Heparan sulfates from bat and human lung and their binding to the spike protein of SARS-CoV-2 virus

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has resulted in a pandemic and continues to spread at an unprecedented rate around the world. Although a vaccine has recently been approved, there are currently few effective therapeutics to fight its associated disease in humans, COVID-19. SARS-CoV-2 and the related severe acute respiratory syndrome (SARS-CoV-1), and Middle East respiratory syndrome (MERS-CoV) result from zoonotic respiratory viruses that have bats as the primary host and an as yet unknown secondary host. While each of these viruses has different protein-based cell-surface receptors, each rely on the glycosaminoglycan, heparan sulfate as a co-receptor. In this study we compare, for the first time, differences and similarities in the structure of heparan sulfate in human and bat lungs. Furthermore, we show that the spike glycoprotein of COVID-19 binds 3.5 times stronger to human lung heparan sulfate than bat lung heparan sulfate.

Interactions of heparin with key glycoproteins of human respiratory syncytial virus

Introduction: The unexpected surge of respiratory syncytial virus (RSV) cases following pandemic phase of COVID-19 has drawn much public attention. Drawing on the latest antiviral research, revisiting this heightened annual outbreak of respiratory disease could lead to new treatments. The ability of sulfated polysaccharides to compete for a variety of viruses binding to cell surface heparan sulfate, suggests several drugs that might have therapeutic potential for targeting RSV–glycosaminoglycan interactions. Methods: In the current study, the binding affinity and kinetics of two RSV glycoproteins (RSV-G protein and RSV-F protein) to heparin were investigated by surface plasmon resonance. Furthermore, solution competition studies using heparin oligosaccharides of different lengths indicated that the binding of RSV-G protein to heparin is size-dependent, whereas RSV-F protein did not show any chain length preference. Results and discussion: The two RSV glycoproteins have slightly different preferences for heparin sulfation patterns, but the N-sulfo group in heparin was most critical for the binding of heparin to both RSV-G protein and RSV-F protein. Finally, pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for their inhibition of the RSV-G protein and RSV-F protein–heparin interaction, and both highly negative compounds showed strong inhibition.

Suramin binds and inhibits infection of SARS-CoV-2 through both spike protein-heparan sulfate and ACE2 receptor interactions.

SARS-CoV-2 receptor binding domains (RBDs) interact with both the ACE2 receptor and heparan sulfate on the surface of host cells to enhance SARS-CoV-2 infection. We show that suramin, a polysulfated synthetic drug, binds to the ACE2 receptor and heparan sulfate binding sites on the RBDs of wild-type, Delta, and Omicron variants. Specifically, heparan sulfate and suramin had enhanced preferential binding for Omicron RBD, and suramin is most potent against the live SARS-CoV-2 Omicron variant (B.1.1.529) when compared to wild type and Delta (B.1.617.2) variants in vitro. These results suggest that inhibition of live virus infection occurs through dual SARS-CoV-2 targets of S-protein binding and previously reported RNA-dependent RNA polymerase inhibition and offers the possibility for this and other polysulfated molecules to be used as potential therapeutic and prophylactic options against COVID-19.

Safety and immunogenicity of a protein subunit COVID-19 vaccine (ZF2001) in healthy children and adolescents aged 3-17 years in China: a randomised, double-blind, placebo-controlled, phase 1 trial and an open-label, non-randomised, non-inferiority, phase 2 trial.

BACKGROUND: ZF2001 is a recombinant protein subunit vaccine against SARS-CoV-2 that has been approved for use in China, Colombia, Indonesia, and Uzbekistan in adults aged 18 years or older, but not yet in children and adolescents younger than 18 years. We aimed to evaluate the safety and immunogenicity of ZF2001 in children and adolescents aged 3-17 years in China. METHODS: The randomised, double-blind, placebo-controlled, phase 1 trial and the open-label, non-randomised, non-inferiority, phase 2 trial were done at the Xiangtan Center for Disease Control and Prevention (Hunan Province, China). Healthy children and adolescents aged 3-17 years, without a history of SARS-CoV-2 vaccination, without a history of COVID-19, without COVID-19 at the time of the study, and without contact with patients with confirmed or suspected COVID-19 were included in the phase 1 and phase 2 trials. In the phase 1 trial, participants were divided into three groups according to age (3-5 years, 6-11 years, and 12-17 years). Each group was randomly assigned (4:1), using block randomisation with five blocks, each with a block size of five, to receive three 25 µg doses of the vaccine, ZF2001, or placebo intramuscularly in the arm 30 days apart. The participants and investigators were masked to treatment allocation. In the phase 2 trial, participants received three 25 µg doses of ZF2001 30 days apart and remained stratified by age group. For phase 1, the primary endpoint was safety and the secondary endpoint was immunogenicity (humoral immune response on day 30 after the third vaccine dose: geometric mean titre [GMT] of prototype SARS-CoV-2 neutralising antibodies and seroconversion rate, and geometric mean concentration [GMC] of prototype SARS-CoV-2 receptor-binding domain [RBD]-binding IgG antibodies and seroconversion rate). For phase 2, the primary endpoint was the GMT of SARS-CoV-2 neutralising antibodies with seroconversion rate on day 14 after the third vaccine dose, and the secondary endpoints included the GMT of RBD-binding antibodies and seroconversion rate on day 14 after the third vaccine dose, the GMT of neutralising antibodies against the omicron BA.2 subvariant and seroconversion rate on day 14 after the third vaccine dose, and safety. Safety was analysed in participants who received at least one dose of the vaccine or placebo. Immunogenicity was analysed in the full-analysis set (ie, participants who received at least one dose and had antibody results) by intention to treat and in the per-protocol set (ie, participants who completed the whole vaccination course and had antibody results). Non-inferiority in the phase 2 trial (neutralising antibody titre of participants from this trial aged 3-17 years vs that of participants aged 18-59 years from a separate phase 3 trial) for clinical outcome assessment was based on the geometric mean ratio (GMR) and was considered met if the lower bound of the 95% CI for the GMR was 0·67 or greater. These trials are registered with ClinicalTrials.gov, NCT04961359 (phase 1) and NCT05109598 (phase 2). FINDINGS: Between July 10 and Sept 4, 2021, 75 children and adolescents were randomly assigned to receive ZF2001 (n=60) or placebo (n=15) in the phase 1 trial and were included in safety and immunogenicity analyses. Between Nov 5, 2021, and Feb 14, 2022, 400 participants (130 aged 3-7 years, 210 aged 6-11 years, and 60 aged 12-17 years) were included in the phase 2 trial and were included in the safety analysis; six participants were excluded from the immunogenicity analyses. 25 (42%) of 60 participants in the ZF2001 group and seven (47%) of 15 participants in the placebo group in phase 1, and 179 (45%) of 400 participants in phase 2, had adverse events within 30 days after the third vaccination, without a significant difference between groups in phase 1. Most adverse events were grade 1 or 2 (73 [97%] of 75 in the phase 1 trial, and 391 [98%] of 400 in the phase 2 trial). One participant in the phase 1 trial and three in the phase 2 trial who received ZF2001 had serious adverse events. One serious adverse event (acute allergic dermatitis) in the phase 2 trial was possibly related to the vaccine. In the phase 1 trial, on day 30 after the third dose, in the ZF2001 group, seroconversion of neutralising antibodies against SARS-CoV-2 was observed in 56 (93%; 95% CI 84-98) of 60 participants, with a GMT of 176·5 (95% CI 118·6-262·8), and seroconversion of RBD-binding antibodies was observed in all 60 (100%; 95% CI 94-100) participants, with a GMC of 47·7 IU/mL (95% CI 40·1-56·6). In the phase 2 trial, on day 14 after the third dose, seroconversion of neutralising antibodies against SARS-CoV-2 was seen in 392 (99%; 95% CI 98-100) participants, with a GMT of 245·4 (95% CI 220·0-273·7), and seroconversion of RBD-binding antibodies was observed in all 394 (100%; 99-100) participants, with a GMT of 8021 (7366-8734). On day 14 after the third dose, seroconversion of neutralising antibodies against the omicron subvariant BA.2 was observed in 375 (95%; 95% CI 93-97) of 394 participants, with a GMT of 42·9 (95% CI 37·9-48·5). For the non-inferiority comparison of participants aged 3-17 years with those aged 18-59 years for SARS-CoV-2 neutralising antibodies, the adjusted GMR was 8·6 (95% CI 7·0-10·4), with the lower bound of the GMR greater than 0·67. INTERPRETATION: ZF2001 is safe, well tolerated, and immunogenic in children and adolescents aged 3-17 years. Vaccine-elicited sera can neutralise the omicron BA.2 subvariant, but with reduced activity. The results support further studies of ZF2001 in children and adolescents. FUNDING: Anhui Zhifei Longcom Biopharmaceutical and the Excellent Young Scientist Program from National Natural Science Foundation of China. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Structural Characteristics of Heparin Binding to SARS-CoV-2 Spike Protein RBD of Omicron Sub-Lineages BA.2.12.1, BA.4 and BA.5.

The now prevalent Omicron variant and its subvariants/sub-lineages have led to a significant increase in COVID-19 cases and raised serious concerns about increased risk of infectivity, immune evasion, and reinfection. Heparan sulfate (HS), located on the surface of host cells, plays an important role as a co-receptor for virus-host cell interaction. The ability of heparin and HS to compete for binding of the SARS-CoV-2 spike (S) protein to cell surface HS illustrates the therapeutic potential of agents targeting protein-glycan interactions. In the current study, phylogenetic tree of variants and mutations in S protein receptor-binding domain (RBD) of Omicron BA.2.12.1, BA.4 and BA.5 were described. The binding affinity of Omicron S protein RBD to heparin was further investigated by surface plasmon resonance (SPR). Solution competition studies on the inhibitory activity of heparin oligosaccharides and desulfated heparins at different sites on S protein RBD-heparin interactions revealed that different sub-lineages tend to bind heparin with different chain lengths and sulfation patterns. Furthermore, blind docking experiments showed the contribution of basic amino acid residues in RBD and sulfo groups and carboxyl groups on heparin to the interaction. Finally, pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for inhibition on the interaction of heparin and S protein RBD of Omicron BA.2.12.1, BA.4/BA.5, and both showed much stronger inhibition than heparin.

A randomized, double-blind, placebo-controlled phase 1 and phase 2 clinical trial to evaluate efficacy and safety of a SARS-CoV-2 vaccine SCoK in adults.

BACKGROUND: To determine an appropriate dose of, and immunization schedule for, a vaccine SCoK against COVID-19 for an efficacy study; herein, we conducted randomized controlled trials to assess the immunogenicity and safety of this vaccine in adults. METHODS: These randomized, double-blind, placebo-controlled phase 1 and 2 trials of vaccine SCoK were conducted in Binhai District, Yan City, Jiangsu Province, China. Younger and older adult participants in phase 1 and 2 trials were sequentially recruited into different groups to be intramuscularly administered 20 or 40 µg vaccine SCoK or placebo. Participants were enrolled into our phase 1 and 2 studies to receive vaccine or placebo. RESULTS: No serious vaccine-related adverse events were observed in either trial. In both trials, local and systemic adverse reactions were absent or mild in most participants. In our phase 1 and 2 studies, the vaccine induced significantly increased neutralizing antibody responses to pseudovirus and live SARS-CoV-2. The vaccine induced significant neutralizing antibody responses to live SARS-CoV-2 on day 14 after the last immunization, with NT50s of 80.45 and 92.46 in participants receiving 20 and 40 µg doses, respectively; the seroconversion rates were 95.83% and 100%. The vaccine SCoK showed a similar safety and immunogenicity profiles in both younger participants and older participants. The vaccine showed better immunogenicity in phase 2 than in phase 1 clinical trial. Additionally, the incidence of adverse reactions decreased significantly in phase 2 clinical trial. The vaccine SCoK was well tolerated and immunogenic.

[Spatiotemporal Distribution and Risk Assessment of Pharmaceuticals in Typical Drinking Water Sources in the Middle Reaches of the Yangtze River].

The seasonal variation and spatial distribution of pharmaceuticals in typical drinking water sources in the middle reaches of the Yangtze River were analyzed using the solid-phase extraction and high-performance liquid chromatography-tandem mass spectrometry methods. Combined with the risk entropy method, the corresponding ecological risks for aquatic organisms were evaluated. The results showed that 80% of the target pharmaceuticals were detected in the drinking water sources, with average concentrations of 0.07-13.00 ng·L-1. The concentrations of the target pharmaceuticals were lower than or comparable with those in other drinking water sources reported in China. The spatiotemporal distribution of different pharmaceuticals varied. Generally, the detection level in winter was higher than that in summer, and there was no significant difference between that upstream and that downstream. This might be mainly attributed to seasonal/regional use and emissions of the pharmaceuticals, the impact of flow rate on dilution, and the impact of temperature on biodegradation. Compared with those before the COVID-19 epidemic, the detection concentrations of the target pharmaceuticals were relatively low. The reason for this might be that the prevention and control of the epidemic reduced the use and emission of the pharmaceuticals to a certain extent, and the high rainfall and runoff strengthened the dilution of water flow. The target pharmaceuticals, especially antibiotics, posed medium or low risks to aquatic organisms (especially algae). Considering the ecological risks and genotoxicity of pharmaceuticals and the potential risks of antibiotic-resistant genes, it is suggested to strengthen the investigation, evaluation, treatment, and control of pharmaceuticals in the water environment.

Efficacy and Safety of the RBD-Dimer-Based Covid-19 Vaccine ZF2001 in Adults.

BACKGROUND: The ZF2001 vaccine, which contains a dimeric form of the receptor-binding domain of severe acute respiratory syndrome coronavirus 2 and aluminum hydroxide as an adjuvant, was shown to be safe, with an acceptable side-effect profile, and immunogenic in adults in phase 1 and 2 clinical trials. METHODS: We conducted a randomized, double-blind, placebo-controlled, phase 3 trial to investigate the efficacy and confirm the safety of ZF2001. The trial was performed at 31 clinical centers across Uzbekistan, Indonesia, Pakistan, and Ecuador; an additional center in China was included in the safety analysis only. Adult participants (≥18 years of age) were randomly assigned in a 1:1 ratio to receive a total of three 25-µg doses (30 days apart) of ZF2001 or placebo. The primary end point was the occurrence of symptomatic coronavirus disease 2019 (Covid-19), as confirmed on polymerase-chain-reaction assay, at least 7 days after receipt of the third dose. A key secondary efficacy end point was the occurrence of severe-to-critical Covid-19 (including Covid-19-related death) at least 7 days after receipt of the third dose. RESULTS: Between December 12, 2020, and December 15, 2021, a total of 28,873 participants received at least one dose of ZF2001 or placebo and were included in the safety analysis; 25,193 participants who had completed the three-dose regimen, for whom there were approximately 6 months of follow-up data, were included in the updated primary efficacy analysis that was conducted at the second data cutoff date of December 15, 2021. In the updated analysis, primary end-point cases were reported in 158 of 12,625 participants in the ZF2001 group and in 580 of 12,568 participants in the placebo group, for a vaccine efficacy of 75.7% (95% confidence interval [CI], 71.0 to 79.8). Severe-to-critical Covid-19 occurred in 6 participants in the ZF2001 group and in 43 in the placebo group, for a vaccine efficacy of 87.6% (95% CI, 70.6 to 95.7); Covid-19-related death occurred in 2 and 12 participants, respectively, for a vaccine efficacy of 86.5% (95% CI, 38.9 to 98.5). The incidence of adverse events and serious adverse events was balanced in the two groups, and there were no vaccine-related deaths. Most adverse reactions (98.5%) were of grade 1 or 2. CONCLUSIONS: In a large cohort of adults, the ZF2001 vaccine was shown to be safe and effective against symptomatic and severe-to-critical Covid-19 for at least 6 months after full vaccination. (Funded by the National Science and Technology Major Project and others; ClinicalTrials.gov number, NCT04646590.).

Immunogenicity and safety of a recombinant fusion protein vaccine (V-01) against coronavirus disease 2019 in healthy adults: a randomized, double-blind, placebo-controlled, phase II trial.

BACKGROUND: Innovative coronavirus disease 2019 (COVID-19) vaccines, with elevated global manufacturing capacity, enhanced safety and efficacy, simplified dosing regimens, and distribution that is less cold chain-dependent, are still global imperatives for tackling the ongoing pandemic. A previous phase I trial indicated that the recombinant COVID-19 vaccine (V-01), which contains a fusion protein (IFN-PADRE-RBD-Fc dimer) as its antigen, is safe and well tolerated, capable of inducing rapid and robust immune responses, and warranted further testing in additional clinical trials. Herein, we aimed to assess the immunogenicity and safety of V-01, providing rationales of appropriate dose regimen for further efficacy study. METHODS: A randomized, double-blind, placebo-controlled phase II clinical trial was initiated at the Gaozhou Municipal Centre for Disease Control and Prevention (Guangdong, China) in March 2021. Both younger (n = 440; 18-59 years of age) and older (n = 440; ≥60 years of age) adult participants in this trial were sequentially recruited into two distinct groups: two-dose regimen group in which participants were randomized either to follow a 10 or 25 µg of V-01 or placebo given intramuscularly 21 days apart (allocation ratio, 3:3:1, n = 120, 120, 40 for each regimen, respectively), or one-dose regimen groups in which participants were randomized either to receive a single injection of 50 µg of V-01 or placebo (allocation ratio, 3:1, n = 120, 40, respectively). The primary immunogenicity endpoints were the geometric mean titers of neutralizing antibodies against live severe acute respiratory syndrome coronavirus 2, and specific binding antibodies to the receptor binding domain (RBD). The primary safety endpoint evaluation was the frequencies and percentages of overall adverse events (AEs) within 30 days after full immunization. RESULTS: V-01 provoked substantial immune responses in the two-dose group, achieving encouragingly high titers of neutralizing antibody and anti-RBD immunoglobulin, which peaked at day 35 (161.9 [95% confidence interval [CI]: 133.3-196.7] and 149.3 [95%CI: 123.9-179.9] in 10 and 25 µg V-01 group of younger adults, respectively; 111.6 [95%CI: 89.6-139.1] and 111.1 [95%CI: 89.2-138.4] in 10 and 25 µg V-01 group of older adults, respectively), and remained high at day 49 after a day-21 second dose; these levels significantly exceed those in convalescent serum from symptomatic COVID-19 patients (53.6, 95%CI: 31.3-91.7). Our preliminary data show that V-01 is safe and well tolerated, with reactogenicity predominantly being absent or mild in severity and only one vaccine-related grade 3 or worse AE being observed within 30 days. The older adult participants demonstrated a more favorable safety profile compared with those in the younger adult group: with AEs percentages of 19.2%, 25.8%, 17.5% in older adults vs. 34.2%, 23.3%, 26.7% in younger adults at the 10, 25 µg V-01 two-dose group, and 50 µg V-01 one-dose group, respectively. CONCLUSIONS: The vaccine candidate V-01 appears to be safe and immunogenic. The preliminary findings support the advancement of the two-dose, 10 µg V-01 regimen to a phase III trial for a large-scale population-based evaluation of safety and efficacy. TRIAL REGISTRATION: http://www.chictr.org.cn/index.aspx (No. ChiCTR2100045107, http://www.chictr.org.cn/showproj.aspx?proj=124702).

Potential Anti-SARS-CoV-2 Activity of Pentosan Polysulfate and Mucopolysaccharide Polysulfate.

With the increased prevalence of new SARS-CoV-2 variants of concern, such as Delta and Omicron, the COVID-19 pandemic has become an ongoing human health disaster, killing millions worldwide. SARS-CoV-2 invades its host through the interaction of its spike (S) protein with a host cell receptor, angiotensin-converting enzyme 2 (ACE2). In addition, heparan sulfate (HS) on the surface of host cells plays an important role as a co-receptor for this viral pathogen-host cell interaction. Our previous studies demonstrated that many sulfated glycans, such as heparin, fucoidans, and rhamnan sulfate have anti-SARS-CoV-2 activities. In the current study, a small library of sulfated glycans and highly negatively charged compounds, including pentosan polysulfate (PPS), mucopolysaccharide polysulfate (MPS), sulfated lactobionic acid, sulodexide, and defibrotide, was assembled and evaluated for binding to the S-proteins and inhibition of viral infectivity in vitro. These compounds inhibited the interaction of the S-protein receptor-binding domain (RBD) (wild type and different variants) with immobilized heparin, a highly sulfated HS, as determined using surface plasmon resonance (SPR). PPS and MPS showed the strongest inhibition of interaction of heparin and S-protein RBD. The competitive binding studies showed that the IC50 of PPS and MPS against the S-protein RBD binding to immobilized heparin was ~35 nM and ~9 nM, respectively, much lower than the IC50 for soluble heparin (IC50 = 56 nM). Both PPS and MPS showed stronger inhibition than heparin on the S-protein RBD or spike pseudotyped lentiviral particles binding to immobilized heparin. Finally, in an in vitro cell-based assay, PPS and MPS exhibited strong antiviral activities against pseudotyped viral particles of SARS-CoV-2 containing wild-type or Delta S-proteins.

Design of a mutation-integrated trimeric RBD with broad protection against SARS-CoV-2.

The continuous emergence of SARS-CoV-2 variants highlights the need of developing vaccines with broad protection. Here, according to the immune-escape capability and evolutionary convergence, the representative SARS-CoV-2 strains carrying the hotspot mutations were selected. Then, guided by structural and computational analyses, we present a mutation-integrated trimeric form of spike receptor-binding domain (mutI-tri-RBD) as a broadly protective vaccine candidate, which combined heterologous RBDs from different representative strains into a hybrid immunogen and integrated immune-escape hotspots into a single antigen. When compared with a homo-tri-RBD vaccine candidate in the stage of phase II trial, of which all three RBDs are derived from the SARS-CoV-2 prototype strain, mutI-tri-RBD induced significantly higher neutralizing antibody titers against the Delta and Beta variants, and maintained a similar immune response against the prototype strain. Pseudo-virus neutralization assay demonstrated that mutI-tri-RBD also induced broadly strong neutralizing activities against all tested 23 SARS-CoV-2 variants. The in vivo protective capability of mutI-tri-RBD was further validated in hACE2-transgenic mice challenged by the live virus, and the results showed that mutI-tri-RBD provided potent protection not only against the SARS-CoV-2 prototype strain but also against the Delta and Beta variants.

The antigenicity of SARS-CoV-2 Delta variants aggregated 10 high-frequency mutations in RBD has not changed sufficiently to replace the current vaccine strain.

Emerging SARS-CoV-2 variants are the most serious problem for COVID-19 prophylaxis and treatment. To determine whether the SARS-CoV-2 vaccine strain should be updated following variant emergence like seasonal flu vaccine, the changed degree on antigenicity of SARS-CoV-2 variants and H3N2 flu vaccine strains was compared. The neutralization activities of Alpha, Beta and Gamma variants' spike protein-immunized sera were analysed against the eight current epidemic variants and 20 possible variants combining the top 10 prevalent RBD mutations based on the Delta variant, which were constructed using pseudotyped viruses. Meanwhile, the neutralization activities of convalescent sera and current inactivated and recombinant protein vaccine-elicited sera were also examined against all possible Delta variants. Eight HA protein-expressing DNAs elicited-animal sera were also tested against eight pseudotyped viruses of H3N2 flu vaccine strains from 2011-2019. Our results indicate that the antigenicity changes of possible Delta variants were mostly within four folds, whereas the antigenicity changes among different H3N2 vaccine strains were approximately 10-100-fold. Structural analysis of the antigenic characterization of the SARS-CoV-2 and H3N2 mutations supports the neutralization results. This study indicates that the antigenicity changes of the current SARS-CoV-2 may not be sufficient to require replacement of the current vaccine strain.

Anti-SARS-CoV-2 Activity of Rhamnan Sulfate from Monostroma nitidum.

The COVID-19 pandemic is a major human health concern. The pathogen responsible for COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), invades its host through the interaction of its spike (S) protein with a host cell receptor, angiotensin-converting enzyme 2 (ACE2). In addition to ACE2, heparan sulfate (HS) on the surface of host cells also plays a significant role as a co-receptor. Our previous studies demonstrated that sulfated glycans, such as heparin and fucoidans, show anti-COVID-19 activities. In the current study, rhamnan sulfate (RS), a polysaccharide with a rhamnose backbone from a green seaweed, Monostroma nitidum, was evaluated for binding to the S-protein from SARS-CoV-2 and inhibition of viral infectivity in vitro. The structural characteristics of RS were investigated by determining its monosaccharide composition and performing two-dimensional nuclear magnetic resonance. RS inhibition of the interaction of heparin, a highly sulfated HS, with the SARS-CoV-2 spike protein (from wild type and different mutant variants) was studied using surface plasmon resonance (SPR). In competitive binding studies, the IC50 of RS against the S-protein receptor binding domain (RBD) binding to immobilized heparin was 1.6 ng/mL, which is much lower than the IC50 for heparin (~750 ng/mL). RS showed stronger inhibition than heparin on the S-protein RBD or pseudoviral particles binding to immobilized heparin. Finally, in an in vitro cell-based assay, RS showed strong antiviral activities against wild type SARS-CoV-2 and the delta variant.

Responses of aquatic organisms downstream from WWTPs to disinfectants and their by-products during the COVID-19 pandemic, Wuhan.

The outbreak of COVID-19 has led to the large-scale usage of chlorinated disinfectants in cities. Disinfectants and disinfection by-products (DBPs) enter rivers through urban drainage and surface runoff. We investigated the variations in residual chlorine, DBPs, and different aquatic organisms in the Hanjiang, Fuhe, and Qinglinghe Rivers in Wuhan during the COVID-19 pandemic. The sampling sites were from the wastewater treatment plant outlets to the downstream drinking water treatment plant intakes. Total residual chlorine and DBPs (dichloromethane and trichloromethane) detected in the river water ranged from 0 to 0.84 mg/L and 0 to 0.034 mg/L, respectively. The residual chlorine and DBPs showed a gradual reduction pattern related to water flow, and the concentration at intakes did not exceed the Chinese drinking water source quality standards. Phytoplankton and zooplankton densities were not significantly correlated with residual chlorine and DBPs. The fluctuations in phytoplankton resource use efficiency (RUE) and zooplankton RUE in the Fuhe River, with the highest residual chlorine, and the Qinglinghe River with the highest DBPs, were higher than those in the Hanjiang River. For benthic macroinvertebrates, the number of functional feeding groups in the Hanjiang River was higher than that in the Fuhe and Qinglinghe Rivers. The water and sediment bacterial communities in the Hanjiang River differed significantly from those in the Fuhe and Qingling Rivers. The denitrification function involved in N metabolism was stronger in the Fuhe and Qinglinghe Rivers. Structural equation modelling revealed that residual chlorine and DBPs impacted the diversity of benthos through direct and indirect effects on plankton. Although large-scale chlorine-containing disinfectants use occurred during the investigation, it did not harm the density of the detected aquatic organisms in water sources. With the regular use of chlorinated disinfectants for indoor and outdoor environments in response to the SARS-CoV-2 globally, it is still necessary to study the long-term and accumulated responses of water ecosystems exposed to chlorine-containing disinfectants.

An ensemble learning method based on ordinal regression for COVID-19 diagnosis from chest CT.

Coronavirus disease 2019 (COVID-19) has brought huge losses to the world, and it remains a great threat to public health. X-ray computed tomography (CT) plays a central role in the management of COVID-19. Traditional diagnosis with pulmonary CT images is time-consuming and error-prone, which could not meet the need for precise and rapid COVID-19 screening. Nowadays, deep learning (DL) has been successfully applied to CT image analysis, which assists radiologists in workflow scheduling and treatment planning for patients with COVID-19. Traditional methods use cross-entropy as the loss function with a Softmax classifier following a fully-connected layer. Most DL-based classification methods target intraclass relationships in a certain class (early, progressive, severe, or dissipative phases), ignoring the natural order of different phases of the disease progression,i.e.,from an early stage and progress to a late stage. To learn both intraclass and interclass relationships among different stages and improve the accuracy of classification, this paper proposes an ensemble learning method based on ordinal regression, which leverages the ordinal information on COVID-19 phases. The proposed method uses multi-binary, neuron stick-breaking (NSB), and soft labels (SL) techniques, and ensembles the ordinal outputs through a median selection. To evaluate our method, we collected 172 confirmed cases. In a 2-fold cross-validation experiment, the accuracy is increased by 22% compared with traditional methods when we use modified ResNet-18 as the backbone. And precision, recall, andF1-score are also improved. The experimental results show that our proposed method achieves a better classification performance than the traditional methods, which helps establish guidelines for the classification of COVID-19 chest CT images.

Segmenting lung lesions of COVID-19 from CT images via pyramid pooling improved Unet.

Segmenting lesion regions of Coronavirus Disease 2019 (COVID-19) from computed tomography (CT) images is a challenge owing to COVID-19 lesions characterized by high variation, low contrast between infection lesions and around normal tissues, and blurred boundaries of infections. Moreover, a shortage of available CT dataset hinders deep learning techniques applying to tackling COVID-19. To address these issues, we propose a deep learning-based approach known as PPM-Unet to segmenting COVID-19 lesions from CT images. Our method improves an Unet by adopting pyramid pooling modules instead of the conventional skip connection and then enhances the representation of the neural network by aiding the global attention mechanism. We first pre-train PPM-Unet on COVID-19 dataset of pseudo labels containing1600 samples producing a coarse model. Then we fine-tune the coarse PPM-Unet on the standard COVID-19 dataset consisting of 100 pairs of samples to achieve a fine PPM-Unet. Qualitative and quantitative results demonstrate that our method can accurately segment COVID-19 infection regions from CT images, and achieve higher performance than other state-of-the-art segmentation models in this study. It offers a promising tool to lay a foundation for quantitatively detecting COVID-19 lesions.

Safety and immunogenicity of a recombinant tandem-repeat dimeric RBD-based protein subunit vaccine (ZF2001) against COVID-19 in adults: two randomised, double-blind, placebo-controlled, phase 1 and 2 trials.

BACKGROUND: Although several COVID-19 vaccines have been developed so far, they will not be sufficient to meet the global demand. Development of a wider range of vaccines, with different mechanisms of action, could help control the spread of SARS-CoV-2 globally. We developed a protein subunit vaccine against COVID-19 using a dimeric form of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein as the antigen. We aimed to assess the safety and immunogenicity of this vaccine, ZF2001, and determine the appropriate dose and schedule for an efficacy study. METHODS: We did two randomised, double-blind, placebo-controlled, phase 1 and phase 2 trials. Phase 1 was done at two university hospitals in Chongqing and Beijing, China, and phase 2 was done at the Hunan Provincial Center for Disease Control and Prevention in Xiangtan, China. Healthy adults aged 18-59 years, without a history of SARS-CoV or SARS-CoV-2 infection, an RT-PCR-positive test result for SARS-CoV-2, a history of contact with confirmed or suspected COVID-19 cases, and severe allergies to any component of the vaccine were eligible for enrolment. In phase 1, participants were randomly assigned (2:2:1) to receive three doses of the vaccine (25 µg or 50 µg) or placebo intramuscularly, 30 days apart. In phase 2, participants were randomly assigned (1:1:1:1:1:1) to receive the vaccine (25 µg or 50 µg) or placebo intramuscularly, 30 days apart, in either a two-dose schedule or a three-dose schedule. Investigators, participants, and the laboratory team were masked to group allocation. For phase 1, the primary outcome was safety, measured by the occurrence of adverse events and serious adverse events. For phase 2, the primary outcome was safety and immunogenicity (the seroconversion rate and the magnitude, in geometric mean titres [GMTs], of SARS-CoV-2-neutralising antibodies). Analyses were done on an intention-to-treat and per-protocol basis. These trials are registered with ClinicalTrials.gov (NCT04445194 and NCT04466085) and participant follow-up is ongoing. FINDINGS: Between June 22 and July 3, 2020, 50 participants were enrolled into the phase 1 trial and randomly assigned to receive three doses of placebo (n=10), the 25 µg vaccine (n=20), or the 50 µg vaccine (n=20). The mean age of participants was 32·6 (SD 9·4) years. Between July 12 and July 17, 2020, 900 participants were enrolled into the phase 2 trial and randomly assigned to receive two doses of placebo (n=150), 25 µg vaccine (n=150), or 50 µg vaccine (n=150), or three doses of placebo (n=150), 25 µg vaccine (n=150), or 50 µg vaccine (n=150). The mean age of participants was 43·5 (SD 9·2) years. In both phase 1 and phase 2, adverse events reported within 30 days after vaccination were mild or moderate (grade 1 or 2) in most cases (phase 1: six [60%] of ten participants in the placebo group, 14 [70%] of 20 in the 25 µg group, and 18 [90%] of 20 in the 50 µg group; phase 2: 37 [25%] of 150 in the two-dose placebo group, 43 [29%] of 150 in the two-dose 25 µg group, 50 [33%] of 150 in the two-dose 50 µg group, 47 [31%] of 150 in the three-dose placebo group, 72 [48%] of 150 in the three-dose 25 µg group, and 65 [43%] of 150 in the three-dose 50 µg group). In phase 1, two (10%) grade 3 or worse adverse events were reported in the 50 µg group. In phase 2, grade 3 or worse adverse events were reported by 18 participants (four [3%] in the two-dose 25 µg vaccine group, two [1%] in the two-dose 50 µg vaccine group, two [1%] in the three-dose placebo group, four [3%] in the three-dose 25 µg vaccine group, and six [4%] in the three-dose 50 µg vaccine group), and 11 were considered vaccine related (two [1%] in the two-dose 25 µg vaccine group, one [1%] in the two-dose 50 µg vaccine group, one [1%] in the three-dose placebo group, two [1%] in the three-dose 25 µg vaccine group, and five [3%] in the three-dose 50 µg vaccine group); seven participants reported serious adverse events (one [1%] in the two-dose 25 µg vaccine group, one [1%] in the two-dose 50 µg vaccine group, two [1%] in the three-dose placebo group, one [1%] in the three-dose 25 µg vaccine group, and two [1%] in the three-dose 50 µg vaccine group), but none was considered vaccine related. In phase 2, on the two-dose schedule, seroconversion rates of neutralising antibodies 14 days after the second dose were 76% (114 of 150 participants) in the 25 µg group and 72% (108 of 150) in the 50 µg group; on the three-dose schedule, seroconversion rates of neutralising antibodies 14 days after the third dose were 97% (143 of 148 participants) in the 25 µg group and 93% (138 of 148) in the 50 µg group. In the two-dose groups in phase 2, the SARS-CoV-2-neutralising GMTs 14 days after the second dose were 17·7 (95% CI 13·6-23·1) in the 25 µg group and 14·1 (10·8-18·3) in the 50 µg group. In the three-dose groups in phase 2, the SARS-CoV-2-neutralising GMTs 14 days after the third dose were 102·5 (95% CI 81·8-128·5) in the 25 µg group and 69·1 (53·0-90·0) in the 50 µg group. INTERPRETATION: The protein subunit vaccine ZF2001 appears to be well tolerated and immunogenic. The safety and immunogenicity data from the phase 1 and 2 trials support the use of the 25 µg dose in a three-dose schedule in an ongoing phase 3 trial for large-scale evaluation of ZF2001's safety and efficacy. FUNDING: National Program on Key Research Project of China, National Science and Technology Major Projects of Drug Discovery, Strategic Priority Research Program of the Chinese Academy of Sciences, and Anhui Zhifei Longcom Biopharmaceutical. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Heparan sulfates from bat and human lung and their binding to the spike protein of SARS-CoV-2 virus.

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has resulted in a pandemic and continues to spread at an unprecedented rate around the world. Although a vaccine has recently been approved, there are currently few effective therapeutics to fight its associated disease in humans, COVID-19. SARS-CoV-2 and the related severe acute respiratory syndrome (SARS-CoV-1), and Middle East respiratory syndrome (MERS-CoV) result from zoonotic respiratory viruses that have bats as the primary host and an as yet unknown secondary host. While each of these viruses has different protein-based cell-surface receptors, each rely on the glycosaminoglycan, heparan sulfate as a co-receptor. In this study we compare, for the first time, differences and similarities in the structure of heparan sulfate in human and bat lungs. Furthermore, we show that the spike glycoprotein of COVID-19 binds 3.5 times stronger to human lung heparan sulfate than bat lung heparan sulfate.