Hyaluronan with Different Molecular Weights Can Affect the Gut Microbiota and Pathogenetic Progression of Post-Intensive Care Syndrome Mice in Different Ways.

Post-intensive care syndrome (PICS) poses a serious threat to the health of intensive care unit (ICU) survivors, and effective treatment options are currently lacking. With increasing survival rates of ICU patients worldwide, there is a rising interest in developing methods to alleviate PICS symptoms. This study aimed to explore the potential of using Hyaluronan (HA) with different molecular weights as potential drugs for treating PICS in mice. Cecal ligation and puncture (CLP) were used to establish a PICS mice model, and high molecular weight HA (HMW-HA) or oligo-HA were used as therapeutic agents. Pathological and physiological changes of PICS mice in each group were monitored. 16S rRNA sequencing was performed to dissect gut microbiota discrepancies. The results showed that both molecular weights of HA could increase the survival rate of PICS mice at the experimental endpoint. Specifically, 1600 kDa-HA can alleviate PICS in a short time. In contrast, 3 kDa-HA treatment decreased PICS model survivability in the early stages of the experiment. Further, via 16S rRNA sequence analysis, we observed the changes in the gut microbiota in PICS mice, thereby impairing intestinal structure and increasing inflammation. Additionally, both types of HA can reverse this change. Moreover, compared to 1600 kDa-HA, 3 kDa-HA can significantly elevate the proportion of probiotics and reduce the abundance of pathogenic bacteria (Desulfovibrionaceae and Enterobacteriaceae). In conclusion, HA holds the advantage of being a potential therapeutic drug for PICS, but different molecular weights can lead to varying effects. Moreover, 1600 kDa-HA showed promise as a protective agent in PICS mice, and caution should be taken to its timing when considering using 3 kDa-HA.

Comparison between the effects of ultrasound guided intra-articular injections of platelet-rich plasma (PRP), high molecular weight hyaluronic acid, and their combination in hip osteoarthritis: a randomized clinical trial.

BACKGROUND: Intra articular (IA) injection of platelet-rich plasma (PRP) and hyaluronic acid (HA) are of the new methods in the management of hip osteoarthritis (OA). The aim of this study was to compare the effectiveness of IA injections of PRP, HA and their combination in patients with hip OA. HA and PRP are two IA interventions that can be used in OA in the preoperative stages. Due to the different mechanisms of action, these two are proposed to have a synergistic effect by combining. METHODS: This is a randomized clinical trial with three parallel groups. In this study, patients with grade 2 and 3 hip OA were included, and were randomly divided into three injection groups: PRP, HA and PRP + HA. In either group, two injections with 2 weeks' interval were performed into the hip joint under ultrasound guidance. Patients were assessed before the intervention, 2 months and 6 months after the second injection, using the visual analog scale (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and Lequesne questionnaires. RESULTS: One hundred five patients were enrolled randomly in HA, PRP and PRP + HA groups. All three groups showed significant improvement in WOMAC, VAS, and Lequesne at 2 months and 6 months compared with baseline. Comparison of the 3 groups demonstrated significant differences regarding WOMAC and Lequesne total scores and the activities of daily living (ADL) subscale of Lequesne (P = 0.041, 0.001 and 0.002, respectively), in which the observed improvement at 6th month was significantly higher in the PRP + HA and PRP groups compared to the HA group. CONCLUSION: Although all 3 interventions were associated with improvement of pain and function in patients with hip OA, the therapeutic effects of PRP and PRP + HA injections lasted longer (6 months), and the effects of these two interventions on patients' performance, disability, and ADL were superior to HA in the long run. Moreover, the addition of HA to PRP was not associated with a significant increase in the therapeutic results. TRIAL REGISTRATION: The study was registered at Iranian Registry of Clinical Trials (IRCT) website http://www.irct.ir/ , a WHO Primary Register setup, with the registration number of IRCT20130523013442N30 on 29/11/2019.

Low molecular weight cytotoxic components (DAMPs) form the post-COVID-19 syndrome.

We studied the role of cytotoxic components (DAMPs) formed in the body of patients with COVID-19 in ensuring the long-term preservation of post-COVID-19 manifestations and the possibility of creating an experimental model by transferring DAMPs to rats. In patients with post-COVID-19 syndrome (PCS) 2 months after SARS-CoV-2 infection we determined the presence of cytotoxic components in the blood serum (Terasaki test, Dunaliella viridis test and content of DAMPs). In post-COVID-19 syndrome patients with a high content of serum cytotoxic oligopeptide fraction (selective group, n = 16) we determined the number of leukocytes, lymphocytes, neutrophil granulocytes and monocytes in the blood, the content of C-reactive protein (CRP), the concentration of C3 and C4 complement components and circulating immune complexes, the serum content of IL-6, IL -10, IL-18, TNF-α, phagocytic activity of neutrophils, presence of neutrophil traps and autoantibodies ANA. It has been shown that in patients with PCS, there are components with cytotoxicity in the blood serum, form specific immunopathological patterns, which are characterized by: an increased content of CRP, complement system components C3 and C4 and cytokines (TNF-α, IL-6, IL-10, IL-18) activation, the formation of a wide range of autoantibodies ANA, the low efficiency of endocytosis in oxygen-independent phagocytosis; their phagocytic activity reaches its functional limit, and against this background, activation of neutrophil traps occurs, which can contribute to further induction of DAMPs. This self-sustaining cell-killing activation provided long-term preservation of PCS symptoms. The transfer of blood serum components from selective group patients with PCS to rats was accompanied by the appearance of cytotoxic components in them which induced sensitization and immunopathological reactions. Preventive administration of a biologically active substance with polyfunctional properties MF to experimental animals "corrected" the initial functional state of the body's immune-metabolic system and eliminated or facilitated immuno-inflammatory reactions.

Inhibitory effects of fucoidan from Laminaria japonica against some pathogenic bacteria and SARS-CoV-2 depend on its large molecular weight.

Fucoidan is a highly sulfated polysaccharide with a wide range of bioactivities, including anti-pathogenic activity. However, the relationship between structure and activity of fucoidan in inhibiting pathogen infections remains unclear. Here, different-molecular-weight fucoidans were prepared by photocatalytic degradation followed by membrane ultrafiltration, and their chemical structures and anti-pathogenic microbiota activity were compared. Results showed that photocatalytic degradation could effectively degrade fucoidan while its structure block and sulfate groups were not destroyed obviously. Fucoidan (90.8 kDa) of 5 mg/mL could inhibit the growth of S. aureus, S. typhimurium and E. coli, but its degradation products, Dfuc1 (19.2 kDa) and Dfuc2 (5.5 kDa), demonstrated lower inhibitory effect. In addition, compared to Dfuc1 and Dfuc2, fucoidan showed stronger capability to prevent the adhesion of S. aureus, L. monocytogenes, V. parahaemolyticus and S. typhimurium to HT-29 cells. Moreover, the inhibitory effect against SARS-CoV-2 and the binding activity to S protein were also positively correlated to molecular weight. These results indicate that natural fucoidan with higher molecular weight are more effective to inhibit these pathogenic bacteria and SARS-CoV-2, providing a better understanding of the relationship between structure and activity of fucoidan against pathogenic microbiota.

Formation of Larger Molecular Weight Disinfection Byproducts from Acetaminophen in Chlorine Disinfection.

Acetaminophen is widely used to treat mild to moderate pain and to reduce fever. Under the worldwide COVID-19 pandemic, this over-the-counter pain reliever and fever reducer has been drastically consumed, which makes it even more abundant than ever in municipal wastewater and drinking water sources. Chlorine is the most widely used oxidant in drinking water disinfection, and chlorination generally causes the degradation of organic compounds, including acetaminophen. In this study, a new reaction pathway in the chlorination of acetaminophen, i.e., oxidative coupling reactions via acetaminophen radicals, was investigated both experimentally and computationally. Using an ultraperformance liquid chromatograph coupled to an electrospray ionization-triple quadrupole mass spectrometer, we detected over 20 polymeric products in chlorinated acetaminophen samples, some of which have structures similar to the legacy pollutants "polychlorinated biphenyls". Both C-C and C-O bonding products were found, and the corresponding bonding processes and kinetics were revealed by quantum chemical calculations. Based on the product confirmation and intrinsic reaction coordinate computations, a pathway for the formation of the polymeric products in the chlorination of acetaminophen was proposed. This study suggests that chlorination may cause not only degradation but also upgradation of a phenolic compound or contaminant.

Low molecular weight chitooligosaccharide inhibits infection of SARS-CoV-2 in vitro.

AIMS: The discovery of antiviral substances to respond to COVID-19 is a global issue, including the field of drug development based on natural materials. Here, we showed that chitosan-based substances have natural antiviral properties against SARS-CoV-2 in vitro. METHODS AND RESULTS: The molecular weight of chitosan-based substances was measured by the gel permeation chromatography analysis. In MTT assay, the chitosan-based substances have low cytotoxicity to Vero cells. The antiviral effect of these substances was confirmed by quantitative viral RNA targeting the RdRp and E genes and plaque assay. Among the substances tested, low molecular weight chitooligosaccharide decreased the fluorescence intensity of SARS-CoV-2 nucleocapsid protein of the virus-infected cells in a dose-dependent manner. CONCLUSIONS: In conclusion, the chitooligosaccharide, a candidate for natural treatment, has antiviral effects against the SARS-CoV-2 virus in vitro. SIGNIFICANCE AND IMPACT OF STUDY: In this study, it was suggested for the first time that chitosan-based substances such as chitooligosaccharide can have an antiviral effect on SARS-CoV-2 in vitro.

Analysis of Recombinant Adenovirus Vectors by Ion Trap Charge Detection Mass Spectrometry: Accurate Molecular Weight Measurements beyond 150 MDa.

Adenovirus is one of the largest nonenveloped, double-stranded DNA viruses. It is widely used as a gene therapy vector and has recently received a lot of attention as a novel vaccine platform for SARS-CoV-2. Human adenovirus 5 (HAdV5) contains over 2500 protein molecules and has a 36 kbp genome. Adenovirus is well beyond the range of conventional mass spectrometry, and it was unclear how well such a large complex could be desolvated. Here, we report molecular weight (MW) distributions measured for HAdV5 and for 11 recombinant AdV vectors with genomes of varying lengths. The MW distributions were recorded using ion trap charge detection mass spectrometry (CDMS), a single-particle technique where m/z and charge are measured for individual ions. The results show that ions as large as 150 MDa can be effectively desolvated and accurate MW distributions obtained. The MW distribution for HAdV5 contains a narrow peak at 156.1 MDa, assigned to the infectious virus. A smaller peak at 129.6 MDa is attributed to incomplete particles that have not packaged a genome. The ions in the 129.6 MDa peak have a much lower average charge than those in the peak at 156.1 MDa. This is attributed to the empty particles missing some or all of the fibers that decorate the surface of the virion. The MW measured for the mature virus (156.1 MDa) is much larger than that predicted from sequence masses and copy numbers of the constituents (142.5 MDa). Measurements performed for recombinant AdV as a function of genome length show that for every 1 MDa increase in the genome MW, the MW of the mature virus increases by around 2.3 MDa. The additional 1.3 MDa is attributed to core proteins that are copackaged with the DNA. This observation suggests that the discrepancy between the measured and expected MWs for mature HAdV5 is due to an underestimate in the copy numbers of the core proteins.

Structural characterization and SARS-CoV-2 inhibitory activity of a sulfated polysaccharide from Caulerpa lentillifera.

Caulerpa lentillifera (Bryopsidophyceae, Chlorophyta) is an edible seaweed attracting great attention for its expansion of farming scale and increasing consumption in these years. In the present study, a sulfated polysaccharide (CLSP-2) was isolated and separated from C. lentillifera, and its chemical structure was elucidated by a series of chemical and spectroscopic methods. Among these methods, mild acid hydrolysis and photocatalytic degradation were applied to release mono- and oligo-saccharide fragments which were further identified by HPLC-MSn analysis, affording the information of the sugar sequences and the sulfate substitution in CLSP-2. Results indicated that the backbone of CLSP-2 was constructed of →6)-ß-Manp-(1→ with sulfated branches at C2, which were comprised of prevalent →3)-ß-Galp4S-(1→, →3)-ß-Galp2,4S-(1→, and minor Xyl. In addition, the virus neutralization assay revealed that CLSP-2 could effectively protect HeLa cells against SARS-CoV-2 infection with an IC50 of 48.48 µg/mL. Hence, the present study suggests CLSP-2 as a promising agent against SARS-CoV-2.

A reverse vaccinology and immunoinformatics approach for designing a multiepitope vaccine against SARS-CoV-2.

Since 2019, the world was involved with SARS-CoV-2 and consequently, with the announcement by the World Health Organization that COVID-19 was a pandemic, scientific were an effort to obtain the best approach to combat this global dilemma. The best way to prevent the pandemic from spreading further is to use a vaccine against COVID-19. Here, we report the design of a recombinant multi-epitope vaccine against the four proteins spike or crown (S), membrane (M), nucleocapsid (N), and envelope (E) of SARS-CoV-2 using immunoinformatics tools. We evaluated the most antigenic epitopes that bind to HLA class 1 subtypes, along with HLA class 2, as well as B cell epitopes. Beta-defensin 3 and PADRE sequence were used as adjuvants in the structure of the vaccine. KK, GPGPG, and AAY linkers were used to fuse the selected epitopes. The nucleotide sequence was cloned into pET26b(+) vector using restriction enzymes XhoI and NdeI, and HisTag sequence was considered in the C-terminal of the construct. The results showed that the proposed candidate vaccine is a 70.87 kDa protein with high antigenicity and immunogenicity as well as non-allergenic and non-toxic. A total of 95% of the selected epitopes have conservancy with similar sequences. Molecular docking showed a strong binding between the vaccine structure and tool-like receptor (TLR) 7/8. The docking, molecular dynamics, and MM/PBSA analysis showed that the vaccine established a stable interaction with both structures of TLR7 and TLR8. Simulation of immune stimulation by this vaccine showed that it evokes immune responses related to humoral and cellular immunity.

Analysis of the avian coronavirus spike protein reveals heterogeneity in the glycans present.

Infectious bronchitis virus (IBV) is an economically important coronavirus, causing damaging losses to the poultry industry worldwide as the causative agent of infectious bronchitis. The coronavirus spike (S) glycoprotein is a large type I membrane protein protruding from the surface of the virion, which facilitates attachment and entry into host cells. The IBV S protein is cleaved into two subunits, S1 and S2, the latter of which has been identified as a determinant of cellular tropism. Recent studies expressing coronavirus S proteins in mammalian and insect cells have identified a high level of glycosylation on the protein's surface. Here we used IBV propagated in embryonated hens' eggs to explore the glycan profile of viruses derived from infection in cells of the natural host, chickens. We identified multiple glycan types on the surface of the protein and found a strain-specific dependence on complex glycans for recognition of the S2 subunit by a monoclonal antibody in vitro, with no effect on viral replication following the chemical inhibition of complex glycosylation. Virus neutralization by monoclonal or polyclonal antibodies was not affected. Following analysis of predicted glycosylation sites for the S protein of four IBV strains, we confirmed glycosylation at 18 sites by mass spectrometry for the pathogenic laboratory strain M41-CK. Further characterization revealed heterogeneity among the glycans present at six of these sites, indicating a difference in the glycan profile of individual S proteins on the IBV virion. These results demonstrate a non-specific role for complex glycans in IBV replication, with an indication of an involvement in antibody recognition but not neutralisation.

Low molecular weight fucoidan attenuating pulmonary fibrosis by relieving inflammatory reaction and progression of epithelial-mesenchymal transition.

Diffuse alveolar injury and pulmonary fibrosis (PF) are the main causes of death of Covid-19 cases. In this study a low molecular weight fucoidan (LMWF) with unique structural was obtained from Laminaria japonica, and its anti- PF and anti-epithelial-mesenchymal transition (EMT) bioactivity were investigated both in vivo and in vitro. After LWMF treatment the fibrosis and inflammatory factors stimulated by Bleomycin (BLM) were in lung tissue. Immunohistochemical and Western-blot results found the expression of COL2A1, ß-catenin, TGF-ß, TNF-α and IL-6 were declined in mice lung tissue. Besides, the phosphorylation of PI3K and Akt were inhibited by LMWF. In addition, the progression of EMT induced by TGF-ß1 was inhibited by LMWF through down-regulated both TGF-ß/Smad and PI3K/AKT signaling pathways. These data indicate that unique LMWF can protect the lung from fibrosis by weakening the process of inflammation and EMT, and it is a promising therapeutic option for the treatment of PF.

Structural features of an acidic polysaccharide with the potential of promoting osteoblast differentiation from Lycium ruthenicum Murr.

The enhanced osteoblast differentiation is beneficial to the prevention of osteoporosis. In this study, a homogeneous polysaccharide (LRP-S2A) with the potential of promoting osteoblast differentiation was obtained from the fruits of Lycium ruthenicum, a traditional herb for treatment of postmenopausal metabolic disorders. Structural identification indicated that LRP-S2A, with a relative molecular weight of 2.65 × 106 Da and an uronic acid content of 41.8%, contained Rha, Ara, Gal, Glc and GlcA in a molar ratio of 1.00 : 2.07 : 0.57 : 2.59 : 4.33 and was composed of a backbone consisting of 6-O-Me-α-(1→4)-D-GlcpA, 2-O-acetyl-α-(1→4)-D-Glcp, α-(1→2,4)-L-Rhap, ß-(1→3)-D-Galp andα-(1→3,5)-L-Araf, and some branches consisting of 6-O-Me-α-(1→4)-D-GlcpA and terminal α-L-Araf. These results suggested that LRP-S2A with the potential of promoting osteoblast differentiation was a new acidic polysaccharide.

Impact of proactive low-molecular weight heparin therapy on outcomes in COVID-1.

OBJECTIVES: Low molecular weight heparin (LMWH) may provide beneficial effects on outcomes of COVID-19. We aimed to examine the impact of LMWH treatment on clinical outcomes (duration of hospitalization, admission to intensive care unit, the requirement for mechanical ventilation, and death) of COVID-19 patients with normal D-dimer levels at admission. BACKGROUND: Coronavirus disease-2019 (COVID-19) predisposes patients to arterial and venous thrombosis. METHODS: In this retrospective, multicentre and observational study we analysed the data of 308 confirmed COVID-19 patients with normal D-dimer levels at initial admission. After propensity score matching (PSM) patients were grouped; Group 1; patients who received LMWH with D-dimer ≤0.5 mg/L, Group 2; patients who received LMWH after D-dimer levels exceeded 0.5 mg/L, and Group 3; patients who did not receive LMWH. RESULTS: After PSM, each group comprised 40 patients. The patients in Group1 had the best clinical outcomes compared to the other groups. Group 3 had the worst clinical outcomes (p<0.005). The benefit of LMWH increased with early prophylactic therapy especially when started while the D-dimer levels were ≤0.5 mg/L. CONCLUSION: Our results strongly suggest that proactive LMWH therapy improves clinical outcomes in hospitalized COVID-19 patients even with normal D-dimer levels (≤ 0.5 mg/L) (Tab. 3, Fig. 2, Ref. 34).

Microchip-based structure determination of low-molecular weight proteins using cryo-electron microscopy.

Interest in cryo-Electron Microscopy (EM) imaging has skyrocketed in recent years due to its pristine views of macromolecules and materials. As advances in instrumentation and computing algorithms spurred this progress, there is renewed focus to address specimen-related challenges. Here we contribute a microchip-based toolkit to perform complementary structural and biochemical analysis on low-molecular weight proteins. As a model system, we used the SARS-CoV-2 nucleocapsid (N) protein (48 kDa) due to its stability and important role in therapeutic development. Cryo-EM structures of the N protein monomer revealed a flexible N-terminal "top hat" motif and a helical-rich C-terminal domain. To complement our structural findings, we engineered microchip-based immunoprecipitation assays that led to the discovery of the first antibody binding site on the N protein. The data also facilitated molecular modeling of a variety of pandemic and common cold-related coronavirus proteins. Such insights may guide future pandemic-preparedness protocols through immuno-engineering strategies to mitigate viral outbreaks.

[BCG, muramylpeptides, trained immunity (part II): a low molecular weight alternative to multicomponent bacterial immunostimulants for prevention of respiratory infections during a pandemic].

During a pandemic, nonspecific immunoprophylaxis of SARS-CoV-2 infection and other acute respiratory infections (ARI), which can worsen the course of COVID-19, is increasingly in demand in addition to specific immunization. BCG vaccine appears to be one of the candidate immunostimulants in this regard. At the same time, other microbe-derived preparations capable of inducing a state of trained immunity deserve attention. BCG and other bacterial immunostimulatory agents containing a large number of biologically active subunits have long been considered as objects of search for promising pharmacological substances. The review analyzes the linkages between BCG, mycobacterial adjuvants, bacterial lysates, trained immunity, muramylpeptides (MPs) and NOD2 receptors in light of the choice of a low molecular weight alternative to multicomponent bacterial immunostimulants for ARI prevention during the COVID-19 pandemic. The search for key molecules by which bacteria stimulate innate and adaptive immune responses proceeds in a spiral. On different loops of this spiral, MPs have repeatedly reproduced the nonspecific effects of multicomponent bacterial adjuvants, vaccines and immunostimulants. MPs and peptidoglycans containing MPs determine the adjuvant properties of the cell walls of mycobacteria and their peptide-glycolipid fraction (wax D). MPs were able to replace Mycobacterium tuberculosis in complete Freunds adjuvant. MPs determine the NOD2-dependent ability of BCG to induce trained immunity. Probably, MPs provide NOD2-mediated long-term prophylactic action of bacterial lysates. All of the above has prompted revisiting the previously obtained evidence of the efficacy of glucosaminylmuramyl dipeptide (GMDP) as a NOD2 agonist in treatment/prevention of respiratory infections. We speculate here that MPs, in particular GMDP, at rational dosing regimens will be able to reproduce many aspects of the nonspecific effects of BCG and multicomponent bacterial immunostimulants in preventing ARI during the COVID-19 pandemic and in the post-pandemic period.

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.

EDTA-K2 Improves the Detection Sensitivity of SARS-CoV-2 IgM and IgG Antibodies by Chelating Colloidal Gold in the Immunochromatographic Assay.

OBJECTIVE: The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is now rapidly spreading globally. Serological tests are an important method to assist in the diagnosis of COVID-19, used for epidemiological investigations. In this study, we aimed to investigate the impact of different types of vacuum collection tubes on the detection of SARS-CoV-2 IgM and IgG antibodies, using the colloidal gold immunochromatographic assay (GICA). PATIENTS AND METHODS: A total of 112 patients with COVID-19 and 200 healthy control subjects with no infection were enrolled in this study. Their serum and plasma were collected into four different types of vacuum blood collection tubes. SARS-CoV-2 IgM and IgG specific antibodies in the plasma and serum were then detected by GICA and chemiluminescence assay (CA), respectively. In addition, the particle sizes of different colloidal gold solutions in the presence of different anticoagulants and coagulants were evaluated by both laser diffraction (Malvern) and confocal laser microscope, respectively. RESULTS: Our results revealed that anticoagulated plasma with EDTA-K2 improved the positive detection rate of SARS-CoV-2 IgM antibodies. Furthermore, our results shown that the detection results by GICA and CA were highly consistent, especially, the results of EDTA-K2 anticoagulated plasma detected by GICA was more consistent with CA results. We confirmed that EDTA-K2 could improve the detection sensitivity of SARS-CoV-2 IgG antibodies by chelating excessive colloidal gold compared with sodium citrate or lithium heparin, these methodologies did not appear to cause false positives. Colloidal gold particles could be chelated and aggregated by EDTA-K2, but not by sodium citrate, lithium heparin and coagulants. CONCLUSION: GICA is widely used to detect antibodies for the advantages of convenient, fast, low cost, suitable for screening large sample and require minimal equipment. In this study, we found that EDTA-K2 amplified the positive antibody signal by chelating colloidal gold and improved the detection sensitivity of SARS-CoV-2 IgM and IgG antibodies when using the GICA. Therefore, we suggested that EDTA-K2 anticoagulated plasma was more suitable for the detection of SARS-CoV-2 antibodies.

Viral Proteins under 70 Kilodaltons in Size Prevent the Development of Long-Lasting B-Cell Immune Memory and IgG2a Prevention in COVID-19 Vaccines.

Coronavirus disease 2019 (COVID-19) consists of a severe involvement of the lower respiratory tract leading to an acute respiratory syndrome. But there exist other infectious respiratory syndromes that have the same initial respiratory symptoms, show similar pattern in the size of the antigenic proteins and release comparable cytokines pathways, but with an unlike response magnitude. Here we propose that COVID-19 disease wrong response in the host immune system can be explained in the perspective of the antigen viral size. In COVID-19 sepsis, the < 70 kDa antigens activate the B-cell receptor (BCR), which modulates the shift in the pattern of T-helper 1 (Th1) to Th2 cytokines, increases the release of interleukin-10 (IL-10) and the up-regulation of the membrane form of tumor necrosis factor alpha (TNF-α), promoting the production of immunoglobulin G1 (IgG1)- and IgG3-neutralizing antibodies, but failing in IgG2a production and in developing long-lasting B-cell immune memory. The sustained infected cells lysis overfeeds high levels of viral proteins < 70 kDa, increases B-cell activation and, in the shift from a Th1 to a Th2 immune response, can trigger a cytokine storm. The continuous BCR activation increases IL-10 release that can lead to cytokine storm, apoptosis, and immune paralysis. Here, we propose a new vaccine design using the polymerization of viral antigens that could be ready in short time, would be cheap and easy to develop because it is based on classic technologies available in every country, is safe because it does not employ genetic material, and would able to promote long-lasting B-cell immune memory and IgG2a production.

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic, or prophylactic. As with other betacoronaviruses, attachment and entry of SARS-CoV-2 are mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin-converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third-generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in antiviral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH, and 6-O-desulfated enoxaparin with 50% inhibitory concentrations (IC50s) of 5.99 µg/liter, 1.08 mg/liter, 1.77 µg/liter, and 5.86 mg/liter, respectively. In summary, several sulfated polysaccharides show potent anti-SARS-CoV-2 activity and can be developed for prophylactic as well as therapeutic purposes.IMPORTANCE The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, in late 2019 and its subsequent spread to the rest of the world has created a pandemic situation unprecedented in modern history. While ACE2 has been identified as the viral receptor, cellular polysaccharides have also been implicated in virus entry. The SARS-CoV-2 spike glycoprotein (SGP) binds to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we report structure-based differences in antiviral activity and affinity to SGP for several sulfated polysaccharides, including both well-characterized FDA-approved drugs and novel marine sulfated polysaccharides, which can be developed for prophylactic as well as therapeutic purposes.