scholarly journals Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron

2021 ◽  
Author(s):  
I-Ting Teng ◽  
Alexandra F. Nazzari ◽  
Misook Choe ◽  
Tracy Liu ◽  
Matheus Oliveira de Souza ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Serum Antibody ◽  
Region Of Interest ◽  
Molecular Probes ◽  
Single Step ◽  
Terminal Sequence ◽  
Specific Amino Acid ◽  
Protease Cleavage ◽  
Isolation And Characterization

Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccines, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring the vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS-CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies.

scholarly journals Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes

2020 ◽  
Author(s):  
Tongqing Zhou ◽  
I-Ting Teng ◽  
Adam S. Olia ◽  
Gabriele Cerutti ◽  
Jason Gorman ◽  
...  
Keyword(s):  
Region Of Interest ◽  
Molecular Probes ◽  
Terminal Sequence ◽  
Protease Cleavage ◽  
Isolation And Characterization ◽  
Cryo Electron Microscopy ◽  
Specific Protease ◽  
A Site ◽  
Biotinylated Probes

SummaryBiotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes.

Purification and partial amino acid sequence of thuricin S, a new anti-Listeriabacteriocin from Bacillus thuringiensis

2007 ◽  
Vol 53 (2) ◽  
pp. 284-290 ◽  
Author(s):  
Sonia Chehimi ◽  
François Delalande ◽  
Sophie Sablé ◽  
Mohamed-Rabeh Hajlaoui ◽  
Alain Van Dorsselaer ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Proteinase K ◽  
Terminal Sequence ◽  
Partial Amino Acid Sequence ◽  
Ph Values ◽  
Heat Stable ◽  
Isolation And Characterization ◽  
Terminal Amino ◽  
Edman Sequencing

We report the isolation and characterization of a new bacteriocin, thuricin S, produced by the Bacillus thuringiensis subsp. entomocidus HD198 strain. This antibacterial activity is sensitive to proteinase K, is heat-stable, and is stable at a variety of pH values (3–10.5). The monoisotopic mass of thuricin S purified by high perfomance liquid chromatography, as determined with mass spectrometry ESI-TOF-MS, is 3137.61 Da. Edman sequencing and NanoESI-MS/MS experiments provided the sequence of the 18 N-terminal amino acids. Interestingly, thuricin S has the same N-terminal sequence (DWTXWSXL) as bacthuricin F4 and thuricin 17, produced by B. thuringiensis strains BUPM4 and NEB17, respectively, and could therefore be classified as a new subclass IId bacteriocin.

scholarly journals Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms

Science ◽  
2020 ◽  
Vol 370 (6519) ◽  
pp. 950-957 ◽  
Author(s):  
M. Alejandra Tortorici ◽  
Martina Beltramello ◽  
Florian A. Lempp ◽  
Dora Pinto ◽  
Ha V. Dang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Viral Escape ◽  
Angiotensin Converting Enzyme 2 ◽  
Effector Functions ◽  
Human Antibodies ◽  
Binding Domains ◽  
Isolation And Characterization ◽  
Cryo Electron Microscopy ◽  
Escape Mutants

Efficient therapeutic options are needed to control the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has caused more than 922,000 fatalities as of 13 September 2020. We report the isolation and characterization of two ultrapotent SARS-CoV-2 human neutralizing antibodies (S2E12 and S2M11) that protect hamsters against SARS-CoV-2 challenge. Cryo–electron microscopy structures show that S2E12 and S2M11 competitively block angiotensin-converting enzyme 2 (ACE2) attachment and that S2M11 also locks the spike in a closed conformation by recognition of a quaternary epitope spanning two adjacent receptor-binding domains. Antibody cocktails that include S2M11, S2E12, or the previously identified S309 antibody broadly neutralize a panel of circulating SARS-CoV-2 isolates and activate effector functions. Our results pave the way to implement antibody cocktails for prophylaxis or therapy, circumventing or limiting the emergence of viral escape mutants.

scholarly journals New Member of the V1V2-Directed CAP256-VRC26 Lineage That Shows Increased Breadth and Exceptional Potency

2015 ◽  
Vol 90 (1) ◽  
pp. 76-91 ◽  
Author(s):  
Nicole A. Doria-Rose ◽  
Jinal N. Bhiman ◽  
Ryan S. Roark ◽  
Chaim A. Schramm ◽  
Jason Gorman ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Broadly Neutralizing Antibodies ◽  
New Members ◽  
Isolation And Characterization ◽  
The Family ◽  
Clade C ◽  
Potential Clinical Utility ◽  
Hiv 1 ◽  
Viral Isolates

ABSTRACT The epitopes defined by HIV-1 broadly neutralizing antibodies (bNAbs) are valuable templates for vaccine design, and studies of the immunological development of these antibodies are providing insights for vaccination strategies. In addition, the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of 12 V1V2-directed neutralizing antibodies, CAP256-VRC26, isolated from an HIV-1 clade C-infected donor at years 1, 2, and 4 of infection (N. A. Doria-Rose et al., Nature 509:55–62, 2014, http://dx.doi.org/10.1038/nature13036 ). Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. Thirteen antibodies were isolated from B cell culture, and eight were isolated using trimeric envelope probes for differential single B cell sorting. One of the new antibodies displayed a 10-fold greater neutralization potency than previously published lineage members. This antibody, CAP256-VRC26.25, neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency. Among the viruses neutralized, the median 50% inhibitory concentration was 0.001 μg/ml. All 33 lineage members targeted a quaternary epitope focused on V2. While all known bNAbs targeting the V1V2 region interact with the N160 glycan, the CAP256-VRC26 antibodies showed an inverse correlation of neutralization potency with dependence on this glycan. Overall, our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent. IMPORTANCE Studies of HIV-1 broadly neutralizing antibodies (bNAbs) provide valuable information for vaccine design, and the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of V1V2-directed neutralizing antibodies from an HIV-1 clade C-infected donor. Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. One of the new antibodies, CAP256-VRC26.25, displayed a 10-fold greater neutralization potency than previously described lineage members. It neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency: the median 50% inhibitory concentration was 0.001 μg/ml. Our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent.

scholarly journals Therapeutic antibodies, targeting the SARS-CoV-2 spike N-terminal domain, protect lethally infected K18-hACE2 mice

2021 ◽  
Author(s):  
Tal Noy-Porat ◽  
Adva Mechaly ◽  
Yinon Levy ◽  
Efi Makdasi ◽  
Ron Alcalay ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Phage Display Library ◽  
Host Cells ◽  
Terminal Domain ◽  
Isolation And Characterization ◽  
Recent Emergence ◽  
Therapeutic Monoclonal Antibodies

AbstractSince the onset of the current COVID-19 pandemic, high priority is given to the development of neutralizing antibodies, as a key approach for the design of therapeutic strategies to countermeasure and eradicate the disease. Previously, we reported the development of human therapeutic monoclonal antibodies (mAbs) exhibiting very high protective ability. These mAbs recognize epitopes on the spike receptor binding domain (RBD) of SARS-CoV-2 that is considered to represent the main rout of receptor engagement by the SARS-CoV-2 virus. The recent emergence of viral variants emphasizes the notion that efficient antibody treatments need to rely on mAbs against several distinct key epitopes in order to circumvent the occurrence of therapy escape-mutants. Here we report the isolation and characterization of 12 neutralizing mAbs, identified by screening a phage-display library constructed from lymphatic cells collected from severe COVID-19 patients. The antibodies target three distinct epitopes on the spike N-terminal domain (NTD) of SARS-CoV-2, one of them defining a major site of vulnerability of the virus. Extensive characterization of these mAbs suggests a neutralization mechanism which relies both on amino-acid and N-glycan recognition on the virus, and involvement of receptors other than the hACE2 on the target cell. Two of the selected mAbs, which demonstrated superior neutralization potency in vitro, were further evaluated in vivo, demonstrating their ability to fully protect K18-hACE2 transgenic mice even when administered at low doses and late after infection. The study demonstrates the high potential of the mAbs for therapy of SARS-CoV-2 infection and underlines the possible role of the NTD in mediating infection of host cells via alternative cellular portals other than the canonical ACE2 receptor.

Isolation and characterization of the principal caseins in rat milk

1980 ◽  
Vol 47 (1) ◽  
pp. 97-102 ◽  
Author(s):  
Jean-Pierre Pelissier ◽  
Ahmed Yahia ◽  
Jean-Marc Chobert ◽  
Bruno Ribadeau Dumas
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Molecular Weight Determination ◽  
Terminal Sequence ◽  
Sequence Determination ◽  
Sialic Acid Content ◽  
Isolation And Characterization ◽  
The Individual ◽  
Similar Peptide

SummaryThe 4 major caseins, A1, A2, B1, B2, from rat milk have been isolated and analysed. From molecular weight determination, amino acid and phosphorus analyses and N-terminal sequence determination, A1 and A2 are concluded to possess similar peptide chains as do B1 and B2, with the individual fractions within each of these 2 groups differing only in their sialic acid content.Mol. wts of approximately 22000 and 38000 were found for A1–A2 and B1–B2 respectively. The amino acid sequence of the first 13 residues of A1–A2 has been partly established. Components A1 and A2 appeared to be homologous with bovine β-casein, whereas B1 and B2 were different from any known casein, especially in their molecular weight.

scholarly journals Cultivation and Serological Characterization of a Human Theiler's-Like Cardiovirus Associated with Diarrheal Disease

2010 ◽  
Vol 84 (9) ◽  
pp. 4407-4414 ◽  
Author(s):  
C. Y. Chiu ◽  
A. L. Greninger ◽  
E. C. Chen ◽  
T. D. Haggerty ◽  
J. Parsonnet ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Diarrheal Disease ◽  
Genotype 3 ◽  
Isolation And Characterization ◽  
Serological Characterization ◽  
Genotype 2 ◽  
Encephalomyelitis Virus ◽  
Blocking Antibodies

ABSTRACT Cardioviruses (e.g., Theiler's murine encephalomyelitis virus [TMEV]) are members of the Picornaviridae family that cause myocarditis and encephalitis in rodents. Recently, several studies have identified human cardioviruses, including Saffold virus (SAFV) and a related virus named human TMEV-like cardiovirus (HTCV). At least eight cardiovirus genotypes are now recognized, with SAFV and most strains of HTCV belonging to genotypes 1 and 2, respectively; genotype 2 strains are the most common in the population. Although a genotype 3 cardiovirus has recently been cultured (SAFV-3), the genotype 1 and 2 cardioviruses have been difficult to propagate in vitro, hindering efforts to understand their seroprevalence and pathogenicity. Here we present the isolation and characterization of a genotype 2 human cardiovirus (HTCV-UC6). Notably, successful cultivation of HTCV-UC6 from stool required the addition of cytokine-blocking antibodies to interrupt downstream antiviral pathways. Unlike SAFV-3, HTCV-UC6 exhibited slow replication kinetics and demonstrated only a moderate cytopathic effect. Serologic assays revealed that 91% of U.S. adults carry antibodies to the genotype 2 cardioviruses, of which 80% generate neutralizing antibodies, in agreement with previous data showing that cardiovirus infection is widespread in humans. We also demonstrate an acute cardiovirus seroconversion event in a child with diarrhea and vomiting, thus reporting for the first time evidence linking cardiovirus infection to diarrheal disease in humans.

scholarly journals Isolation and characterization of two sequence-specific endonucleases from Anabaena variabilis

1976 ◽  
Vol 159 (2) ◽  
pp. 317-322 ◽  
Author(s):  
K Murray ◽  
S G Hughes ◽  
J S Brown ◽  
S A Bruce
Keyword(s):  
Anabaena Variabilis ◽  
Bacteriophage Λ ◽  
Terminal Sequence ◽  
Isolation And Characterization

Two endonucleases, AvaI and AvaII, were isolated from Anabaena variabilis on the basis of their ability to make a limited number of breaks at specific points in bacteriophage λ DNA. Neither enzyme has cofactor requirements beyond Mg2+. Endonuclease AvaI makes eight breaks in the phage λ chromosome at which the 5'-terminal sequence is pPy-C-G-N. AvaII endonuclease cuts phage λ DNA more extensively, yielding fragments with the 5'-terminal sequence G-T-C-N or G-A-C-N. Neither enzyme generates cohesive ends.

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