Journal: Immunity

Article Title: Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease

doi: 10.1016/j.immuni.2023.02.005

Figure Lengend Snippet: Binding and neutralization properties of S2 stem-helix mAbs (A) Dot plots showing ELISA binding (OD 405 ) reactivity of immune sera from COVID-19-convalescent donors (n = 15), 2× spike mRNA-vaccinated donors (n = 10), 3× spike mRNA-vaccinated donors (n = 9), and SARS-CoV-2 recovered-vaccinated donors (n = 15) to 25-mer peptides corresponding to spike S2 stem-helix regions of human β-(sarbecoviruses: SARS-CoV-1 or 2; merbecovirus: MERS-CoV; embecoviruses: HCoV-HKU1 and HCoV-OC43) and α-(HCoV-NL63 and HCoV-229E) coronaviruses. (B) Correlation between binding of recovered-vaccinated sera to SARS-CoV-2 stem-helix peptide and other β-CoV (MERS-CoV, HCoV-HKU1, and HCoV-OC43) stem-helix peptides. Responses for binding to two stem-helix peptides were compared by non-parametric Spearman correlation two-tailed test with 95% confidence interval, and the Spearman correlation coefficient (r) and p values are indicated. (C) A total of 40 S2 stem-helix mAbs were isolated from 9 SARS-CoV-2 recovered-vaccinated donors (CC9, CC24, CC25, CC67, CC68, CC84, CC92, CC95, and CC99). MAbs were isolated by single B cell sorting using SARS-CoV-2 and MERS-CoV S proteins as baits. Heatmap showing IGHV germline gene usage (colored: VH1-46 [green], VH3-23 [plum], and other VH genes [gray]), IGLV germline gene usage (colored: VK3-20 [light blue], VL1-51 [yellow orange], and other VL genes [gray]), lineage information (unique [cyan] and expanded [tangerine] lineages), and V-gene nucleotide somatic hypermutations (SHMs). EC 50 ELISA binding titers of mAbs with β- and α-HCoV spike S2 stem-helix region peptides are shown. IC 50 neutralization of mAbs against pseudoviruses of clade 1a (SARS-CoV-2 and Pang17), clade 1b (SARS-CoV-1, WIV1, and SHC014) sarbecoviruses, and MERS-CoV. Spike S2 stem-helix bnAbs, CC40.8, S2P6, and CV3-25, were used as controls for binding and neutralization assays. (D) Out of 40 stem-helix bnAbs isolated, 32 were unique clones. All 32 unique mAbs neutralized all 5 ACE2-utilizing sarbecoviruses tested. 23 out of the 32 unique mAbs exhibited cross-neutralization against MERS-CoV. See also Figure S1 and Tables S1–S3 .

Article Snippet: N-terminal biotinylated peptides corresponding to the spike stem-helix regions of SARS-CoV-1/2 (PLQPELDSFKEELDKYFKNHTSPDV), MERS-CoV (PLLGNSTGIDFQDELDEFFKNVSTSIP), HCoV-HKU1 (HSVPKLSDFESELSHWFKNQTSIAP), HCoV-OC43 (TSIPNLPDFKEELDQWFKNQTSVAP), HCoV-229E (TIVPEYIDVNKTLQELSYKLPNYTV) and HCoV-NL63 (TVIPDYVDVNKTLQEFAQNLPKYVK) were synthesized at A&A Labs (Synthetic Biomolecules).

Techniques: Binding Assay, Neutralization, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Isolation, FACS, Clone Assay

Journal: Immunity

Article Title: Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease

doi: 10.1016/j.immuni.2023.02.005

Figure Lengend Snippet: Neutralization of SARS-CoV-2 VOCs by S2 spike stem-helix bnAbs (A) Schema showing SARS-CoV-2 spike domains and subdomains of S1 and S2 subunits and spike amino acid changes and deletions in VOCs. Spike regions are labeled (NTD, N-terminal domain; RBD, receptor-binding domain; CTD1, C-terminal domain 1; CTD2, C-terminal domain 2; S1/S2, S1/S2 furin cleavage site; S2′, S2′ TMPRSS2 or cathepsin B/L cleavage site; FP, fusion peptide; HR1, heptad repeat 1; CH, central helix region; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane anchor); the amino acid substitutions are indicated on each VOC spike. The symbols for single mutation, insertion, and deletion are indicated. S2 stem helix is unchanged on all SARS-CoV-2 VOCs. (B) Neutralization of SARS-CoV-2 (WT) and major SARS-CoV-2 variants (Alpha; Beta; Gamma; Delta; and Omicron subvariants BA.1, BA.2, BA.2.12.1, XBB, BA.2.75, BA.2.75.2, BA.4/5, BA.4.6, and BQ.1.1) by 10 select S2 stem-helix bnAbs. (C) IC 50 neutralization titers of select S2 stem-helix bnAbs against SARS-CoV-2 (WT) and the major SARS-CoV-2 variants. The IC 50 neutralization fold-change of S2 stem-helix bnAbs with SARS-CoV-2 variants compared with the WT virus. Spike RBD nAb CC12.1 was used as control.

Article Snippet: N-terminal biotinylated peptides corresponding to the spike stem-helix regions of SARS-CoV-1/2 (PLQPELDSFKEELDKYFKNHTSPDV), MERS-CoV (PLLGNSTGIDFQDELDEFFKNVSTSIP), HCoV-HKU1 (HSVPKLSDFESELSHWFKNQTSIAP), HCoV-OC43 (TSIPNLPDFKEELDQWFKNQTSVAP), HCoV-229E (TIVPEYIDVNKTLQELSYKLPNYTV) and HCoV-NL63 (TVIPDYVDVNKTLQEFAQNLPKYVK) were synthesized at A&A Labs (Synthetic Biomolecules).

Techniques: Neutralization, Labeling, Binding Assay, Mutagenesis, Virus, Control

Journal: Immunity

Article Title: Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease

doi: 10.1016/j.immuni.2023.02.005

Figure Lengend Snippet: Immunogenetic and kinetic properties of S2 β-CoV spike stem-helix bnAbs (A and B) Pie plots showing IGHV and IGKV/IGLV gene usage distribution of isolated stem-helix mAbs. Enriched heavy (IGHV1-46 [green] and IGHV3-23 [plum]) (A) and light (IGKV3-20 [sky blue] and IGLV1-51 [yellow]) (B) gene families were colored. Dot plots showing % nucleotide mutations (SHMs) in the heavy (VH) or light (VL) chains of mAbs. The mAbs were grouped by neutralization against sarbecoviruses (SARS) or sarbecoviruses + MERS-CoV (SARS + MERS). (C and D) CDRH3 (C) or CDRL3 (D) length distributions of isolated mAbs across SARS or SARS + MERS bnAb groups, compared with human baseline germline reference. MAbs with 10 and 11 amino acid CDRH3s or mAbs with 11 amino acid CDRL3s were enriched in isolated S2 stem-helix bnAbs, compared with baseline germline reference and are indicated by arrows. (E) Sequence conservation logos of 11 amino acid-long CDRL3-bearing stem-helix bnAbs (n = 18) showed enrichment of certain V-J-gene-encoded residues, compared with the human baseline reference. Enriched residues (corresponding to a PPxF motif) were indicated by arrows. The PPxF CDRL3 motif was shown to be important for S2 stem epitope recognition by structural studies below. (F) BLI binding kinetics of S2 stem-helix mature bnAbs and their inferred germline (iGL) versions to SARS-CoV-2 and MERS-CoV stem-helix peptides. Maximum binding responses, dissociation constants (K D App ), and on-rate (k on ) and off-rate constants (k off ) for each antibody-protein interaction were compared. K D App , k on , and k off values were calculated only for antibody-antigen interactions where a maximum binding response of 0.2 nm was obtained. Statistical comparisons between two groups were performed using a Mann-Whitney two-tailed test ( ∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, p > 0.05). (G) IC 50 neutralization of S2 stem-helix bnAb iGLs with SARS-CoV-2 and MERS-CoV pseudoviruses. See also Figures S2 , , E, and S6F and Table S3 .

Article Snippet: N-terminal biotinylated peptides corresponding to the spike stem-helix regions of SARS-CoV-1/2 (PLQPELDSFKEELDKYFKNHTSPDV), MERS-CoV (PLLGNSTGIDFQDELDEFFKNVSTSIP), HCoV-HKU1 (HSVPKLSDFESELSHWFKNQTSIAP), HCoV-OC43 (TSIPNLPDFKEELDQWFKNQTSVAP), HCoV-229E (TIVPEYIDVNKTLQELSYKLPNYTV) and HCoV-NL63 (TVIPDYVDVNKTLQEFAQNLPKYVK) were synthesized at A&A Labs (Synthetic Biomolecules).

Techniques: Isolation, Neutralization, Sequencing, Binding Assay, MANN-WHITNEY, Two Tailed Test

Journal: Immunity

Article Title: Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease

doi: 10.1016/j.immuni.2023.02.005

Figure Lengend Snippet: Fine epitope specificities of S2 β-CoV spike stem-helix bnAbs (A) ELISA-based epitope mapping of S2 stem-helix bnAbs with alanine scan peptides (25-mer) from the SARS-CoV-2 stem helix. Heatmap shows fold-changes in EC 50 binding titers of mAb binding to SARS-CoV-2 stem-helix peptide alanine mutants, compared with the WT peptide. SARS-CoV-2 stem-helix residue positions targeted (2-fold or higher decrease in EC 50 binding titer compared with WT stem peptide) are indicated in different colors. Three hydrophobic residues, F 1148 , L 1152 , and F 1156 , were commonly targeted by stem-helix bnAbs and form the core of the bnAb epitope. Association of heavy-chain (IGHV1-46 and IGHV3-23) and light-chain (IGKV3-20 and IGLV1-51) gene usage and CDRL3 length are shown for the mAbs. (B) Heatmap summary of BLI competition epitope binning of S2 stem-helix bnAbs with human S2 bnAbs of known epitope specificities (CC25.106, CC95.108, CC68.109, CC99.103, S2P6, CC48.8, and CV3-25). The BLI competition was performed with SARS-CoV-2 S protein, and the competition levels are indicated as bright red (very strong), red (strong), orange (moderate), light blue (weak), and gray (very weak). See also Table S4 .

Article Snippet: N-terminal biotinylated peptides corresponding to the spike stem-helix regions of SARS-CoV-1/2 (PLQPELDSFKEELDKYFKNHTSPDV), MERS-CoV (PLLGNSTGIDFQDELDEFFKNVSTSIP), HCoV-HKU1 (HSVPKLSDFESELSHWFKNQTSIAP), HCoV-OC43 (TSIPNLPDFKEELDQWFKNQTSVAP), HCoV-229E (TIVPEYIDVNKTLQELSYKLPNYTV) and HCoV-NL63 (TVIPDYVDVNKTLQEFAQNLPKYVK) were synthesized at A&A Labs (Synthetic Biomolecules).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Residue

Journal: Immunity

Article Title: Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease

doi: 10.1016/j.immuni.2023.02.005

Figure Lengend Snippet: IGHV1-46 public antibodies target highly conserved residues in betacoronaviruses (A) Epitope residues in SARS-CoV-2, HCoV-HKU1, and MERS-CoV. Stem helices of these viruses are shown in ribbon representation. Epitope residues involved in interaction with public antibodies are shown as sticks with amino acid positions labeled. Glycan molecules (sticks, white) were modeled (based on structure in PDB: 7LM8 ) to show potential spatial restrictions at this epitope site. Green, SARS-CoV-2; navy blue, HCoV-HKU1; orange, MERS-CoV. (B) IGHV1-46 antibodies bind the stem helix in two distinct binding modes, namely, mode 1 and mode 2. The stem helices of betacoronavirus spikes are shown in ribbon mode and aligned in the same orientation. Epitope residues are shown in yellow sticks and antibodies in surface representation colored by their heavy and light chains; lavender, IGHV1-46; beige, IGLV1-51; light gray, IGKV3-20. The antibody approach angle is almost 180° rotated between IGHV1-46 + IGKV3-20 and IGHV1-46 + IGLV1-51 antibodies. (C) Epitope residues of CC25.106 are involved in the spike fusion activity. Epitope residues are shown as yellow sticks. The epitope location is shown in the prefusion and postfusion structures. Key epitope residues are buried in the stem-helix bundle (green) in prefusion spike (left, PDB: 6XR8 ) or buried in interaction with the coiled-coil central helices (cyan) in the postfusion spike (right, PDB: 6XRA ). Insets in the left and right corners show the overall structure of prefusion and postfusion spike trimer. Ribbon model in the middle shows CC25.106 in complex with SARS-CoV-2 stem helix (green) with antibody in lavender, heavy chain, and beige, light chain. Arrowhead indicates a glycosylation site. CH (cyan), central helix; HR1 (pink), heptad repeat 1; HR2 (magenta), heptad repeat 2. See also Figures S4 A, , , and and Tables S2 and .

Article Snippet: N-terminal biotinylated peptides corresponding to the spike stem-helix regions of SARS-CoV-1/2 (PLQPELDSFKEELDKYFKNHTSPDV), MERS-CoV (PLLGNSTGIDFQDELDEFFKNVSTSIP), HCoV-HKU1 (HSVPKLSDFESELSHWFKNQTSIAP), HCoV-OC43 (TSIPNLPDFKEELDQWFKNQTSVAP), HCoV-229E (TIVPEYIDVNKTLQELSYKLPNYTV) and HCoV-NL63 (TVIPDYVDVNKTLQEFAQNLPKYVK) were synthesized at A&A Labs (Synthetic Biomolecules).

Techniques: Labeling, Glycoproteomics, Binding Assay, Activity Assay

Journal: Immunity

Article Title: Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease

doi: 10.1016/j.immuni.2023.02.005

Figure Lengend Snippet: Antibody germline-encoded residues interact with the stem helix Key epitope residues and their interacting paratope residues are shown in sticks. Dashed lines represent polar interactions. Antibodies are shown in ribbon representation and stem helices in backbone tubes with side chains as sticks. SARS-CoV-2 stem helix is shown in green and MERS-CoV in orange. ∗ indicates somatically hypermutated residue. (A) CC25.106 interacts with SARS-CoV-2 stem helix. Lavender, heavy chain; beige, light chain. V H Y 33 , I 50 , N 56 , and K 52 interact with F 1148 , E 1151 , L 1152 , and Y 1155 of the stem helix. IGHV1-46 germline-encoded residues are involved in key interactions with the stem helix. (B) CC25.106 light chain interacts with SARS-CoV-2 stem helix. V L N 51 , K 66 , W 91 , and Y 32 form key interactions with L 1152 , D 1153 , F 1156 , and N 1158 of the stem helix. All of these residues are encoded by the IGLV1-51 germline gene. (C) CC99.103 heavy chain interacts with the MERS-CoV stem helix. The epitope sites are similar between CC25.106 and CC99.103, but their heavy chains bind in opposite directions with respect to the hydrophobic core. (D) CC99.103 light chain interacts with MERS-CoV stem helix. V L Y 32 , Y 91 , S 93 , and F 96 interact with F 1231 , E 1234 , and L 1235 of MERS-CoV. V L P 95 and P 95a at the tip of CDRL3 β-turn interact with F 1238 . The shortest distance between V L P 95a and F 1238 is 3.5 Å. All of these paratope residues interacting with the stem helix are encoded by IGKV3-20 except for V L F 96 , which is encoded by IGKJ3 germline. See also Figures S4 B, C, , and and Table S5 .

Article Snippet: N-terminal biotinylated peptides corresponding to the spike stem-helix regions of SARS-CoV-1/2 (PLQPELDSFKEELDKYFKNHTSPDV), MERS-CoV (PLLGNSTGIDFQDELDEFFKNVSTSIP), HCoV-HKU1 (HSVPKLSDFESELSHWFKNQTSIAP), HCoV-OC43 (TSIPNLPDFKEELDQWFKNQTSVAP), HCoV-229E (TIVPEYIDVNKTLQELSYKLPNYTV) and HCoV-NL63 (TVIPDYVDVNKTLQEFAQNLPKYVK) were synthesized at A&A Labs (Synthetic Biomolecules).

Techniques: Residue

Journal: Immunity

Article Title: Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease

doi: 10.1016/j.immuni.2023.02.005

Figure Lengend Snippet: Prophylactic treatment of aged mice with S2 stem-helix bnAbs protected against challenge with diverse betacoronaviruses (A) Three S2 stem-helix bnAbs (CC25.106, CC68.109, and CC99.103), individually, or a DEN3 control antibody were administered intraperitoneally (i.p.) at 300 μg per animal (∼10 mg/kg) into 12 groups of aged mice (10 animals per group). Each group of animals was challenged intranasally (i.n.) 12 h after antibody infusion with one of 3 mouse-adapted (MA) betacoronaviruses (MA10-SARS-2 = SARS-CoV-2, 1 × 10 3 plaque-forming units [PFUs] per mouse; MA15-SARS1 = SARS-CoV-1, 1 × 10 3 PFU per mouse; M35c4-MERS = MERS-CoV, 1 × 10 5 PFU per mouse). As a control, groups of mice were exposed to PBS in the absence of virus. (B, F, and J) Percent weight change in S2 stem-helix bnAbs or DEN3 control antibody-treated animals after challenge with mouse-adapted betacoronaviruses. Percent weight change was calculated from day 0 starting weight for all animals. Data are presented as mean values ± SEM. Statistical significance was calculated with Dunnett’s multiple comparisons test between each experimental group and the DEN3 control Ab group. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, not significant (p > 0.05). A one-way ANOVA was used. (C, G, and K) Day 2 post-infection hemorrhage (Gross Pathology score) scored at tissue harvest in mice prophylactically treated with S2 stem-helix bnAbs or DEN3 control mAb (n = 5 individuals for each group). Data are presented as mean values ± SEM. (D, H, and L) Day 2 post-infection pulmonary function (shown as Penh score) was measured by whole-body plethysmography in mice prophylactically treated with S2 stem-helix bnAbs or DEN3 control mAb (n = 5 individuals for each group). Data were shown as box-and-whisker plots showing data points from minimum to maximum. (E, I, and M) Lung virus titers (PFU per lung) were determined by plaque assay of lung tissues collected at days 2 or 4/5 after infection (n = 5 individuals per time point for each group). Data are shown as dot plots with bar heights representing the mean. See also Table S6 .

Article Snippet: N-terminal biotinylated peptides corresponding to the spike stem-helix regions of SARS-CoV-1/2 (PLQPELDSFKEELDKYFKNHTSPDV), MERS-CoV (PLLGNSTGIDFQDELDEFFKNVSTSIP), HCoV-HKU1 (HSVPKLSDFESELSHWFKNQTSIAP), HCoV-OC43 (TSIPNLPDFKEELDQWFKNQTSVAP), HCoV-229E (TIVPEYIDVNKTLQELSYKLPNYTV) and HCoV-NL63 (TVIPDYVDVNKTLQEFAQNLPKYVK) were synthesized at A&A Labs (Synthetic Biomolecules).

Techniques: Control, Virus, Infection, Whisker Assay, Plaque Assay

Journal: Immunity

Article Title: Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease

doi: 10.1016/j.immuni.2023.02.005

Figure Lengend Snippet:

Article Snippet: N-terminal biotinylated peptides corresponding to the spike stem-helix regions of SARS-CoV-1/2 (PLQPELDSFKEELDKYFKNHTSPDV), MERS-CoV (PLLGNSTGIDFQDELDEFFKNVSTSIP), HCoV-HKU1 (HSVPKLSDFESELSHWFKNQTSIAP), HCoV-OC43 (TSIPNLPDFKEELDQWFKNQTSVAP), HCoV-229E (TIVPEYIDVNKTLQELSYKLPNYTV) and HCoV-NL63 (TVIPDYVDVNKTLQEFAQNLPKYVK) were synthesized at A&A Labs (Synthetic Biomolecules).

Techniques: Recombinant, Staining, Reverse Transcription, Luciferase, Membrane, Software, Expressing, Binding Assay

Journal: British Journal of Pharmacology

Article Title: Biological actions of green tea catechins on cardiac troponin C

doi: 10.1111/j.1476-5381.2010.00942.x

Figure Lengend Snippet: Role of Tn in determining the differential sensitivity to EGCg in cardiac and fast skeletal muscle. (A) Effect of EGCg on force-pCa relationships in skinned fast skeletal muscle fibres. The data represent the means ± SE of measurements on 5 fibres. (B) SDS-PAGE analysis of skinned cardiac muscle fibres in which endogenous cTn was exchanged with fsTn. Note that the decrease in cTnI was not apparent due to the presence of other protein(s) with similar mobility. Densitometric analyses of TnC isoforms in skinned fibres indicated that 83.5 ± 3.0% (n = 7 fibres) of endogenous Tn was exchanged with fsTn. LC1 and LC2, ventricular myosin light chains 1 and 2, respectively. (C) Effect of EGCg on force-pCa relationships in skinned cardiac muscle fibres exchanged with fsTn. The data represent the means ± SE of measurements on 7 fibres. (D) EGCg-induced decrease in the Ca2+-sensitivity (ΔpCa50) in skinned cardiac muscle fibres exchanged with fsTn. The data represent the means ± SE of measurements on 7 fibres. ***P < 0.001 versus fast skeletal muscle (Dunnett's multiple comparison test). EGCg, (-)-Epigallocatechin-3-gallate; fsTn, fast skeletal muscle troponin; Tn, troponin.

Article Snippet: Binding of cTnI N-terminal peptide to cTnC A synthetic peptide for the N-terminal helix region of cTnI (AKKKSKISASRKLQLKTLLLQIAKQELE) was purchased from Greiner Bio-One (Tokyo, Japan).

Techniques: SDS Page, Comparison

Journal: British Journal of Pharmacology

Article Title: Biological actions of green tea catechins on cardiac troponin C

doi: 10.1111/j.1476-5381.2010.00942.x

Figure Lengend Snippet: Binding of EGCg to cTn subunits. (A) Binding of EGCg to cTnC, cTnI and cTnT were directly determined from the frequency changes (ΔF) of QCM upon cumulative addition of EGCg. The data represent the means ± SE of 3–4 measurements. The EGCg-cTnC binding data were fitted to a hyperbolic one-site binding equation, and a best-fitted curve was obtained with a KD of 14.6 µM. (B) Effects of EGCg on the binding of a cTnI N-terminal peptide (cTnI35–62) to cTnC. Binding of the cTnI N-terminal peptide (cTnI35–62) to cTnC was directly determined from ΔF of QCM upon cumulative addition of the peptide in the absence or presence of 300 µM EGCg. The data represent the means ± SE of 3 measurements. *P < 0.05, versus –EGCg (unpaired t-test). The data were fitted to a hyperbolic one-site binding equation, and best-fitted curves were obtained with KD's of 9.1 and 1.4 µM for cTnC and cTnC+EGCg, respectively. EGCg, (-)-Epigallocatechin-3-gallate; QCM, quartz crystal microbalance.

Article Snippet: Binding of cTnI N-terminal peptide to cTnC A synthetic peptide for the N-terminal helix region of cTnI (AKKKSKISASRKLQLKTLLLQIAKQELE) was purchased from Greiner Bio-One (Tokyo, Japan).

Techniques: Binding Assay