Journal: Nature Communications

Article Title: Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

doi: 10.1038/s41467-021-27611-y

Figure Lengend Snippet: a ELISA screen for identification of potential SARS-CoV-2 RBD binders. Negative control wells containing expression media, and positive control wells containing VNAR E06 (anti-serum albumin) and rabbit monoclonal CR3022-RB (anti-SARS-CoV-2 Spike) are indicated. b Primary screen for identification of neutralizing VNAR domains. Concentration-dependent neutralization of pseudotyped SARS-CoV-2 (black) or SARS-CoV-1 (red) in HEK293T cells transiently expressing ACE2. Data represents mean ± s.e.m. relative luminescence units (RLUs) from n = 3 independent biological experiments. c Left , rank-ordered IC 50 values for neutralizing VNARs from panel ( b ). VNARs with IC 50 < 10 nM (dashed line) were selected for further characterization. Upper right, depiction of primary sequences of selected VNARs, relative length of complementarity determining regions (CDR1, CDR3) and location of cysteine residues (teal) are shown. d Phylogenetic tree of selected virus taxa, divergent lineages of betacoronaviruses are shown. Glycoproteins encoded by the indicated viruses (*) were used to generate pseudoviruses. e Heatmap summarizing IC 50 values for neutralization of the indicated pseudovirus with the indicated VNAR antibody. Values are derived from experiments described in ( f ). f Secondary validation of selected neutralizing VNAR domains. Concentration-dependent neutralization of viral particles pseudotyped with glycoproteins natively encoded by either SARS-CoV-2 (black), SARS-CoV-1 (red), WIV1-CoV (blue). MERS-CoV (green), or VSV (purple) in Calu-3 cells. Cell viability was also assessed in the presence of increasing concentrations of VNARs (yellow). Data represents mean ± s.e.m. RLU values from n = 3 independent biological experiments. g Concentration-dependent neutralization of replication-competent authentic SARS-CoV-2, strain USA_WA1/2020 in Vero E6 cells. Data represents mean ± s.e.m. RFU values from n = 3 independent biological experiments.

Article Snippet: Plasmids used for generating pseudovirus stocks were sourced as follows: plasmid encoding an Env -defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.R-E − ) , was obtained through the NIH AIDS Reagent Program; plasmids encoding SARS-CoV-1 and SARS-CoV-2 Spike were from Fang Li (Addgene plasmid #145031 and #145032, respectively) ; WIV1-CoV Spike was from Alejandro Balazs (Addgene plasmid #164439) ; MERS-CoV Spike was from Sino Biological (VG40069-NF); and VSV-G was from Bob Weinberg (Addgene plasmid #8454).

Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Expressing, Positive Control, Concentration Assay, Neutralization, Derivative Assay

Journal: Nature Communications

Article Title: Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

doi: 10.1038/s41467-021-27611-y

Figure Lengend Snippet: a Sequence alignment between SARS-CoV-1, SARS-CoV-2, and the MERS RBDs. Residues different from SARS-CoV-2 are highlighted in red and the interaction interface for VNAR-3B4 is marked with a blue box. Bolded letters indicate residues critical for the interaction between the RBD and 3B4 with arrows indicating the residues that form a backbone beta-sheet. The sequence alignment is numbered above according to SARS-CoV-2. b Surface representations of SARS-CoV-1 (pink, above) and MERS (green, below) with variant residues colored red. The ACE2 binding interface is highlighted in purple for SARS-CoV-1 and the DPP4 binding interface in orange for MERS. The homology-modeled interaction interface for 3B4 is colored blue for both structures. c Zoomed in view of the modeled interaction interface between 3B4 (blue) and SARS-CoV-1 (pink, above) and MERS (green, below), with 3B4 colored blue in both pictures. Interacting residues are highlighted as in Fig. , showing the backbone interactions in black dashes and sidechain to backbone or sidechain to sidechain interactions shown as yellow dashes. Insets show the orientation of the zoomed in view. d Overlays of the 3B4 interface from modeled RBDs and their matching RBDs obtained by x-ray crystallography. The panel above shows the modeled SARS-CoV-1 RBD, colored pink, aligned with the crystal structure of SARS-CoV-1 RBD bound to ACE2 (PDB id: 2AJF), colored magenta. The panel below shows the modeled MERS RBD, colored light green, aligned with the crystal structure of MERS RBD bound to DPP4 (PDB id: 4L72), colored dark green.

Article Snippet: Plasmids used for generating pseudovirus stocks were sourced as follows: plasmid encoding an Env -defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.R-E − ) , was obtained through the NIH AIDS Reagent Program; plasmids encoding SARS-CoV-1 and SARS-CoV-2 Spike were from Fang Li (Addgene plasmid #145031 and #145032, respectively) ; WIV1-CoV Spike was from Alejandro Balazs (Addgene plasmid #164439) ; MERS-CoV Spike was from Sino Biological (VG40069-NF); and VSV-G was from Bob Weinberg (Addgene plasmid #8454).

Techniques: Sequencing, Variant Assay, Binding Assay

Journal: Scientific Reports

Article Title: Unbiased approach to identify and assess efficacy of human SARS-CoV-2 neutralizing antibodies

doi: 10.1038/s41598-022-19780-7

Figure Lengend Snippet: Rapid Discovery of Neutralizing Antibodies. ( A ) The G-MAB phage display library was panned for SARS-CoV-2 Spike S1 subunit-binding scFv fragments. Following confirmation of binding activity and blocking of S1:ACE2 interactions by candidate scFvs, the most potent of these candidates were converted to IgG1 antibodies bearing the LALA Fc modification. Candidate nAbs were characterized for binding of Spike S1 subunit and neutralization of related clinical SARS-CoV-2 isolates. Affinity maturation of potent nAbs was carried out in parallel to biophysical profiling, cell line development, and evaluation of protective efficacy for the parental nAbs, S1D2 and S7E3 to yield STI-2020 and STI-5041, respectively. Artwork credit: William SooHoo. ( B ) Affinity measurements of STI-2020 and STI-5041 for Spike S1 domain from the following isolates and VOCs: USA/WA-1/2020(WA-1) isolate, D614G 2020001 isolate, B.1.1.7 VOC (Alpha), B.1.351 VOC (Beta). The antibody affinities were measured using SPR on a BIAcore T200 instrument using a 1:1 binding model. ( C ) Spike protein derived from the WA-1 and 2020001 (D614G) SARS-CoV-2 isolates were independently expressed on the surface of HEK 293 cells. Serially-diluted STI-2020 or STI-5041 were assayed for Spike protein binding by flow cytometry. To quantify antibody binding, mean fluorescent intensity was measured for each dilution tested and the EC 50 value was calculated for each nAb. Representative replicate experiments are shown. ( D ) STI-2020 and STI-5041 were evaluated in neutralizing test for potency against SARS-CoV-2 USA/WA-1/2020, 2020001 (D614G), B.1.1.7 VOC (Alpha), B.1.351 VOC (Beta) and B.1.617.2 (Delta).

Article Snippet: For the pseudovirus neutralization study, codon optimized SARS-CoV-2 Wuhan Spike carrying the D614G amino acid change (Sino Biological, Cat #VG40589-UT(D614G)) was modified to remove the last 21 amino acids at the C-terminus (SpikeΔ21) and was used as the parental clone.

Techniques: Binding Assay, Activity Assay, Blocking Assay, Modification, Neutralization, Derivative Assay, Protein Binding, Flow Cytometry

Journal: Scientific Reports

Article Title: Unbiased approach to identify and assess efficacy of human SARS-CoV-2 neutralizing antibodies

doi: 10.1038/s41598-022-19780-7

Figure Lengend Snippet: Pharmacokinetic and bioavailability of Neutralizing Antibody. ( A ) Neutralization of SARS-CoV-2 Spike-pseudotyped VSV by STI-2020 and STI-5041. VSVΔG-luciferase was pseudotyped with the indicated spike variant, incubated with STI-2020 or STI-5041 at a range of 0.0005–10 µg/mL for 30 min, then added to 293-ACE2 target cells. Absolute IC 50 was calculated from luciferase values and are indicated. Experiments were performed at least three independent times and data presented as the mean ± SD. ( B ) Epitope binning was performed as described in the Materials and Methods. The sensorgram shows STI-5041 can bind to SARS-CoV-2 S1 and STI-2020 complex (blue line) and indicates that STI-5041 and STI-2020 bind to distinct epitopes. ( C–H ) Biodistribution: Concentration of STI-2020 ( C , D ) or 5041 ( E , F ) in serum and lung lavage or lysates of spleens, lungs, small intestines, and large intestines collected from female CD-1 mice administered STI-2020 IV at doses of 0.5 mg/kg (Dark blue circle), 0.05 mg/kg (Sky blue circle), or 0.005 mg/kg (light blue circle) or IN at doses of 2.5 mg/kg (black circle), 0.5 mg/kg (Maroon circle), 0.05 mg/kg (Red Circle), and 0.005 mg/kg (Rose circle) or STI-5041 administered IV at doses of 2 mg/kg (Dark blue circle), 0.2 mg/kg (Sky blue circle), and 0.02 mg/kg (light blue circle) or IN at doses of 10 mg/kg (Black circle), 2 mg/kg (Maroon circle), 0.2 mg/kg (Red circle), and 0.02 mg/kg (Rose circle) at 24 h post-administration as compared to samples collected from untreated mice. Values represent mean ± SEM (n = 3 animals no treatment group, n = 5 in treatment groups). Significant differences are denoted by * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001. Pharmacokinetics: Concentration of STI-2020 ( G ) or STI-5041 ( H ) in lungs and isolated serum collected from female CD-1 mice administered STI-2020 or STI-5041 intranasally (IN) at a dose of 5 mg/kg or 20 mg/kg, respectively. Samples from treated mice were collected at the indicated timepoint post-administration; antibody concentrations were quantified by ELISA and compared to samples collected from untreated mice. Overlay of antibody concentrations in lung tissue vs. serum following IN administration of a 5 mg/kg or 20 mg/kg dose. Values represent mean ± SD (n = 3 animals no treatment group, n = 6 per time point in treatment groups).

Article Snippet: For the pseudovirus neutralization study, codon optimized SARS-CoV-2 Wuhan Spike carrying the D614G amino acid change (Sino Biological, Cat #VG40589-UT(D614G)) was modified to remove the last 21 amino acids at the C-terminus (SpikeΔ21) and was used as the parental clone.

Techniques: Neutralization, Luciferase, Variant Assay, Incubation, Concentration Assay, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Unbiased approach to identify and assess efficacy of human SARS-CoV-2 neutralizing antibodies

doi: 10.1038/s41598-022-19780-7

Figure Lengend Snippet: Efficacy of Intravenous (IV) Delivery of Neutralizing Antibodies in the golden (Syrian) Hamster Model of COVID-19. Female hamsters were inoculated with SARS-CoV-2 USA/WA-1/2020 ( A , B ) or SARS-CoV-2 Beta variant ( C , D ) on day 0. One-hour post-infection, animals were administered a single intravenous dose of Isotype control IgG (500 µg) or, For A and B, STI-2020 (100 µg, 300 µg, or 500 µg); For C and D, Isotype control IgG (1,000 µg) or STI-5041 (500 µg, or 1,000 µg). Weight changes were recorded for up to 11 days. ( A ) Average % weight change ± SEM was plotted for each group. Days in which there was a significant difference in average % weight change compared to Isotype control IgG 500 µg-treated animals are denoted by * ( p -value < 0.05). ( B ) Lung tissues collected from five animals per group and virus titers were determined on day 5. A broken line indicates the detection limit of the assay (< 1.5 TCID 50 /g). ( C ) Average % weight change ± SEM was plotted for each group. Days in which there was a significant difference in average % weight change for STI-5041 at 500 µg or 1,000 µg compared to Isotype control IgG 1000 µg-treated animals are denoted by * ( p -value < 0.05). ( D ) Lung tissues collected from five animals per group administered Isotype control IgG (1,000 µg) or STI-5041 (500 µg or 1,000 µg) and virus titers were determined on day 5.

Article Snippet: For the pseudovirus neutralization study, codon optimized SARS-CoV-2 Wuhan Spike carrying the D614G amino acid change (Sino Biological, Cat #VG40589-UT(D614G)) was modified to remove the last 21 amino acids at the C-terminus (SpikeΔ21) and was used as the parental clone.

Techniques: Variant Assay, Infection, Control, Virus

Journal: Scientific Reports

Article Title: Unbiased approach to identify and assess efficacy of human SARS-CoV-2 neutralizing antibodies

doi: 10.1038/s41598-022-19780-7

Figure Lengend Snippet: Efficacy of Intranasal (IN) Delivery of Neutralizing Antibodies in the golden (Syrian) Hamster Model of COVID-19. ( A , B ) Female hamsters were inoculated with SARS-CoV-2 USA/WA-1/2020, and then administrated with 500 µg or 400 µg Isotype control antibody or 500 µg or 400 µg STI-2020 intranasally at 12 h post-infection. ( A ) Average % weight change ± SEM was plotted for each group. Days in which there was a significant difference in average % weight change for STI-5020 at 500 µg compared to Isotype control IgG 500 µg-treated animals are denoted by * ( p -value < 0.05). ( B ) Upper panels show representative figures of nasal turbinates and nasal septum at 5 d.p.i. Average ± SEM of OE thickness on the nasal septum in the lower graph for STI-5020 at 400 µg compared to Isotype control IgG 400 µg-treated animals are denoted with * ( p -value < 0.05). ( C ) Hamsters were inoculated with SARS-CoV-2 Beta variant, and then administrated with 500 µg Isotype control antibody or 100 µg, 300 µg, or 500 µg STI-5041 intranasally at 12 h post-infection. Average % weight change ± SEM was plotted for each group.

Article Snippet: For the pseudovirus neutralization study, codon optimized SARS-CoV-2 Wuhan Spike carrying the D614G amino acid change (Sino Biological, Cat #VG40589-UT(D614G)) was modified to remove the last 21 amino acids at the C-terminus (SpikeΔ21) and was used as the parental clone.

Techniques: Control, Infection, Variant Assay