Highly Sensitive Poly-N-isopropylacrylamide Microgel-based Electrochemical Biosensor for the Detection of SARS-COV-2 Spike Protein

Objective Late 2019 witnessed the outbreak and widespread transmission of coronavirus disease 2019(COVID-19),a new,highly contagious disease caused by novel severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Consequently,considerable attention has been paid to the development of new diagnostic tools for the early detection of SARS-CoV-2.Methods In this study,a new poly-N-isopropylacrylamide microgel-based electrochemical sensor was explored to detect the SARS-CoV-2 spike protein(S protein)in human saliva.The microgel was composed of a copolymer of N-isopropylacrylamide and acrylic acid,and gold nanoparticles were encapsulated within the microgel through facile and economical fabrication.The electrochemical performance of the sensor was evaluated through differential pulse voltammetry.Results Under optimal experimental conditions,the linear range of the sensor was 10-13-10-9 mg/m L,whereas the detection limit was 9.55 fg/mL.Furthermore,the S protein was instilled in artificial saliva as the infected human saliva model,and the sensing platform showed satisfactory detection capability.Conclusion The sensing platform exhibited excellent specificity and sensitivity in detecting spike protein,indicating its potential application for the time-saving and inexpensive detection of SARS-CoV-2.

Identification of an HLA-A~* 0201-restricted CD8^+ T-cell epitope SSp-1 of SARS-CoV spike protein

A novel coronavirus, severe acute respiratory syndrome (SA RS)-associated coronavirus (SARS-CoV), has been identified as the causal agent of SARS. Spike (S) protein is a major structural glycoprotein of the SARS virus and a potential target for SARS-specific cell-mediated immune responses. A pa nel of S protein-derived peptides was tested for their binding affinity to HLA -A *0201 molecules. Peptides with high affinity for HLA-A *0201 were then as se ssed for their capacity to elicit specific immune responses mediated by cytotoxi c T lymphocytes (CTLs) both in vivo, in HLA-A2.1/K b transgenic mice, a nd in vitro, from peripheral blood lymphocytes (PBLs) harvested from healthy HLA-A 2.1 + donors. SARS-CoV protein-derived peptide-1 (SSp-1 RLNEVAKNL), induced pepti de-specific CTLs both in vivo (transgenic mice) and in vitro (human PBL s), which specifically released interferon-gamma (IFN-gamma) upon stimulation with SSp-1-pulsed autologous dendritic cells (DCs) or T2 cells. SSp-1-specif ic CTLs also lysed major histocompatibility complex (MHC)-matched tumor cell lines engineered to express S proteins. HLA-A *0201-SSp-1 tetramer staining re vealed the presence of significant populations of SSp-1-specific CTLs in SSp- 1-induced CD8 + T cells. We propose that the newly identified epitope SSp-1 w ill help in the characterization of virus control mechanisms and immunopathology in SARS-CoV infection, and may be relevant to the development of immunotherape utic approaches for SARS.

CLONING SEGMENT SPIKE PROTEIN GENE OF SARS-COV AND ITS EXPRESSION IN ESCHERICHIA COLI

Objective Expressing and purifying the segment of SARS-CoV spike protein in E.Coli. Methods The target gene was obtained by RT-PCR. The PCR product was cloned into pEGM- T Easy Vector, sequencing and double restriction digestion ( BamHⅠ,PstⅠ) were performed. The target gene was subcloned into PQE30 expression vector. The gene was expressed in the E.coli strain M15 cells induced by IPTG. The protein was purified with a nickel HiTrap chelating metal affinity column. Results The recombinant expression plasmid was successfully constructed and the protein was well expressed in E. coli strain M15 cells. The ideal pure protein was obtained by purification. Western blotting analysis suggested the protein could act with the convalescent sera of lab confirmed SARS patients. Conclusion The segment of SARS-CoV spike protein was well expressed and purified, and can be applied in diagnosis and immunological research of SARS.

The Immunity Induced by Recombinant Spike Proteins of SARS Coronavirus in Balb/c Mice

The immune effect of two recombinant protein fragments of spike protein in severe acute respiratory syndrome coronavirus (SARS CoV) was investigated in Balb/c mice. Two partial spike gene fragments S1 (322-1464 bp) and S2 (2170-2814 bp) of SARS coronavirus were amplified by RT-PCR, and cloned into pET-23a prokaryotic expression vector, then transformed into competent Escherichia E.coli BL21 (DE3)(pLysS) respectively. Recombinant proteins were expressed and puri- fied by Ni2+ immobilized metal ion affinity chromatography. The purified proteins mixed with com- plete Freund adjuvant were injected into Balb/c mice three times at a two-week interval. High titer antibody was detected in the serum of immunized Balb/c mice, and mice immunized with S1 protein produced high titer IgG1, IgG2a, IgG2b and IgG3, while those immunized with S2 protein produced high titer IgG1, IgG2a, but lower titer IgG2b and IgG3. Serum IFN-γ concentration was increased significantly but the concentrations of IL-2, IL-4 and IL-10 had no significant change. And a marked increase was observed in the number of spleen CD8+ T cells. The results showed that recombinant proteins of SARS coronavirus spike protein induced hormonal and cellular immune response in Balb/c mice.

Mutations in spike protein and allele variations in ACE2 impact targeted therapy strategies against SARS-CoV-2

Coronavirus disease 2019(COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2(SARS-Co V-2), has spread rapidly worldwide with high rates of transmission and substantial mortality. To date, however, no effective treatments or enough vaccines for COVID-19 are available. The roles of angiotensin converting enzyme 2(ACE2) and spike protein in the treatment of COVID-19 are major areas of research. In this study, we explored the potential of ACE2 and spike protein as targets for the development of antiviral agents against SARS-Co V-2. We analyzed clinical data, genetic data, and receptor binding capability.Clinical data revealed that COVID-19 patients with comorbidities related to an abnormal reninangiotensin system exhibited more early symptoms and poorer prognoses. However, the relationship between ACE2 expression and COVID-19progression is still not clear. Furthermore, if ACE2 is not a good targetable protein, it would not be applicable across a wide range of populations. The spike-S1 receptor-binding domain that interacts with ACE2 showed various amino acid mutations based on sequence analysis. We identified two spike-S1 point mutations(V354 F and V470 A) by receptorligand docking and binding enzyme-linked immunosorbent assays. These variants enhanced the binding of the spike protein to ACE2 receptors and were potentially associated with increased infectivity. Importantly, the number of patients infected with the V354 F and V470 A mutants has increased with the development of the SARS-Co V-2 pandemic. These results suggest that ACE2 and spike-S1 are likely not ideal targets for the design of peptide drugs to treat COVID-19 in different populations.

Mass Spectrometry Analysis of Newly Emerging Coronavirus HCoV-19 Spike Protein and Human ACE2 Reveals Camouflaging Glycans and Unique Post-Translational Modifications

The coronavirus disease 2019(COVID-19)pandemic has led to worldwide efforts to understand the biological traits of the newly identified human coronavirus(HCoV-19)virus.In this mass spectrometry(MS)-based study,we reveal that out of 21 possible glycosites in the HCoV-19 spike protein(S protein),20 are completely occupied by N-glycans,predominantly of the oligomannose type.All seven glycosylation sites in human angiotensin I converting enzyme 2(hACE2)were found to be completely occupied,mainly by complex N-glycans.However,glycosylation did not directly contribute to the binding affinity between HCoV-19 S protein and hACE2.Additional post-translational modification(PTM)was identified,including multiple methylated sites in both proteins and multiple sites with hydroxylproline in hACE2.Refined structural models of HCoV-19 S protein and hACE2 were built by adding N-glycan and PTMs to recently published cryogenic electron microscopy structures.The PTM and glycan maps of HCoV-19 S protein and hACE2 provide additional structural details for studying the mechanisms underlying host attachment and the immune response of HCoV-19,as well as knowledge for developing desperately needed remedies and vaccines.

Identification of an HLA-A*0201-restricted CD8^+ T-cell epitope SSp-1 of SARS-CoV spike protein

A novel coronavirus, severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV), has been identified as the causal agent of SARS. Spike (S) protein is a major structural glycoprotein of the SARS virus and a potential target for SARS-specific cell-mediated immune responses. A panel of S protein-derived peptides was tested for their binding affinity to HLA-A*0201 molecules. Peptides with high affinity for HLA-A*0201 were then assessed for their capacity to elicit specific immune responses mediated by cytotoxic T lymphocytes (CTLs) both in vivo, in HLA-A2.1/K b transgenic mice, and in vitro, from peripheral blood lymphocytes (PBLs) harvested from healthy HLA-A2.1(+) donors. SARS-CoV protein-derived peptide-1 (SSp-1 RLNEVAKNL), induced peptide-specific CTLs both in vivo (transgenic mice) and in vitro (human PBLs), which specifically released interferon-gamma (IFN-γ) upon stimulation with SSp-1-pulsed autologous dendritic cells (DCs) or T2 cells. SSp-1-specific CTLs also lysed major histocompatibility complex (MHC)-matched tumor cell lines engineered to express S proteins. HLA-A*0201-SSp-1 tetramer staining revealed the presence of significant populations of SSp-1-specific CTLs in SSp-1-induced CD8+ T cells. We propose that the newly identified epitope SSp-1 will help in the characterization of virus control mechanisms and immunopathology in SARS-CoV infection, and may be relevant to the development of immunotherapeutic approaches for SARS.

<i>Morinda citrifolia</i>(Noni) Fruit Juice Inhibits SARS-CoV-2 Spike Protein Binding of Angiotensin-Converting Enzyme 2 (ACE2)

COVID-19 is a global pandemic that has claimed millions of lives. This disease is caused by a coronavirus, SARS-CoV-2, which requires the binding of its spike protein to angiotensin-converting enzyme 2 (ACE2) for infection of the host cell. Morinda citrifolia (noni) fruit juice has antiviral activity that involves enhancement of immune system function. SARS-CoV-2 spike-ACE2 interaction experiments were carried out to further investigate the antiviral properties of noni juice and its major iridoids. Noni juice inhibited binding by approximately 69%. Scandoside was the most active of the three iridoids evaluated, reducing average spike protein-ACE2 interaction by 79.25%. The iridoids worked synergistically towards inhibiting spike protein binding when assayed together, improving activity by more than 22% above the expected level. But the modest activity of the most abundant iridoid, deacetylasperulosidic acid, indicates that other phytochemicals (i.e. scopoletin, quercetin, rutin and kaempferol) are also involved. Our results suggest that the presence of several biological active phytochemicals in noni juice enhances resistance to SARS-CoV-2 by interfering with its ability to bind ACE2. This is a new and significant anti-viral mechanism of noni juice that does not directly involve its immunomodulatory properties.

Receptor-binding domain of SARS-Cov spike protein: Soluble expression in E.coli, purification and functional characterization

瞄准：日冕病毒的尖铁蛋白质为病毒绑定，熔化和入口负责，并且是中和抗体的主要 inducer。这篇论文是发现严重尖锐呼吸联系症候群的日冕病毒(SARS-Cov ) 的一个可溶、功能的 recombinant 受体绑定领域，并且分析它的受体绑定能力。方法：三个熔化标签(谷胱甘肽 S-transferase， GST；thioredoxin， Trx；麦芽糖绑定蛋白质， MBP ) ，它最好贡献增加的溶解度并且到便于异种蛋白质的合适的合拢，被用来在埃希氏杆菌属关口 i 获得 RBD 蛋白质的可溶、功能的表示(BL21 (DE3 ) 和 Rosetta-gamiB (DE3 ) 拉紧) 。净化的可溶的 RBD 蛋白质的受体绑定能力然后被 ELISA 和流动血细胞计数试金检测。结果：当在许多不同文化和正式就职条件下面在 BL21 (DE3 ) 和 Rosetta-gamiB (DE3 ) 作为 TrxA 标签形式熔化了时， SARS-Cov 尖铁蛋白质的 RBD 被表示为包括身体。并且当 RBD 被表示时，当 MBP 标注了形式， SDS 页上没有可见表示乐队。仅仅 GST 标注了 RBD 是可溶的在 BL21 (DE3 ) 表示了，并且蛋白质被巢菜净化主要层析系统。ELISA 数据证明那 GST/RBD 抗原带着 anti-RBD 老鼠有阳性反应单音的同种细胞的抗体 1A5。进一步的流动血细胞计数试金表明了 RBD ACE2 的有约束力的能力的高效率(变换血管收缩素的酶 2 ) 积极 Vero E6 房间。并且 ACE2 与 SARS-Cov 尖铁蛋白质作为反省的细胞的受体被证明一个起始亲密关系的相互作用。到 Vero E6 房间的 GST 和 GST/RBD 绑定的几何平均数分别地是 77.08 和 352.73。结论：在这篇论文，我们得到标注 RBD 蛋白质在 E.coli BL21 (DE3 ) 表示了的足够的可溶的 N 终端 GST；从 ELISA 和流动血细胞计数试金的数据证明 recombinant 蛋白质高效地对 ACE2 积极 Vero E6 房间功能、有约束力。并且从 E.coli 导出的 recombinant RBD 能习惯于与受体开发对块 S 蛋白质绑定疫苗的子单元并且到抵销 SARS-Cov 感染。

Identification of SARS-CoV-2 Spike Protein Inhibitors from Urtica dioica to Develop Herbal-Based Therapeutics Against COVID-19

Objective: The high transmission rate and mutations of SARS-Co V-2 have made it a global pandemic, and the shortage of any effective clinical treatment has created such a commotion. There are some synthetic antiviral drugs, such as remdesivir and lopinavir that are being repurposed to treat SARS-Co V-2, but all of these demonstrate extreme side effects in humans. Hence, promoting herbal-based drug development has become crucial as they are cost-effective and have lesser or no side effects. Urtica dioica is abundant in the Himalayan region and the compounds present in it have shown significant antiviral and anti-SARS activity. Therefore, molecular docking studies were performed to identify SARS-Co V-2 spike protein inhibitors from U. dioica to combat the COVID-19 disease. Materials and Methods: Compounds from U. dioica were screened using the bioinformatic approach, and subsequently, these compounds were docked with the S1 subunit of the COVID-19 spike protein(PDB ID: 6YOR). Molecular docking was carried out using the Py Rx software(0.8 version) and further examined by employing the Discovery Studio Visualizer. Results: About all the selected compounds showed significant binding energy(e.g., beta-sitosterol:-10.3 kcal/mol) in contrast to the control chloroquine phosphate. This binding was observed with the spike protein residues that were common in the old strain and the more contagious newly modified B.1.1.7 strain of SARS-Co V-2. Conclusions: Thus, our study can be used in effective drug development against SARS-Co V-2 and its mutant strains also.

Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies

Coronavirus disease 2019(COVID-19),caused by the novel human coronavirus SARS-CoV-2,is currently a major threat to public health worldwide.The viral spike protein binds the host receptor angiotensin-converting enzyme 2(ACE2)via the receptor-binding domain(RBD),and thus is believed to be a major target to block viral entry.Both SARS-CoV-2 and SARS-CoV share this mechanism.Here we functionally analyzed the key amino acid residues located within receptor binding motif of RBD that may interact with human ACE2 and available neutralizing antibodies.The in vivo experiments showed that immunization with either the SARS-CoV RBD or SARS-CoV-2 RBD was able to induce strong clade-specific neutralizing antibodies in mice;however,the cross-neutralizing activity was much weaker,indicating that there are distinct antigenic features in the RBDs of the two viruses.This finding was confirmed with the available neutralizing monoclonal antibodies against SARS-CoV or SARS-CoV-2.It is worth noting that a newly developed SARS-CoV-2 human antibody,HA001,was able to neutralize SARS-CoV-2,but failed to recognize SARS-CoV.Moreover,the potential epitope residues of HA001 were identified as A475 and F486 in the SARS-CoV-2 RBD,representing new binding sites for neutralizing antibodies.Overall,our study has revealed the presence of different key epitopes between SARS-CoV and SARSCoV-2,which indicates the necessity to develop new prophylactic vaccine and antibody drugs for specific control of the COVID-19 pandemic although the available agents obtained from the SARS-CoV study are unneglectable.

Using the spike protein feature to predict infection risk and monitor the evolutionary dynamic of coronavirus

Background:Coronavirus can cross the species barrier and infect humans with a severe respiratory syndrome.SARS-CoV-2 with potential origin of bat is still circulating in China.In this study,a prediction model is proposed to evaluate the infection risk of non-human-origin coronavirus for early warning.Methods:The spike protein sequences of 2666 coronaviruses were collected from 2019 Novel Coronavirus Resource(2019nCoVR)Database of China National Genomics Data Center on Jan 29,2020.A total of 507 human-origin viruses were regarded as positive samples,whereas 2159 non-human-origin viruses were regarded as negative.To capture the key information of the spike protein,three feature encoding algorithms(amino acid composition,AAC;parallel correlation-based pseudo-amino-acid composition,PC-PseAAC and G-gap dipeptide composition,GGAP)were used to train 41 random forest models.The optimal feature with the best performance was identified by the multidimensional scaling method,which was used to explore the pattern of human coronavirus.Results:The 10-fold cross-validation results showed that well performance was achieved with the use of the GGAP(g=3)feature.The predictive model achieved the maximum ACC of 98.18%coupled with the Matthews correlation coefficient(MCC)of 0.9638.Seven clusters for human coronaviruses(229E,NL63,OC43,HKU1,MERS-CoV,SARS-CoV,and SARS-CoV-2)were found.The cluster for SARS-CoV-2 was very close to that for SARS-CoV,which suggests that both of viruses have the same human receptor(angiotensin converting enzyme II).The big gap in the distance curve suggests that the origin of SARS-CoV-2 is not clear and further surveillance in the field should be made continuously.The smooth distance curve for SARS-CoV suggests that its close relatives still exist in nature and public health is challenged as usual.Conclusions:The optimal feature(GGAP,g=3)performed well in terms of predicting infection risk and could be used to explore the evolutionary dynamic in a simple,fast and large-scale manner.The study may be beneficial for the surveillance of the genome mutation of coronavirus in the field.

SARS-CoV-2 spike protein binds to bacterial lipopolysaccharide and boosts proinflammatory activity

There is a link between high lipopolysaccharide(LPS)levels in the blood and the metabolic syndrome,and metabolic syndrome predisposes patients to severe COVID-19.Here,we define an interaction between SARS-CoV-2 spike(S)protein and LPS,leading to aggravated inflammation in vitro and in vivo.Native gel electrophoresis demonstrated that SARS-CoV-2 S protein binds to LPS.Microscale thermophoresis yielded a of〜47 nM for the interaction.Computational modeling and all-atom molecular dynamics simulations further substantiated the experimental results,identifying a main LPS-binding site in SARS-CoV-2 S protein.S protein,when combined with low levels of LPS,boosted nuclear factor-kappa B(NF-k B)activation in monocytic THP-1 cells and cytokine responses in human blood and peripheral blood mononuclear cells,respectively.The in vitro inflammatory response was further validated by employing NF-kB reporter mice and in vivo bioimaging.Dynamic light scattering,transmission electron microscopy,and LPS-FITC analyses demonstrated that S protein modulated the aggregation state of LPS,providing a molecular explanation for the observed boosting effect.Taken together,our results provide an interesting molecular link between excessive inflammation during infection with SARS-CoV-2 and comorbidities involving increased levels of bacterial endotoxins.

Comprehensive interactome analysis of the spike protein of swine acute diarrhea syndrome coronavirus

Swine acute diarrhea syndrome coronavirus(SADS‐CoV)is a recently discovered coronavirus that causes severe and acute diarrhea and rapid weight loss in piglets.SADS‐CoV was reported to be capable of infecting cell lines derived from diverse species,including bats,mice,hamsters,rats,chickens,pigs,nonhuman primates,and humans,implying its high risk of cross‐species infection.However,its receptor is still unknown.In this study,the receptor‐binding domain of the SADS‐CoV spike(S)protein was purified and then subjected to affinity purification(AP)‐coupled mass spectrometry(MS)‐based proteomic analysis to identify the interactors of the SADS‐CoV S protein.Forty‐three host proteins were identified,and a Gene Ontology analysis indicated that these interactors can be grouped into categories such as“cell‐cell adhesion”,“translation”“viral transcription”,suggesting that these processes may participate in the SADS‐CoV life cycles.RNA interference‐based screening of these interactors indicated that PPIB and vimentin can affect SADS‐CoV replication.Our study provides an overarching view into the host interactome of the SADS‐CoV S protein and highlights potential targets for the development of therapeutics against SADS‐CoV.

Immune response in Balb/c mice induced by recombinant spike protein of severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a highly aggressive pathogen that caused SARS in 2003 and 2004.^(1-4) Spike protein (S) on the surface of virus particles mediates the attachment of the virus to cell surface receptors and induces the fusion of viral and cellular membranes. According to the epitope analysis and structure of S protein, we used two fragments of S protein S1 (108-488aa) and S2 (723-938aa) expressed in Escherichia coli and immunized Balb/c mice to investigate the immune response to the recombinant proteins in mice.~5

A Strategy for Searching Antigenic Regions in the SARS-CoV Spike Protein

In the face of the worldwide threat of severe acute respiratory syndrome (SARS) to human life, some of the most urgent challenges are to develop fast and accurate analytical methods for early diagnosis of this disease as well as to create a safe anti-viral vaccine for prevention. To these ends, we investigated the antigenicity of the spike protein (S protein), a major structural protein in the SARS-coronavirus (SARS-CoV). Based upon the theoretical analysis for hydrophobicity of the S protein, 18 peptides were synthesized. Using Enzyme-Linked Immunosorbent Assay (ELISA),these peptides were screened in the sera from SARS patients. According to these results, two fragments of the S gene were amplified by PCR and cloned into pET-32a. Both S fragments were expressed in the BL-21 strain and further purified with an affinity chromatography. These recombinant S fragments were confirmed to have positive cross-reactions with SARS sera, either by Western blot or by ELISA. Our results demonstrated that the potential epitope regions were located at Codons 469-882 in the S protein, and one epitope site was located at Codons 599-620. Identification of antigenic regions in the SARS-CoV S protein may be important for the functional studies of this virus or the development of clinical diagnosis.

SARS-CoV-2 spike protein and RNA dependent RNA polymerase as targets for drug and vaccine development: A review

The present pandemic has posed a crisis to the economy of the world and the health sector.Therefore,the race to expand research to understand some good molecular targets for vaccine and therapeutic development for SARS-CoV-2 is inevitable.The newly discovered coronavirus 2019(COVID-19)is a positive sense,single-stranded RNA,and enveloped virus,assigned to the beta CoV genus.The virus(SARS-CoV-2)is more infectious than the previously detected coronaviruses(MERS and SARS).Findings from many studies have revealed that S protein and RdRp are good targets for drug repositioning,novel therapeutic development(antibodies and small molecule drugs),and vaccine discovery.Therapeutics such as chloroquine,convalescent plasma,monoclonal antibodies,spike binding peptides,and small molecules could alter the ability of S protein to bind to the ACE-2 receptor,and drugs such as remdesivir(targeting SARS-CoV-2 RdRp),favipir,and emetine could prevent SASR-CoV-2 RNA synthesis.The novel vaccines such as mRNA1273(Moderna),3LNP-mRNAs(Pfizer/BioNTech),and ChAdOx1-S(University of Oxford/Astra Zeneca)targeting S protein have proven to be effective in combating the present pandemic.Further exploration of the potential of S protein and RdRp is crucial in fighting the present pandemic.

Dissection of SARS Coronavirus Spike Protein into Discrete Folded Fragments

The spike protein of the severe acute respiratory syndrome coronavirus （SARS-CoV） mediates cell fusion by binding to target cell surface receptors. This paper reports a simple method for dissecting the viral protein and for searching for foldable fragments in a random but systematic manner. The method involves digestion by DNase I to generate a pool of short DNA segments, followed by an additional step of reassembly of these segments to produce a library of DNA fragments with random ends but controllable lengths. To rapidly screen for discrete folded polypeptide fragments, the reassembled gene fragments were further cloned into a vector as N-terminal fusions to a folding reporter gene which was a variant of green fluorescent protein. Two foldable fragments were identified for the SARS-CoV spike protein, which coincide with various anti-SARS peptides derived from the hepated repeat （HR） region 2 of the spike protein. The method should be applicable to other viral proteins to isolate antigen or vaccine candidates, thus providing an alternative to the full-length proteins （subunits） or linear short peptides.

SARS-CoV-2 spike protein as a bacterial lipopolysaccharide delivery system in an overzealous inflammatory cascade

Accumulating evidence indicates a potential role for bacterial lipopolysaccharide(LPs)in the overactivation of the immune response during SARS-CoV-2 infection.LPS is recognized by Toll-like receptor 4,mediating proinflammatory effects.We previously reported that LPS directly interacts with SARS-CoV-2 spike(S)protein and enhances proinflammatory activities.Using native gel electrophoresis and hydrogen-deuterium exchange mass spectrometry,we showed that LPS binds to multiple hydrophobic pockets spanning both the S1 and S2 subunits of the S protein.Molecular simulations validated by a microscale thermophoresis binding assay revealed that LPS binds to the S2 pocket with a lower affinity compared to S1,suggesting a role as an intermediate in LPS transfer.Congruently,nuclear factor-kappa B(NF-kB)activation in monocytic THP-1 cells is strongly boosted by S2.Using NF-kB reporter mice followed by bioimaging,a boosting effect was observed for both S1 and S2,with the former potentially facilitated by proteolysis.The Omicron S variant binds to LPS,but with reduced affinity and LPS boosting in vitro and in vivo.Taken together,the data provide a molecular mechanism by which S protein augments LPS-mediated hyperinflammation.

Spike protein-based epitopes predicted against SARS-CoV-2 through literature mining

Background:With the diffusion of SARS-CoV-2 around the world,human health is being threatened.As there is no effective vaccine yet,the development of the vaccine is urgently in progress.Materials and methods:Immunoinformatics methods were applied to predict epitopes from the Spike protein through mining literature associated with B-and T-cell epitopes prediction published or preprinted since the outbreak of the virus till June 1,2020.3D structure of the Spike protein were obtained(PDB ID:6VSB)for prediction of discontinuous B-cell epitopes and localization of epitopes in the hotspot regions.Results:Methods provided by the Immune Epitope Database(IEDB)server were the most frequently used to predict epitopes.Sequence alignment of the epitopes extracted from literature with the Spike protein demonstrated that the epitopes in different studies converged to multiple short hotspot regions.There were three hotspot regions found in RBD of the Spike protein harboring B-cell linear epitopes(‘RQIAPGQTGKIADYNYKLPD’,‘SYGFQPTNGVGYQ’and‘YAWNRKRISNCVA’)predicted to have high antigenicity score.Two T-cell epitopes(‘KPFERDISTEIYQ’and‘NYNYLYRLFR’)predicted to be highly antigenic in the original studies were discovered in the hotspot region.Toxicity and allergenicity analysis confirmed all the five epitopes are of non-toxin,and four of them are of non-allergen.The five epitopes identified in hotspot regions of RBD were found fully exposed based on the 3D structure of the Spike protein.Conclusion:The five epitopes we discovered from literature mining may be potential candidates for diagnostics and vaccine development against SARS-CoV-2.