scholarly journals Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

ACS Omega ◽  
2020 ◽  
Author(s):  
Natalia G. Herrera ◽  
Nicholas C. Morano ◽  
Alev Celikgil ◽  
George I. Georgiev ◽  
Ryan J. Malonis ◽  
...  
Keyword(s):  
Antigenic Analysis ◽  
S Protein

scholarly journals Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis

2020 ◽  
Author(s):  
Natalia G. Herrera ◽  
Nicholas C. Morano ◽  
Alev Celikgil ◽  
George I. Georgiev ◽  
Ryan J. Malonis ◽  
...  
Keyword(s):  
Cell Lines ◽  
Protein Microarray ◽  
The Novel ◽  
Antigenic Analysis ◽  
High Quality ◽  
Health Crisis ◽  
S Protein ◽  
Global Pandemic ◽  
S Proteins

ABSTRACTCoronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F™ and ExpiCHO-S™ cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-S™ cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs, and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.

DNA-Directed Patterning for Versatile Validation and Characterization of a Lipid-Based Nanoparticle Model of SARS-CoV-2

2021 ◽  
Author(s):  
Molly Kozminsky ◽  
Thomas Carey ◽  
Lydia L. Sohn
Keyword(s):  
High Throughput ◽  
Glass Substrate ◽  
Rapid Development ◽  
Neutralizing Antibody ◽  
S Protein ◽  
Biological Relevance ◽  
The Face ◽  
Great Control ◽  
Tetraspanin Cd63

Lipid-based nanoparticles have risen to the forefront of the COVID-19 pandemic—from encapsulation of vaccine components to modeling the virus, itself. Their rapid development in the face of the volatile nature of the pandemic requires high-throughput, highly flexible methods for characterization. DNA-directed patterning is a versatile method to immobilize and segregate lipid-based nanoparticles for subsequent analysis. DNA-directed patterning selectively conjugates oligonucleotides onto a glass substrate and then hybridizes them to complementary oligonucleotides tagged to the liposomes, thereby patterning them with great control and precision. The power of this method is demonstrated by characterizing a novel recapitulative lipid-based nanoparticle model of SARS-CoV-2 —S-liposomes— which present the SARS-CoV-2 spike (S) protein on their surfaces. Patterning of a mixture of S-liposomes and liposomes that display the tetraspanin CD63 into discrete regions of a substrate is used to show that ACE2 specifically binds to S-liposomes. Importantly, DNA-directed patterning of S-liposomes is used to verify the performance of a commercially available neutralizing antibody against the S protein. Ultimately, the introduction of S-liposomes to ACE2-expressing cells demonstrates the biological relevance of DNA-directed patterning. Overall, DNA-directed patterning enables a wide variety of custom assays for the characterization of any lipid-based nanoparticle.

scholarly journals Molecular characterization of porcine epidemic diarrhoea virus (PEDV) in Poland reveals the presence of swine enteric coronavirus (SeCoV) sequence in S gene

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258318
Author(s):  
Marta Antas ◽  
Monika Olech ◽  
Anna Szczotka-Bochniarz
Keyword(s):  
Phylogenetic Analysis ◽  
Molecular Characterization ◽  
Rna Virus ◽  
Economic Losses ◽  
High Morbidity ◽  
Antigenic Analysis ◽  
S Gene ◽  
Porcine Epidemic Diarrhoea Virus ◽  
Diarrhoea Virus

Porcine epidemic diarrhoea (PED) is a highly contagious enteric viral disease of pigs with a high morbidity and mortality rate, which ultimately results in huge economic losses in the pig production sector. The etiological agent of this disease is the porcine epidemic diarrhoea virus (PEDV) which is an enveloped, positive single-stranded RNA virus. The aim of this study was to perform molecular characterization of PEDV to identify the strains circulating in Poland. In this study, 662 faecal samples from 2015 to 2021 were tested with reverse transcription quantitative real-time PCR (RT-qPCR) and the results showed that 3.8% of the tested samples revealed a positive result for PEDV. A phylogenetic analysis of the complete genome and complete S gene sequences showed that Polish PEDV strains belonged to the G1b (S-INDEL) subgroup and were closely related to the European PEDV strains isolated from 2014 to 2019. Furthermore, RDP4 analysis revealed that the Polish PEDV strains harboured a recombinant fragment of ~400 nt in the 5’ end of S gene with PEDV and swine enteric coronavirus (SeCoV) being the major and minor parents, respectively. Antigenic analysis showed that the aa sequences of neutralizing epitopes were conserved among the Polish PEDV strains. Only one strain, #0100/5P, had a unique substitution in the COE epitope. However, Polish PEDV strains showed several substitutions, especially in the COE antigen, as compared to the classical strain CV777. To the best of our knowledge, this is the first report concerning the molecular characterization of porcine epidemic diarrhoea virus strains, as well as the first phylogenetic analysis for PEDV in Poland.

scholarly journals Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins

2020 ◽  
pp. mcp.RA120.002295 ◽  
Author(s):  
Yong Zhang ◽  
Wanjun Zhao ◽  
Yonghong Mao ◽  
Yaohui Chen ◽  
Shisheng Wang ◽  
...  
Keyword(s):  
Insect Cells ◽  
Host Cells ◽  
Protein Subunit ◽  
Genome Sequences ◽  
Complex Type ◽  
Site Specific ◽  
Differential Processing ◽  
S Protein ◽  
Shed Light

The glycoprotein spike (S) on the surface of SARS-CoV-2 is a determinant for viral invasion and host immune response. Herein, we characterized the site-specific N-glycosylation of S protein at the level of intact glycopeptides. All 22 potential N-glycosites were identified in the S-protein protomer and were found to be preserved among the 753 SARS-CoV-2 genome sequences. The glycosites exhibited glycoform heterogeneity as expected for a human cell-expressed protein subunit. We identified masses that correspond to 157 N-glycans, primarily of the complex type. In contrast, the insect cell-expressed S protein contained 38 N-glycans, completely of the high-mannose type. Our results revealed that the glycan types were highly determined by the differential processing of N-glycans among human and insect cells, regardless of the glycosites’ location. Moreover, the N-glycan compositions were conserved among different sizes of subunits. Our study indicate that the S protein N-glycosylation occurs regularly at each site, albeit the occupied N-glycans were diverse and heterogenous. This N-glycosylation landscape and the differential N-glycan patterns among distinct host cells are expected to shed light on the infection mechanism and present a positive view for the development of vaccines and targeted drugs.

scholarly journals Isolation and Characterization of Influenza C Viruses in the Philippines and Japan

2014 ◽  
Vol 53 (3) ◽  
pp. 847-858 ◽  
Author(s):  
Takashi Odagiri ◽  
Yoko Matsuzaki ◽  
Michiko Okamoto ◽  
Akira Suzuki ◽  
Mariko Saito ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Mdck Cells ◽  
Severe Pneumonia ◽  
Respiratory Illness ◽  
The Philippines ◽  
Antigenic Analysis ◽  
Isolation And Characterization ◽  
Gene Sequence Analysis ◽  
Influenza C Virus

From November 2009 to December 2013 in the Philippines, 15 influenza C viruses were isolated, using MDCK cells, from specimens obtained from children with severe pneumonia and influenza-like illness (ILI). This is the first report of influenza C virus isolation in the Philippines. In addition, from January 2008 to December 2013, 7 influenza C viruses were isolated from specimens that were obtained from children with acute respiratory illness (ARI) in Sendai city, Japan. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoprotein showed that 19 strains (12 from the Philippines and 7 from Japan) were similar to the influenza C virus reference strain C/Sao Paulo/378/82 (SP82). Phylogenetic analysis of the HE gene showed that the strains from the Philippines and Japan formed distinct clusters within an SP82-related lineage. The clusters that included the Philippine and Japanese strains were shown to have diverged from a common ancestor around 1993. In addition, phylogenetic analysis of the internal genes showed that all strains isolated in the Philippines and Japan had emerged through reassortment events. The composition of the internal genes of the Philippine strains was different from that of the Japanese strains, although all strains were classified into an SP82-related lineage by HE gene sequence analysis. These observations suggest that the influenza C viruses analyzed here had emerged through different reassortment events; however, the time and place at which the reassortment events occurred were not determined.

scholarly journals Determination of serotyping antigens, clonal analysis and genetic characterization of the 4CMenB vaccine antigen genes in invasive Neisseria meningitidis from Western Canada, 2009 to 2013

2014 ◽  
Vol 63 (11) ◽  
pp. 1490-1499 ◽  
Author(s):  
Dennis K. S. Law ◽  
Jianwei Zhou ◽  
Saul Deng ◽  
Linda Hoang ◽  
Gregory Tyrrell ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Clonal Complex ◽  
Factor H ◽  
Vaccine Antigen ◽  
Western Canada ◽  
Antigenic Analysis ◽  
Clonal Nature ◽  
4Cmenb Vaccine

This study examined invasive Neisseria meningitidis recovered from invasive meningococcal disease (IMD) cases in Western Canada between 2009 and 2013. A total of 161 isolates from individual IMD cases were analysed for serogroup, serotype, serosubtype, PorA genotype, multi-locus sequence type and nucleotide sequence of their 4CMenB vaccine antigen genes. Sixty-nine isolates were serogroup B (MenB), 47 were serogroup Y (MenY), 22 were serogroup C (MenC), 19 were serogroup W (MenW), three were serogroup E and one was non-encapsulated. MenC, MenY and MenW were mainly clonal, represented primarily by clonal complex (cc) 11, cc23 or cc167, and cc22, respectively. In contrast, MenB were composed of eight different ccs together with 11 isolates not assigned to any known cc. Antigenic analysis and PorA genotyping confirmed the heterogeneity of MenB isolates, while such results supported the clonal nature of most MenC, MenY and MenW isolates. Thirty-four (21.1 %) isolates had at least one gene that encoded one matching vaccine protein component of the 4CMenB vaccine (i.e. PorA P1.4; fHbp variant 1.1; NHBA peptide 2; and NadA-1, -2, or -3). An additional 18 isolates had genes that encoded variant 1 or subfamily B factor H binding proteins of this same vaccine.

scholarly journals Fibronectin receptors of phagocytes. Characterization of the Arg-Gly-Asp binding proteins of human monocytes and polymorphonuclear leukocytes.

1988 ◽  
Vol 167 (3) ◽  
pp. 777-793 ◽  
Author(s):  
E J Brown ◽  
J L Goodwin
Keyword(s):  
Surface Molecule ◽  
Human Monocytes ◽  
Ligand Specificity ◽  
Cell Binding ◽  
Antigenic Analysis ◽  
Vitronectin Receptor ◽  
Surface Molecules ◽  
Rgd Sequence ◽  
Single Receptor

We have defined the cell surface molecules of human monocytes and PMN that bind to the chymotryptic cell binding domain of Fn and to a synthetic peptide, KYAVTGRGDS, based on the sequence of Fn, by affinity chromatography. Monocytes express two receptors that differ in their affinity for CBD-Sepharose and peptide-Sepharose, but that both recognize the RGD sequence. Only a single receptor is purified from PMN, which resembles the monocyte surface molecule that binds to peptide-Sepharose. These receptors are not part of the Mac-1, LFA-1, p(150,95) family, but do have homology to the platelet Fn receptor, gpIIb/IIIa. Interestingly, the antigenic crossreactivity between gpIIb/IIIa and the phagocyte receptors purified on peptide-Sepharose is largely in the beta chain of the receptors. The alpha chains appear to be distinct, based on molecular weight, antigenic analysis, and ligand specificity. This receptor also seems to be the surface molecule on monocytes that is critical for phagocytosis enhancement by Fn. Thus, we have defined the phagocyte Fn receptor that transduces the signal for increased phagocytosis by monocytes; it may be a third member of a family of adhesion molecules that includes the gpIIb/IIIa of platelets and the vitronectin receptor of fibroblasts.

scholarly journals Characterization of SARS-CoV-2 Variants N501Y.V1 and N501Y.V2 Spike on Viral Infectivity

2021 ◽  
Vol 11 ◽  
Author(s):  
Haijun Tang ◽  
Long Gao ◽  
Zhao Wu ◽  
Fang Meng ◽  
Xin Zhao ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Viral Infectivity ◽  
Infection Efficiency ◽  
S Protein ◽  
The United Kingdom ◽  
Rapid Spread ◽  
Global Spread ◽  
Vesicular Stomatitis ◽  
The Stability

SARS-coronavirus 2 (SARS-CoV-2), pathogen of coronavirus disease 2019 (COVID-19), is constantly evolving to adapt to the host and evade antiviral immunity. The newly emerging variants N501Y.V1 (B.1.1.7) and N501Y.V2 (B.1.351), first reported in the United Kingdom and South Africa respectively, raised concerns due to the unusually rapid global spread. The mutations in spike (S) protein may contribute to the rapid spread of these variants. Here, with a vesicular stomatitis virus (VSV)-based pseudotype system, we demonstrated that the pseudovirus bearing N501Y.V2 S protein has higher infection efficiency than pseudovirus with wildtype (WT) and D614G S protein. Moreover, pseudovirus with N501Y.V1 or N501Y.V2 S protein has better thermal stability than WT and D614G, suggesting these mutations of variants may increase the stability of SARS-CoV-2 S protein and virion. However, the pseudovirus bearing N501Y.V1 or N501Y.V2 S protein has similar sensitivity to inhibitors of protease and endocytosis with WT and D614G. These findings could be of value in preventing the spread of virus and developing drugs for emerging SARS-CoV-2 variants.

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