Journal: Cell reports. Medicine

Article Title: Safety and immunogenicity of SARS-CoV-2 self-amplifying RNA vaccine expressing an anchored RBD: A randomized, observer-blind phase 1 study.

doi: 10.1016/j.xcrm.2023.101134

Figure Lengend Snippet: Figure 3. SARS-CoV-2 IgG and neutralizing antibody responses (A and B) Serum IgG titers against wild-type SARS- CoV-2 RBD protein (A) and pseudovirus neutralizing antibody titers (ID50) against SARS-CoV-2 variants (B) for non-elderly (left side) and elderly (right side) participants (n = 10 per dose group, and n = 6 for the placebo group). Participants received one injection of VLPCOV-01 (0.3, 1.0, or 3.0 mg), 30 mg BNT162b2, or placebo on day 1 (week 0). Logarithmic values are reported as geometric mean titers for serum IgG and neutralizing antibody against pseudovirus. Bars indicate 95% CIs. (C) The correlation between serum neutralizing antibody titers against pseudovirus Wuhan (wild type) and IgG antibody titers against SARS-CoV-2 RBD protein following booster vaccination. Pearson’s product-moment correlation coefficient and p value were calculated following log trans- formation of source data (r = 0.950, p < 0.001).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-human CD154-FITC (clone: TRAP1) BD Biosciences cat# 555699; RRID: AB_396049 Mouse anti-human CD3-BUV615 (clone: SP34-2) BD Biosciences cat# 751249; RRID: AB_2875266 Mouse anti-human CD4-PE-Cy5.5 (clone: S3.5) Thermo Ficher Scientific cat# MHCD0418; RRID: AB_10376013 Mouse anti-human CD8-BUV563 (clone: RPA-T8) BD Biosciences cat# 612914; RRID: AB_2870200 Mouse anti-human CD27-PE-Cy5 (clone: 1A4CD27) Beckman coulter cat# 6607107 Mouse anti-human CD45RO-BUV805 (clone: UCHL1) BD Biosciences cat# 748367; RRID: AB_2872786 Mouse anti-human IFN-g-BV786 (clone: 4S.B3) BioLegend cat# 502542; RRID: AB_2563882 Mouse anti-human TNF-BV650 (clone: MAb11) BioLegend cat# 502938; RRID: AB_2562741 Rat anti-human IL-13-BV421 (clone: JES10-5A2) BD Biosciences cat# 563580; RRID: AB_2738290 Mouse anti-human IL-21-Ax647 (clone: 3A3-N21) BD Biosciences cat# 560493; RRID: AB_1645421 Mouse anti-human IL-4-PE-Cy7 (clone:8D4-8) BD Biosciences cat# 560672; RRID: AB_1727547 Mouse anti-human IL-17A-BV605 (clone: BL168) BioLegend cat# 512326; RRID: AB_2563887 Rat anti-human IL-2-BUV737 (clone: MQ117H12) BD Biosciences cat# 612836 Mouse anti-human CD107A-BV711 (clone: H4A3) BioLegend cat# 328640; RRID: AB_2565840 Mouse anti-human MIP1b-Alexa700 (clone: D21-1351) BD Biosciences cat# 561278; RRID: AB_10612008 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG1 (AS35) ACROBiosystems cat# SAD-S35 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG2 (AS35) ACROBiosystems cat# SAD-S66 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG3 (AS35) ACROBiosystems cat# SAD-S67 Anti-SARS-CoV-2 Spike RBD Neutralizing Antibody, Human IgG4 (AS35) ACROBiosystems cat# SAD-S68 Mouse anti-Human IgG1 Fc Secondary Antibody, HRP Thermo Fisher Scientific cat# MH1715; RRID: AB_2539710 Mouse Anti-Human IgG2 Fc-BIOT HP6002 SouthernBiotech cat# 9070-08; RRID: AB_2796638 Mouse Anti-Human IgG3 Hinge-BIOT HP6050 SouthernBiotech cat# 9210-08; RRID: AB_2796700 Mouse Anti-Human IgG4 Fc-BIOT HP6025 SouthernBiotech cat# 9200-08; RRID: AB_2796692 Biological samples Human PBMCs This study This study Chemicals, peptides, and recombinant proteins Benzonase Nuclease, Purity >90% MERCK Millipore cat# 70746 (Continued on next page) Cell Reports Medicine 4, 101134, August 15, 2023 e1

Techniques: Injection

Journal: Nature Communications

Article Title: A CRISPR-based ultrasensitive assay detects attomolar concentrations of SARS-CoV-2 antibodies in clinical samples

doi: 10.1038/s41467-022-32371-4

Figure Lengend Snippet: a Schematic illustration of the workflow of the UCAD assay. b Sequence design of the dsDNA template for triggering the collateral cleavage activity of CRISPR-Cas12a, as well as the strategy to split the dsDNA template into TS and NTS probes for UCAD. c Proximity binding of the two UCAD probes to the same anti-RBD antibody through affinity motifs enhances the T m of the duplex domain from 10 to 46 °C and thus enables the stable binding between TS and NTS probes. d , e Kinetic curves for the detection of anti-RBD IgG ( d ) and IgM ( e ) in commercially purchased certified anti-SARS-CoV-2 IgG/IgM positive and negative serum samples using UCAD. f A calibration curve for the detection of anti-SARS-CoV-2 spike protein RBD human mAb (clone: OTIH401) in the range of 1 aM to 1 pM was established by plotting fluorescence intensity at 40 min of the CRISPR-Cas12a reaction as a function of target concentrations, which was also compared with UCAD without RPA amplification. Each concentration has replicated measurements ( n = 2). g , h Detection of anti-RBD IgG ( g ) and IgM ( h ) in certified anti-SARS-CoV-2 IgG/IgM positive serum diluted in negative human serum with dilution factors from 1 to 100,000 using both UCAD and commercial ELISA kits. Each concentration has replicated measurements ( n = 2). Source data are available in the Source Data file.

Article Snippet: SARS-CoV-2 Spike RBD human monoclonal antibody (Cat #: TA190325, clone name: OTIH401) and SARS-CoV-2 N protein human monoclonal antibody (Cat #: TA190323, clone name: OTIH1G5) were purchased from Origene, Beijing, China.

Techniques: Sequencing, Activity Assay, CRISPR, Binding Assay, Fluorescence, Amplification, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: A CRISPR-based ultrasensitive assay detects attomolar concentrations of SARS-CoV-2 antibodies in clinical samples

doi: 10.1038/s41467-022-32371-4

Figure Lengend Snippet: a Detection of the target anti-human RBD and closely related nontargeted anti-MERS-CoV spike protein RBD human mAb (clone: m336), anti-SARS-CoV-2 nucleocapsid (N) protein human mAb, and anti-SARS-CoV-2 spike protein RBD rabbit pAb at a concentration of 10 fM using UCAD. Each sample was measured twice in two independent experiments. b , c Evaluation of the specificity of UCAD for anti-RBD IgG ( b ) and IgM ( c ) against anti-SARS-CoV in clinical sera from three SARS patients in 2003. Each serum sample was measured twice in two independent experiments. The UCAD signals of anti-SARS-CoV positive sera were significantly lower than the anti-SARS-CoV-2 positive serum ( p = 0.0004 for IgG, p = 0.0025 for IgM). Ordinary one-way ANOVA were used to compare the difference between multiple groups. d Schematic illustration of the modularity of UCAD for varying mutants of RBD and the N protein of SARS-CoV-2 by switching the recognition motif on the TS probe. e Heatmap of the detection of 10 fM wild-type (WT), Delta specific, and Omicron specific anti-RBD human mAb by using WT RBD, Delta RBD, and Omicron RBD (B.1.1.529) modified TS probes. f Heatmap of the detection of 10 fM WT RBD human mAb and anti-N protein human mAb with WT RBD and N protein modified TS probes. Source data are available in the Source Data file. ns: p > 0.05, *** p ≤ 0.001.

Article Snippet: SARS-CoV-2 Spike RBD human monoclonal antibody (Cat #: TA190325, clone name: OTIH401) and SARS-CoV-2 N protein human monoclonal antibody (Cat #: TA190323, clone name: OTIH1G5) were purchased from Origene, Beijing, China.

Techniques: Concentration Assay, Modification

Journal: Nature Communications

Article Title: A CRISPR-based ultrasensitive assay detects attomolar concentrations of SARS-CoV-2 antibodies in clinical samples

doi: 10.1038/s41467-022-32371-4

Figure Lengend Snippet: a Schematic illustration of lateral flow readout for UCAD for the detection of anti-SARS-CoV-2 IgG and IgM. b The design of the lateral flow strip that captured all anti-FAM labeled AuNPs at C-line in the absence of the target antibody. In the presence of ant-SARS-CoV-2 IgG or IgM, the FAM-Dig dual-labeled reporters were cleaved by Cas12a-crRNA so that AuNPs could escape the capture at C-line and accumulated at T-line through the immobilized secondary antibody. c Visual detection of 15 positive clinical sera and 15 pre-pandemic sera by UCAD integrated with lateral flow strips.

Article Snippet: SARS-CoV-2 Spike RBD human monoclonal antibody (Cat #: TA190325, clone name: OTIH401) and SARS-CoV-2 N protein human monoclonal antibody (Cat #: TA190323, clone name: OTIH1G5) were purchased from Origene, Beijing, China.

Techniques: Stripping Membranes, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Structures of synthetic nanobody–SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction

doi: 10.1016/j.jbc.2021.101202

Figure Lengend Snippet: Sybodies bind the RBD with K D values in the nanomolar range. The RBD was coupled to a biosensor chip as described in . Graded concentrations (31–500 nM) of each of the indicated sybodies were offered to the coupled surface (from t = 0 to t = 160 s) followed by buffer washout and measurement of net binding (in RU). Experimental curves ( red ) were fit by global analysis using Biacore T200 evaluation software 3.1 (Cytiva) ( black ). The curves shown are representative of at least two determinations. A , B , C , D , and E represent data and curve fits for Sb14, Sb15, Sb16, Sb45, and Sb68, respectively. RBD, receptor-binding domain; RU, resonance units.

Article Snippet: Plasmids pSbinit encoding sybodies Sb14, Sb15, Sb16, Sb45, and Sb68 (Addgene #153522, #153523, #153524, #153526, and #153527, respectively) were originally reported by Walter et al. ( ) and generously made available.

Techniques: Binding Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Structures of synthetic nanobody–SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction

doi: 10.1016/j.jbc.2021.101202

Figure Lengend Snippet: X-ray data collection and refinement statistics

Article Snippet: Plasmids pSbinit encoding sybodies Sb14, Sb15, Sb16, Sb45, and Sb68 (Addgene #153522, #153523, #153524, #153526, and #153527, respectively) were originally reported by Walter et al. ( ) and generously made available.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Structures of synthetic nanobody–SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction

doi: 10.1016/j.jbc.2021.101202

Figure Lengend Snippet: Overall structures of Sb14, Sb16, Sb45, and Sb68 complexed with the SARS-CoV-2 RBD. Ribbon (sybodies) and ribbon plus surface (RBD) representations of the complex of ( A ) Sb16 ( slate ) with the RBD ( gray ) (7KGK); ( B ) Sb45 ( cyan ) with the RBD (7KGJ); ( D ) Sb45 and Sb68 ( purple ) with the RBD (7KLW), and ( E ) Sb14 ( blue ) and Sb68 ( magenta ) with the RBD (7MFU). Sb16–RBD and Sb45–RBD superimposed based on the RBD are shown in panel C , to highlight CDR loops, which are color-coded as CDR1 ( pink ), CDR2 ( orange ), and CDR3 ( red ). The CDR2 of Sb16 and CDR3 of Sb45 interact similarly with the RBD surface. Panel F shows a sequence alignment of the four sybodies. CDR1, complementarity-determining region 1; CDR2, complementarity-determining region 2; CDR3, complementarity-determining region 3; RBD, receptor-binding domain; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Article Snippet: Plasmids pSbinit encoding sybodies Sb14, Sb15, Sb16, Sb45, and Sb68 (Addgene #153522, #153523, #153524, #153526, and #153527, respectively) were originally reported by Walter et al. ( ) and generously made available.

Techniques: Sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Structures of synthetic nanobody–SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction

doi: 10.1016/j.jbc.2021.101202

Figure Lengend Snippet: Interfaces and interactions of sybodies with the RBD. A , Sb16–RBD; ( B ) Sb45–RBD; ( C ) Sb14–RBD; and ( D ) Sb68–RBD. (Individual contacting residues are listed in Table S1 ). CDR1, CDR2, and CDR3 are painted pink , orange , and red , respectively. Additional non-CDR-contacting residues are colored lime . On the RBD surface, the epitopic residues that contact the sybodies are colored according to the sybody CDR. CDR1, complementarity-determining region 1; CDR2, complementarity-determining region 2; CDR3, complementarity-determining region 3; RBD, receptor-binding domain.

Article Snippet: Plasmids pSbinit encoding sybodies Sb14, Sb15, Sb16, Sb45, and Sb68 (Addgene #153522, #153523, #153524, #153526, and #153527, respectively) were originally reported by Walter et al. ( ) and generously made available.

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Structures of synthetic nanobody–SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction

doi: 10.1016/j.jbc.2021.101202

Figure Lengend Snippet: Sybodies clash with ACE2 in RBD complex structures. A , Sb16 ( slate ), Sb45 ( cyan ), Sb14 ( blue ), and Sb68 ( purple )—RBD complexes were superposed on the ACE2–RBD structure ( salmon ) (6M0J) based on the RBD. Views of Sb16 ( B ), Sb45 ( C ), and Sb14 ( D ) are shown alone as well. Sb14 and Sb16 are buried inside ACE2, Sb45 is partially buried in ACE2, and Sb68 has major clashes with two N-glycan sites (N322 and N546) of ACE2 (inset). E , epitopic area (on the RBD) captured by ACE2 ( salmon ) is indicated along with its BSA. ACE2, angiotensin-converting enzyme 2; BSA, buried surface area; RBD, receptor-binding domain.

Article Snippet: Plasmids pSbinit encoding sybodies Sb14, Sb15, Sb16, Sb45, and Sb68 (Addgene #153522, #153523, #153524, #153526, and #153527, respectively) were originally reported by Walter et al. ( ) and generously made available.

Techniques: Glycoproteomics, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Structures of synthetic nanobody–SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction

doi: 10.1016/j.jbc.2021.101202

Figure Lengend Snippet: RBD mutations affect sybody binding. A , SPR binding of each of the indicated sybodies ( across top ) to each of the individual RBD mutants. Inset shows binding of sybodies to the WT RBD (from Fig. 1 ). Experimental tracings are shown in red , curve fits in black , and k d (s −1 ) and K D (M) values as determined from global fitting with BIAeval 2.0 are provided in each panel. B , location of contacts of Sb16, Sb45, and Sb14 is shown. E484, K417, and N501 of the RBD (WT) interact with K32, Y54, and R60 of Sb16, respectively; E484 and N501 of the RBD (WT) interact with R33 and H103 of Sb45, respectively; and E484, K417, and N501 of the RBD (WT) interact with Q39, E35, and Y60 of Sb14, respectively. C , comparison of complex structures with minimized models involving the N501Y mutation. In silico mutagenesis of N501Y was performed using 7KGK (Sb16+RBD), 7KGJ (Sb45+RBD), and 7MFU (Sb14+RBD+Sb68). After amino acid substitution in Coot, local energy minimization (within 15–20 Å of the mutant residue) was performed through three rounds in PHENIX. For the Sb16–RBD complex, when N501 is mutated to Y501, the loop (496–506, from yellow to wheat ) extends about 2.4 Å, but R60 (revealing a double conformation) still forms hydrogen bonds with the Y501 loop; for the Sb45–RBD complex, when N501 is mutated to Y501, the loop (496–506, from yellow to wheat ) extends about 1.0 Å, but H103 of Sb45 would still interact with Y501; for the Sb14–RBD complex, when N501 is mutated to Y501, the loop (496–506, from yellow to wheat ) is extended about 2.0 Å, but T58 and K65 still form hydrogen bonds with Y501. D , the surface charge of Sb16; K32 forms a hydrogen bond with E484 of the RBD with the opposite charge; the surface charge of Sb45, R33 forms a hydrogen bond with E484 of the RBD with the opposite charge; the surface charge of Sb14, Q39 (a neutral residue) interacts with E484 of the RBD. E , surface charge of the WT RBD and surface charge of the RBD with the three mutations (E484, K417N, and N501Y). When E484 is mutated to K484, the surface charge is changed from negative to positive. Therefore, the hydrogen bonds are broken, pushing Sb16 and Sb45 out of contact, whereas because Q39 of Sb14 is not a charged residue, it still may interact with K484 of the mutated RBD. RBD, receptor-binding domain; SPR, surface plasmon resonance.

Article Snippet: Plasmids pSbinit encoding sybodies Sb14, Sb15, Sb16, Sb45, and Sb68 (Addgene #153522, #153523, #153524, #153526, and #153527, respectively) were originally reported by Walter et al. ( ) and generously made available.

Techniques: Binding Assay, Comparison, Mutagenesis, In Silico, Residue, SPR Assay

Journal: Cell Reports

Article Title: PD-1 high CXCR5 – CD4 + peripheral helper T cells promote CXCR3 + plasmablasts in human acute viral infection

doi: 10.1016/j.celrep.2022.111895

Figure Lengend Snippet:

Article Snippet: Anti-SARS-CoV-2 S-RBD protein Human IgM ELISA Kit , Proteintech , KE30004.

Techniques: Purification, Recombinant, Cell Isolation, Enzyme-linked Immunosorbent Assay, DNA Library Preparation, Software

Journal: bioRxiv

Article Title: Synthetic nanobody–SARS-CoV-2 receptor-binding domain structures identify distinct epitopes

doi: 10.1101/2021.01.27.428466

Figure Lengend Snippet: Sybodies bind RBD with K D values in the nanomolar range. RBD (A, B, C) or S protein (E, F, G) was coupled to a biosensor chip as described in Materials and Methods. Graded concentrations (31 to 500 nM) were flowed over the coupled surfaces (from t=0 to t= 120 s, followed by buffer washout) and net RU signal (compared to mock-coupled surface) was measured by SPR for Sb16 (A, E); Sb45 (B, F); and Sb68 (C, G). Summary of triplicate mean ± SD determinations is shown in Tables (D, H). Global analysis (curve fits in red, residuals below the curves) was accomplished using EVILFIT ( , ), and the major components of binding are shown.

Article Snippet: Plasmids pSb-init encoding sybodies Sb16, Sb45. and Sb68 (Addgene #153524, #153526, and #153527, respectively) were originally reported by Walter et al ( ) and generously made available.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Synthetic nanobody–SARS-CoV-2 receptor-binding domain structures identify distinct epitopes

doi: 10.1101/2021.01.27.428466

Figure Lengend Snippet: SEC profiles reveal direct interaction of sybodies with RBD. (A) Sybodies (50 μg) and RBD (50 μg) were injected onto a Shodex-KW-802.5 column (0.75 ml/min) in 100 μl individually and elution times are shown. Sb16 and RBD (B), Sb45 and RBD (C), or Sb68 and RBD (D) were mixed in equal concentrations (50 μg in 100 μl), incubated at 4 °C overnight and then analyzed on the Shodex column.

Article Snippet: Plasmids pSb-init encoding sybodies Sb16, Sb45. and Sb68 (Addgene #153524, #153526, and #153527, respectively) were originally reported by Walter et al ( ) and generously made available.

Techniques: Injection, Incubation

Journal: bioRxiv

Article Title: Synthetic nanobody–SARS-CoV-2 receptor-binding domain structures identify distinct epitopes

doi: 10.1101/2021.01.27.428466

Figure Lengend Snippet: Electron density (as blue) maps (2mFo-DFc) for CDR loops and those residues in contact with RBD: (A) Sb16 on RBD surface, resolution=2.6 Å, R free =0.277; (B) Sb45 on RBD surface, resolution=2.3 Å, R free =0.216; (C) Sb68 on RBD surface, resolution=2.6 Å, R free =0.255; (D) Sb16 alone, resolution=2.1 Å, R free =0.259. Contour at 1.0σ, CDR1 loop as pink, CDR2 loop as orange, CDR3 loop as red, non-CDR residues as lime, and RBD is in background as gray.

Article Snippet: Plasmids pSb-init encoding sybodies Sb16, Sb45. and Sb68 (Addgene #153524, #153526, and #153527, respectively) were originally reported by Walter et al ( ) and generously made available.

Techniques:

Journal: bioRxiv

Article Title: Synthetic nanobody–SARS-CoV-2 receptor-binding domain structures identify distinct epitopes

doi: 10.1101/2021.01.27.428466

Figure Lengend Snippet: Overall structures of Sb16, Sb45 and Sb68 complexes with SARS-CoV-2 RBD. Ribbons (sybodies) and ribbons plus surface (RBD) representations of the complex of (A) Sb16 (slate) with RBD (grey) (7KGK); (B) Sb45 (cyan) with RBD (7KGJ), and (C) Sb45 and Sb68 (purple) with RBD (7KLW). Sb16-RBD and Sb45-RBD, superimposed based on the RBD are shown in (D) to highlight CDR loops, which are color coded as indicated. The CDR2 of Sb16 and CDR3 of Sb45 interact similarly with the RBD surface.

Article Snippet: Plasmids pSb-init encoding sybodies Sb16, Sb45. and Sb68 (Addgene #153524, #153526, and #153527, respectively) were originally reported by Walter et al ( ) and generously made available.

Techniques:

Journal: bioRxiv

Article Title: Synthetic nanobody–SARS-CoV-2 receptor-binding domain structures identify distinct epitopes

doi: 10.1101/2021.01.27.428466

Figure Lengend Snippet: Interface and interaction of (A) Sb16-RBD, (B) Sb45-RBD and (C) Sb68-RBD. (Individual contacting residues are listed in TableS2 in Supplemental Materials). CDR1, CDR2, CDR3 regions are painted pink, orange and red respectively. Additional non-CDR region contacting residues are colored lime. On the RBD surface, the epitopic residues that contact the sybodies are colored according to the sybody CDR.

Article Snippet: Plasmids pSb-init encoding sybodies Sb16, Sb45. and Sb68 (Addgene #153524, #153526, and #153527, respectively) were originally reported by Walter et al ( ) and generously made available.

Techniques:

Journal: bioRxiv

Article Title: Synthetic nanobody–SARS-CoV-2 receptor-binding domain structures identify distinct epitopes

doi: 10.1101/2021.01.27.428466

Figure Lengend Snippet: Sybodies compete with ACE2 for RBD binding. (A) Sb16 (slate), Sb45 (cyan) and Sb68 (purple) – RBD complexes were superposed on the ACE2—RBD structure (salmon) (6M0J) based on the RBD. Sb16 is buried inside ACE2; Sb45 is partially buried in ACE2; and Sb68 has major clashes with two N-glycan sites (N322 and N546) of ACE2. (B) Epitopic areas (on RBD) captured by ACE2 (salmon), BSA = 844 (Å 2 ); (C) by Sb16 (slate), BSA = 1003 (Å 2 ); (D) by Sb45 (cyan), BSA = 976 (Å 2 ); and (E) by Sb68 (purple), BSA = 640 (Å 2 ).

Article Snippet: Plasmids pSb-init encoding sybodies Sb16, Sb45. and Sb68 (Addgene #153524, #153526, and #153527, respectively) were originally reported by Walter et al ( ) and generously made available.

Techniques: Binding Assay, Glycoproteomics

Journal: bioRxiv

Article Title: Synthetic nanobody–SARS-CoV-2 receptor-binding domain structures identify distinct epitopes

doi: 10.1101/2021.01.27.428466

Figure Lengend Snippet: Superposition of complexes on spike models reveals accessibility of sybodies. Superposition of (A) Sb16-RBD (slate) on spike (6XEY: 3-down; 6Z43: 1-up and 2-down; 7A29: 2-up and 1-down; 7JVC: 3-up); (B) Sb45-RBD (cyan); and (C) Sb68-RBD (purple), there is no accessible surface for Sb68 on 3-down of spike; (D) Sb68 and Sb16 on RBD; (E) SB68 and Sb45 on RBD.

Article Snippet: Plasmids pSb-init encoding sybodies Sb16, Sb45. and Sb68 (Addgene #153524, #153526, and #153527, respectively) were originally reported by Walter et al ( ) and generously made available.

Techniques:

Journal: bioRxiv

Article Title: Synthetic nanobody–SARS-CoV-2 receptor-binding domain structures identify distinct epitopes

doi: 10.1101/2021.01.27.428466

Figure Lengend Snippet: Sybodies, nanobodies, and antibodies bind different epitopic regions of the RBD. (A) Definition of Classes according to Barnes et al. and Epitope area according to Xiang et al.. Epitope I to IV are almost the same as Class 1 to 4, except for Epitope V which spans Class 3 and Class 4 (not shown). Color codes are for representative Fab (only shown are the variable domains) or sybody/nanobody. Sb68 falls into Class-4/Epitope-IV overlapping with V HH 72 and HH-E, and CR3022. RBD is in gray and two N-glycans (N165 and N343 in red) are shown. (B) Sb16 clashes with L-chain of B38 (7BZ5) of Class-1 and H-chain of COVA2-39 (7JMP) H-chain Class-2. It falls between Class-1/Epitope-I and Class-2/Epitope-II but almost covers SR4. (C) Sb45 overlaps with H-chain of COVA2-39 (7JMP) of Class-2 and lies in the same orientation as Nb20 (7JVB) - Epitope-II.

Article Snippet: Plasmids pSb-init encoding sybodies Sb16, Sb45. and Sb68 (Addgene #153524, #153526, and #153527, respectively) were originally reported by Walter et al ( ) and generously made available.

Techniques:

Journal: bioRxiv

Article Title: Synthetic nanobody–SARS-CoV-2 receptor-binding domain structures identify distinct epitopes

doi: 10.1101/2021.01.27.428466

Figure Lengend Snippet: Comparison of the ternary sybody structure (Sb45–RBD–Sb68, 7KLW) with the ternary nanobody structure (V HH -E–RBD–V HH -U, 7KN5), Koenig et al. . Sb45 in cyan, Sb68 in purpleblue, RBD in gray, V HH -E in green, V HH -U in blue. CDR1, CDR2, and CDR3 of Sb45, CDR3 of Sb68 are highlighted in pink, orange, and red. Three views are each rotated by 90°. CDR3 and CDR2 loops of Sb45 ride along both sides of the RBD, while V HH -E uses only an extended CDR3 loop on the side. Sb68 is slightly lower than V HH -U while V HH -E is similar to V HH 72.

Article Snippet: Plasmids pSb-init encoding sybodies Sb16, Sb45. and Sb68 (Addgene #153524, #153526, and #153527, respectively) were originally reported by Walter et al ( ) and generously made available.

Techniques: Comparison