBD protein could increase humoral immune response against both WT and current VOCs. These results suggest that the Omicron-specific subunit booster shows its advantages in the immune protection from both WT and current VOCs, and that SARS-CoV-2 vaccines administration using two or more virus lineages as antigens might improve the NAb response.

Glucose-6 phosphatase catalytic subunit inhibits the proliferation of liver cancer cells by inducing cell cycle arrest / 中华肝脏病杂志

Objective: To investigate the effects of glucose-6-phosphatase catalytic subunit (G6PC) recombinant adenovirus on proliferation and cell cycle regulation of liver cancer cells. Methods: Recombinant adenovirus AdG6PC was constructed. Huh7 cells and SK-Hep1 cells were set as Mock, AdGFP and AdG6PC group. Cell proliferation and clone formation assay were used to observe the proliferation of liver cancer cells. Transwell and scratch assay were used to observe the invasion and migration of liver cancer cells. Cell cycle flow cytometry assay was used to analyze the effect of G6PC overexpression on the proliferation cycle of liver cancer cells. Western blot was used to detect the effect of G6PC overexpression on the cell-cycle protein expression in liver cancer cells. Results: The recombinant adenovirus AdG6PC was successfully constructed. Huh7 and SK-Hep1 cells proliferation assay showed that the number of proliferating cells in the AdG6PC group was significantly lower than the other two groups (P < 0.05). Clone formation assay showed that the number of clones was significantly lower in AdG6PC than the other two groups (P < 0.05), suggesting that G6PC overexpression could significantly inhibit the proliferation of liver cancer cells. Transwell assay showed that the number of cell migration was significantly lower in AdG6PC than the other two groups (P < 0.05). Scratch repair rate was significantly lower in AdG6PC than the other two groups (P < 0.05), suggesting that G6PC overexpression can significantly inhibit the invasion and migration of liver cancer cells. Cell cycle flow cytometry showed that G6PC overexpression had significantly inhibited the Huh7 cells G(1)/S phase transition. Western blot result showed that G6PC overexpression had down-regulated the proliferation in cell-cycle related proteins expression. Conclusion: G6PC inhibits the proliferation, cell-cycle related expression, and migration of liver cancer cells by inhibiting the G(1)/S phase transition.

Comparison of postoperative mid-term and long-term quality of life between Billroth-I gastroduodenostomy and Billroth-II gastrojejunostomy after radical distal gastrectomy in patients with gastric cancer: a cohort study based on a case registry database / 中华胃肠外科杂志

Objective: The pattern of digestive tract reconstruction in radical gastrectomy for gastric cancer is still inconclusive. This study aims to compare mid-term and long-term quality of life after radical gastrectomy for distal gastric cancer between Billroth-I (B-I) and Billroth-II (B-II) reconstruction. Methods: A retrospective cohort study was conducted.Clinicopathological and follow-up data of 859 gastric cancer patients were colected cellected from the surgical case registry database of Gastrointestinal Surgery Center of Sichuan University West China Hospital, who underwent radical distal gastric cancer resection between January 2016 and December 2020. Inclusion criteria: (1) gastric cancer confirmed by preoperative gastroscopy and biopsy; (2) elective radical distal major gastrectomy performed according to the Japanese Society for Gastric Cancer treatment guidelines for gastric cancer; (3) TNM staging referenced to the American Cancer Society 8th edition criteria and exclusion of patients with stage IV by postoperative pathology; (4) combined organ resection only involving the gallbladder or appendix; (5) gastrointestinal tract reconstruction modality of B-I or B-II; (6) complete clinicopathological data; (7) survivor during the last follow-up period from December 15, 2021 to January 15, 2022. Exclusion criteria: (1) poor compliance to follow-up; (2) incomplete information on questionnaire evaluation; (3) survivors with tumors; (4) concurrent malignancies in other systems; (5) concurrent psychiatric and neurological disorders that seriously affected the objectivity of the questionnaire or interfered with patient's cognition. Telephone follow-up was conducted by a single investigator from December 2021 to January 2022, and the standardized questionnaire EORTC QLQ-C30 scale (symptom domains, functional domains and general health status) and EORTC QLQ-STO22 scale (5 symptoms of dysphagia, pain, reflux, restricted eating, anxiety; 4 single items of dry mouth, taste, body image, hair loss) were applied to evaluate postoperative quality of life. In 859 patients, 271 were females and 588 were males; the median age was 57.0 (49.5, 66.0) years. The included cases were divided into the postoperative follow-up first year group (202 cases), the second year group (236 cases), the third year group (148 cases), the fourth year group (129 cases) and the fifth year group (144 cases) according to the number of years of postoperative follow-up. Each group was then divided into B-I reconstruction group and B-II reconstruction group according to procedure of digestive tract reconstruction. Except for T-stage in the fourth year group, and age, tumor T-stage and tumor TNM-stage in the fifth year group, whose differences were statistically significant between the B-I and B-II reconstruction groups (all P<0.05), the differences between the B-I and B-II reconstruction groups in terms of demographics, body mass index (BMI), tumor TNM-stage and tumor pathological grading in postoperative follow-up each year group were not statistically significant (all P>0.05), suggesting that the baseline information between B-I reconstruction group and the B-II reconstruction group in postoperative each year group was comparable. Evaluation indicators of quality of life (EORTC QLQ-C30 and EORTC QLQ-STO22 scales) and nutrition-related laboratory tests (serum hemoglobin, albumin, total protein, triglycerides) between the B-I reconstruction group and B-II reconstruction group in each year group were compared. Non-normally distributed continuous variables were presented as median (Q(1),Q(3)), and compared by using the Wilcoxon rank sum test (paired=False). The χ(2) test or Fisher's exact test was used for comparison of categorical variables between groups. Results: There were no statistically significant differences in all indexes EORTC QLQ-30 scale between the B-I reconstruction group and the B-II reconstruction group among all postoperative follow-up year groups (all P>0.05). The EORTC QLQ-STO22 scale showed that significant differences in pain and eating scores between the B-I reconstruction group and the B-II reconstruction group were found in the second year group, and significant differences in eating, body and hair loss scores between the B-I reconstruction group and the B-II reconstruction group were found in the third year group (all P<0.05), while no significant differences of other item scores between the B-I reconstruction group and the B-II reconstruction group were found in postoperative follow-up of all year groups (P>0.05). Triglyceride level was higher in the B-II reconstruction group than that in the B-I reconstruction group (W=2 060.5, P=0.038), and the proportion of patients with hyperlipidemia (triglycerides >1.85 mmol/L) was also higher in the B-II reconstruction group (19/168, 11.3%) than that in the B-I reconstruction group (0/34) (χ(2)=0.047, P=0.030) in the first year group with significant difference. Albumin level was lower in the B-II reconstruction group than that in the B-I reconstruction group (W=482.5, P=0.036), and the proportion of patients with hypoproteinemia (albumin <40 g/L) was also higher in the B-II reconstruction group (19/125, 15.2%) than that in the B-I reconstruction group (0/19) in the fifth year group, but the difference was not statistically significant (χ(2)=0.341, P=0.164). Other nutrition-related clinical laboratory tests were not statistically different between the B-I reconstruction and the B-II reconstruction in each year group (all P>0.05). Conclusions: The effects of both B-I and B-II reconstruction methods on postoperative mid-term and long-term quality of life are comparable. The choice of reconstruction method after radical resection of distal gastric cancer can be based on a combination of patients' condition, sugenos' eoperience and operational convenience.

Reduced infectivity but increased immune escape of the new SARS-CoV-2 variant of concern Omicron

A new detected SARS-CoV-2 variant Omicron (B.1.1.529) had reported from more than 80 countries. In the past few weeks, a new wave of infection driven by Omicron is in progress. Omicron Spike (S) protein pseudotyped virus was used to determine the effect of S mutations on its capacity of infectivity and immune evasion. Our results showed the lower entry efficiency and less cleavage ability of Omicron than D614G variant. Pseudotype-based neutralizing assay was performed to analyze neutralizing antibodies elicited by previously infection or the RBD-based protein subunit vaccine ZF2001 against the Omicron variant. Sera sampled at around one month after symptom onset from 12 convalescents who were previously infected by SARS-CoV-2 original strain shows a more than 20-fold decrease of neutralizing activity against Omicron variant, when compared to D614G variant. Among 12 individuals vaccinated by RBD subunit vaccine, 58.3% (7/12) sera sampled at 15-60 days after 3rd-dose vaccination did not neutralize Omicron. Geometric mean titers (GMTs, 50% inhibitory dose [ID50]) of these sera against Omicron were 9.4-fold lower than against D614G. These results suggested a higher risk of Omicron breakthrough infections and reduced efficiency of the protective immunity elicited by existing vaccines. There are important implications about the modification and optimization of the current epidemic prevention and control including vaccine strategies and therapeutic antibodies against Omicron variant.

Reduced neutralization of SARS-CoV-2 B.1.617 variant by inactivated and RBD-subunit vaccine

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The Spike protein that mediates coronavirus entry into host cells is a major target for COVID-19 vaccines and antibody therapeutics. However, multiple variants of SARS-CoV-2 have emerged, which may potentially compromise vaccine effectiveness. Using a pseudovirus-based assay, we evaluated SARS-CoV-2 cell entry mediated by the viral Spike B.1.617 and B.1.1.7 variants. We also compared the neutralization ability of monoclonal antibodies from convalescent sera and neutralizing antibodies (NAbs) elicited by CoronaVac (inactivated vaccine) and ZF2001 (RBD-subunit vaccine) against B.1.617 and B.1.1.7 variants. Our results showed that, compared to D614G and B.1.1.7 variants, B.1.617 shows enhanced viral entry and membrane fusion, as well as more resistant to antibody neutralization. These findings have important implications for understanding viral infectivity and for immunization policy against SARS-CoV-2 variants.

Ultrapotent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus displayed remarkable efficacy against authentic B.1.351 virus. Each of these 3 mAbs in combination with one neutralizing Ab recognizing non-competing epitope exhibited synergistic effect against authentic SARS-CoV-2 virus. Surprisingly, structural analysis revealed that 58G6 and 13G9, encoded by the IGHV1-58 and the IGKV3-20 germline genes, both recognized the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly bound to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrated prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. These 2 ultrapotent neutralizing Abs can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.

Emerging SARS-CoV-2 variants reduce neutralization sensitivity to convalescent sera and monoclonal antibodies

SARS-CoV-2 Spike-specific antibodies contribute the majority of the neutralizing activity in most convalescent human sera. Two SARS-CoV-2 variants, N501Y.V1 (also known as B.1.1.7 lineage or VOC-202012/01) and N501Y.V2 (B.1.351 lineage), reported from the United Kingdom and South Africa, contain several mutations in the receptor binding domain of Spike and are of particular concern. To address the infectivity and neutralization escape phenotypes potentially caused by these mutations, we used SARS-CoV-2 pseudovirus system to compare the viral infectivity, as well as the neutralization activities of convalescent sera and monoclonal antibodies (mAbs) against SARS-CoV-2 variants. Our results showed that N501Y Variant 1 and Variant 2 increase viral infectivity compared to the reference strain (wild-type, WT) in vitro. At 8 months after symptom onset, 17 serum samples of 20 participants (85%) retaining titers of ID50 >40 against WT pseudovirus, whereas the NAb titers of 8 samples (40%) and 18 samples (90%) decreased below the threshold against N501Y.V1 and N501Y.V2, respectively. In addition, both N501Y Variant 1 and Variant 2 reduced neutralization sensitivity to most (6/8) mAbs tested, while N501Y.V2 even abrogated neutralizing activity of two mAbs. Taken together the results suggest that N501Y.V1 and N501Y.V2 reduce neutralization sensitivity to some convalescent sera and mAbs.

Identification of bis-benzylisoquinoline alkaloids as SARS-CoV-2 entry inhibitors from a library of natural products in vitro

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major public health issue. To screen for antiviral drugs for COVID-19 treatment, we constructed a SARS-CoV-2 spike (S) pseudovirus system using an HIV-1-based lentiviral vector with a luciferase reporter gene to screen 188 small potential antiviral compounds. Using this system, we identified nine compounds, specifically, bis-benzylisoquinoline alkaloids, that potently inhibited SARS-CoV-2 pseudovirus entry, with EC50 values of 0.1-10 M. Mechanistic studies showed that these compounds, reported as calcium channel blockers (CCBs), inhibited Ca2+-mediated membrane fusion and consequently suppressed coronavirus entry. These candidate drugs showed broad-spectrum efficacy against the entry of several coronavirus pseudotypes (SARS-CoV, MERS-CoV, SARS-CoV-2 [S-D614, S-G614, N501Y.V1 and N501Y.V2]) in different cell lines (293T, Calu-3, and A549). Antiviral tests using native SARS-CoV-2 in Vero E6 cells confirmed that four of the drugs (SC9/cepharanthine, SC161/hernandezine, SC171, and SC185/neferine) reduced cytopathic effect and supernatant viral RNA load. Among them, cepharanthine showed the strongest anti-SARS-CoV-2 activity. Collectively, this study offers new lead compounds for coronavirus antiviral drug discovery.

Changes of Humoral Immunity Response in SARS-CoV-2 Convalescent Patients over 8 months

Many countries around the world have all seen a sharp rise in COVID-19 cases as the second wave since the beginning of October 2020. Decline of antibodies response to severe acute respiratory syndrome coronavirus (SARS-CoV-2) that was reported exclusively in the early month increases the risk of reinfection for convalescent individuals. There is a current need to follow the maintenance of special antibodies against SARS-CoV-2. Here, we reported changes of antibodies against SARS-CoV-2 in convalescent patients over 8 months. Antibodies of all 20 participants targeting SARS-CoV-2 spike receptor binding-domain (RBD) had decreased from a mean OD450 value 1.78 to 0.38 over 8 months. The neutralizing antibody (NAb) titers decreased from the mean ID50 value 836 to 170. The NAb titers were significantly correlated with IgG level during 8 months (P<0.001). Furthermore, while RBD-specific IgG existence of 25% (5/20) convalescent plasma was undetectable, the NAb titers of 15% (3/20) convalescent plasma decreased below the threshold. In addition, compared to wild-type SARS-CoV-2 (S-D614), lower titers of neutralizing antibodies against its G614 variant were shown at 8 months after symptom onset. This study has important implications when considering antibody protection against SARS-CoV-2 reinfection.

A key linear epitope for a potent neutralizing antibody to SARS-CoV-2 S-RBD

The spread of SARS-CoV-2 confers a serious threat to the public health without effective intervention strategies1-3. Its variant carrying mutated Spike (S) protein D614G (SD614G) has become the most prevalent form in the current global pandemic4,5. We have identified a large panel of potential neutralizing antibodies (NAbs) targeting the receptor-binding domain (RBD) of SARS-CoV-2 S6. Here, we focused on the top 20 potential NAbs for the mechanism study. Of them, the top 4 NAbs could individually neutralize both authentic SARS-CoV-2 and SD614G pseudovirus efficiently. Our epitope mapping revealed that 16/20 potent NAbs overlapped the same steric epitope. Excitingly, we found that one of these potent NAbs (58G6) exclusively bound to a linear epitope on S-RBD (termed as 58G6e), and the interaction of 58G6e and the recombinant ACE2 could be blocked by 58G6. We confirmed that 58G6e represented a key site of vulnerability on S-RBD and it could positively react with COVID-19 convalescent patients plasma. We are the first, as far as we know, to provide direct evidences of a linear epitope that can be recognized by a potent NAb against SARS-CoV-2 S-RBD. This study paves the way for the applications of these NAbs and the potential safe and effective vaccine design.

A rapid and efficient screening system for neutralizing antibodies and its application for the discovery of potent neutralizing antibodies to SARS-CoV-2 S-RBD

Neutralizing antibodies (Abs) have been considered as promising therapeutics for the prevention and treatment of pathogens. After the outbreak of COVID-19, potent neutralizing Abs to SARS-CoV-2 were promptly developed, and a few of those neutralizing Abs are being tested in clinical studies. However, there were few methodologies detailly reported on how to rapidly and efficiently generate neutralizing Abs of interest. Here, we present a strategically optimized method for precisive screening of neutralizing monoclonal antibodies (mAbs), which enabled us to identify SARS-CoV-2 receptor-binding domain (RBD) specific Abs within 4 days, followed by another 2 days for neutralization activity evaluation. By applying the screening system, we obtained 198 Abs against the RBD of SARS-CoV-2. Excitingly, we found that approximately 50% (96/198) of them were candidate neutralizing Abs in a preliminary screening of SARS-CoV-2 pseudovirus and 20 of these 96 neutralizing Abs were confirmed with high potency. Furthermore, 2 mAbs with the highest neutralizing potency were identified to block authentic SARS-CoV-2 with the half-maximal inhibitory concentration (IC50) at concentrations of 9.88 ng/ml and 11.13 ng/ml. In this report, we demonstrated that the optimized neutralizing Abs screening system is useful for the rapid and efficient discovery of potent neutralizing Abs against SARS-CoV-2. Our study provides a methodology for the generation of preventive and therapeutic antibody drugs for emerging infectious diseases.

Longitudinal dynamics of the neutralizing antibody response to SARS-CoV-2 infection

BackgroundCoronavirus disease 2019 (COVID-19) is a global pandemic with no licensed vaccine or specific antiviral agents for therapy. Little is known about the longitudinal dynamics of SARS-CoV-2-specific neutralizing antibodies (NAbs) in COVID-19 patients. MethodsBlood samples (n=173) were collected from 30 COVID-19 patients over a 3-month period after symptom onset and analyzed for SARS-CoV-2-specific NAbs, using the lentiviral pseudotype assay, coincident with the levels of IgG and proinflammatory cytokines. ResultsSARS-CoV-2-specific NAb titers were low for the first 7-10 d after symtom onset and increased after 2-3 weeks. The median peak time for NAbs was 33 d (IQR 24-59 d) after symptom onset. NAb titers in 93.3% (28/30) of the patients declined gradually over the 3-month study period, with a median decrease of 34.8% (IQR 19.6-42.4%). NAb titers increased over time in parallel with the rise in IgG antibody levels, correlating well at week 3 (r = 0.41, p < 0.05). The NAb titers also demonstrated a significant positive correlation with levels of plasma proinflammatory cytokines, including SCF, TRAIL, and M-CSF. ConclusionsThese data provide useful information regarding dynamic changes in NAbs in COVID-19 patients during the acute and convalescent phases.

Cytokine biomarkers of COVID-19

We used a new strategy to screen cytokines associated with SARS-CoV-2 infection. Cytokines that can classify populations in different states of SARS-CoV-2 infection were first screened in cross-sectional serum samples from 184 subjects by 2 statistical analyses. The resultant cytokines were then analyzed for their interrelationships and fluctuating features in sequential samples from 38 COVID-19 patients. Three cytokines, M-CSF, IL-8 and SCF, which were clustered into 3 different correlation groups and had relatively small fluctuations during SARS-CoV-2 infection, were selected for the construction of a multiclass classification model. This model discriminated healthy individuals and asymptomatic and nonsevere patients with accuracy of 77.4% but was not successful in classifying severe patients. Further searching led to a single cytokine, hepatocyte growth factor (HGF), which classified severe from nonsevere COVID-19 patients with a sensitivity of 84.6% and a specificity of 97.9% under a cutoff value of 1128 pg/ml. The level of this cytokine did not increase in nonsevere patients but was significantly elevated in severe patients. Considering its potent antiinflammatory function, we suggest that HGF might be a new candidate therapy for critical COVID-19. In addition, our new strategy provides not only a rational and effective way to focus on certain cytokine biomarkers for infectious diseases but also a new opportunity to probe the modulation of cytokines in the immune response.

The D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity and decreases neutralization sensitivity to individual convalescent sera

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike (S) protein that mediates SARS-CoV-2 entry into host cells is a major target for vaccines and therapeutics. Thus, insights into its sequence variations are key to understanding the infection and antigenicity of SARS-CoV-2. A dominant mutational variant at position 614 of the S protein (aspartate to glycine, D614G mutation) was observed in the SARS-CoV-2 genome sequence obtained from the Nextstrain database. Using a pseudovirus-based assay, we identified that S-D614 and S-G614 protein pseudotyped viruses share a common receptor, human angiotensin-converting enzyme 2 (ACE2), which could be blocked by recombinant ACE2 with the fused Fc region of human IgG1. However, S-D614 and S-G614 protein demonstrated functional differences. First, S-G614 protein could be cleaved by serine protease elastase-2 more efficiently. Second, S-G614 pseudovirus infected 293T-ACE2 cells significantly more efficiently than did the S-D614 pseudovirus, especially in the presence of elastase-2. Third, an elastase inhibitor approved for clinical use blocked elastase-enhanced S-G614 pseudovirus infection. Moreover, 93% (65/70) convalescent sera from patients with COVID-19 could neutralize both S-D614 and S-G614 pseudoviruses with comparable efficiencies, but about 7% (5/70) convalescent sera showed reduced neutralizing activity against the S-G614 pseudovirus. These findings have important implications for SARS-CoV-2 transmission and immune interventions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Antibody responses to SARS-CoV-2 in COVID-19 patients: the perspective application of serological tests in clinical practice

BackgroundWe aim to investigate the profile of acute antibody response in COVID-19 patients, and provide proposals for the usage of antibody test in clinical practice. MethodsA multi-center cross-section study (285 patients) and a single-center follow-up study (63 patients) were performed to investigate the feature of acute antibody response to SARS-CoV-2. A cohort of 52 COVID-19 suspects and 64 close contacts were enrolled to evaluate the potentiality of the antibody test. ResultsThe positive rate for IgG reached 100% around 20 days after symptoms onset. The median day of seroconversion for both lgG and IgM was 13 days after symptoms onset. Seroconversion of IgM occurred at the same time, or earlier, or later than that of IgG. IgG levels in 100% patients (19/19) entered a platform within 6 days after seroconversion. The criteria of IgG seroconversion and > 4-fold increase in the IgG titers in sequential samples together diagnosed 82.9% (34/41) of the patients. Antibody test aided to confirm 4 patients with COVID-19 from 52 suspects who failed to be confirmed by RT-PCR and 7 patients from 148 close contacts with negative RT-PCR. ConclusionIgM and IgG should be detected simultaneously at the early phase of infection. The serological diagnosis criterion of seroconversion or the >; 4-fold increase in the IgG titer is suitable for a majority of COVID-19 patients. Serologic test is helpful for the diagnosis of SARS-CoV-2 infection in suspects and close contacts.

A Peptide-based Magnetic Chemiluminescence Enzyme Immunoassay for Serological Diagnosis of Corona Virus Disease 2019 (COVID-19)

A respiratory illness has been spreading rapidly in China, since its outbreak in Wuhan city, Hubei province in December 2019. The illness was caused by a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical manifestations related to SARS-CoV-2 infection ranged from no symptom to fatal pneumonia. World Health Organization (WHO) named the diseases associated with SARS-CoV-2 infection as COVID-19. Real time RT-PCR is the only laboratory test available till now to confirm the infection. However, the accuracy of real time RT-PCR depends on many factors, including sampling location and of methods, quality of RNA extraction and training of operators etc.. Variations in these factors might significantly lower the sensitivity of the detection. We developed a peptide-based luminescent immunoassay to detect IgG and IgM. Cut-off value of this assay was determined by the detection of 200 healthy sera and 167 sera from patients infected with other pathogens than SARS-CoV-2. To evaluate the performance of this assay, we detected IgG and IgM in the 276 sera from confirmed patients. The positive rate of IgG and IgM were 71.4% (197/276) and 57.2% (158/276) respectively. By combining with real time RT-PCR detection, this assay might help to enhance the accuracy of diagnosis of SARS-CoV-2 infection.

Application of special acupoints for chronic gastritis in ancient literature of acupuncture and moxibustion / 中国针灸

The application of special acupoints for different primary symptoms of chronic gastritis in ancient literature of acupuncture and moxibustion was analyzed and summarized. With keywords, electronic and manual retrieval of ancient literature being performed to establish a database, the association rules were performed with SPSS Modeler 18. As a result, ① among all the 2243 items included, 109 items mentioned special acupoints (excluding those only mentioned crossing acupoint). The frequency of special acupoints was 2554 (71.7%), and the special acupoints on the spleen meridian, the stomach meridian, the conception vessel were frequently used. The frequency of five- point was highest, followed by front- points. ② The special acupoints for four main primary symptoms of chronic gastritis, "stomachache" "vomiting and regurgitation" "belching and acid regurgitation" and "epigastric fullness", included Zusanli (ST 36), Neiguan (PC 6), Zhongwan (CV 12), Gongsun (SP 4), Taibai (SP 3). In addition, the back- points were also selected to treat the primary symptoms other than "epigastric fullness". Zhangmen (LR 13) was added to treat "belching and acid regurgitation". The combination of Neiguan (PC 6) and Gongsun (SP 4) showed the strongest correlation; due to different primary symptoms, the combination had different emphasis.

Sirt6-induced autophagy restricted TREM-1-mediated pyroptosis in ox-LDL-treated endothelial cells: relevance to prognostication of patients with acute myocardial infarction.

Inflammation mediated by myeloid cells trigger receptors 1 (TREM-1) is important for atherosclerosis development, while sirtuin 6 (Sirt6) levels decrease in atheroscleoritc plaque. Here we demonstrate that oxidatively modified low density lipoprotein (ox-LDL)-treated endothelial cells (ECs) exhibited increased TREM-1-mediated pyroptosis and decreased Sirt6-induced autophagy. We show that high sTREM-1 and low sSirt6 levels were independent predictors of boosted endothelial microparticles (EMPs) on admission, and were associated with increased risk for all-cause mortality and major adverse cardiovascular events (MACE) at median 24 months (interquartile range, 18-26) follow-up in acute myocardial infarction (AMI) patients. Additionally, blockage of Sirt6-induced autophagy led to augmented TREM-1-mediated pyroptosis, whereas Sirt6 overexpression attenuated ECs inflammation and pyroptosis following ox-LDL treatment. Our findings indicate that TREM-1 and in a reversed trend Sirt6 appeared to be markers of endothelial inflammation with potential for use in risk stratification.