Journal: bioRxiv

Article Title: Spike Protein Targeting “Nano-Glue” that Captures and Promotes SARS-CoV-2 Elimination

doi: 10.1101/2021.04.13.439641

Figure Lengend Snippet: a) The schema of the CIPS blocking the infection of SARS-CoV-2 to the host cells. b) The antiviral effect of CIPS on SARS-CoV-2 infection with Vero-E6 cells was measured after 48 h virus post-infection with/without CIPS. Real-time PCR data for SARS-CoV-2 ORF1ab from one out of three experiments are presented as mean ± SEM of 3-5 biological replicates. Cell viability of CIPS was evaluated after 48 h. c) Representative images of Vero-E6 cells 48 h post-infection with SARS-CoV-2. Ctrl: uninfected cells; Virus: cells infected with SARS-CoV-2; Virus + CIPS: cells infected with SARS-CoV-2 treated with 24 pM CIPS. d) Antiviral effect of CIPS on Vero-E6 cells with SARS-CoV-2 post-infection. Vero-E6 cells were firstly challenged with SARS-CoV-2 (12000 pfu) for 2 h, and continuously cultured in fresh or CIPS containing medium for another 48 h. Intracellular SARS-CoV-2 was evaluated by qPCR. e-h) The anti-SARS-CoV-2 capacity of CIPS on human airway epithelial organoids derived from human nasal tissue. (e) SARS-CoV-2 replication (ORF1ab) was observed upon infection on the cultured airway epithelium in the presence or absence of 24 pM CIPS. Data presented as mean ± SEM (n=3). ***, p<0.005 by Student’s t- test. (f) Representative image of H&E staining of cultured tissue cross-sections. Ctrl: uninfected tissue; Virus: tissue infected with SARS-CoV-2; Virus + CIPS: tissue infected with SARS-CoV-2 treated with 24 pM CIPS. (g) The thickness of the human respiratory epithelium section that measured by ImageJ. Data are representative of best epithelium section, and presented as mean±SEM of technical triplicates. *, p<0.05 and **, p<0.01 by Student’s t-test. (h) Cell distribution in the human respiratory epithelium after SARS-CoV-2 infection. Cross-section of the human respiratory epithelial tissue in control culture conditions (Ctrl), upon exposure to SARS-CoV-2 (Virus) and exposed to SARS-CoV-2 together with CIPS (Virus+CIPS). Green: tubulin staining the ciliated columnar epithelial cells; Red: MU5AC staining of mucus-producing goblet cells; Blue: DAPI staining of nuclei. Scale bar = 20 μm. The white arrows show the sites of tissue damage.

Article Snippet: The sensor was then immersed into the solution of different proteins, the receptor binding domain of the SARS-CoV-2 spike protein (RBD, from DIMA Biotechnology Ltd, China), human serum albumin (HSA, from Solarbio Science & Technology Co., Ltd. China), fibrinogen (FG, from Sigma-Aldrich), immunoglobulin G (IgG, from Sigma-Aldrich), transferrin (Tf, from Sigma-Aldrich), high density lipoprotein (HDL, from Yiyuan biotechnology Ltd, Guangzhou, China)) at different concentrations (12.5-200 nM) and then their binding was assessed with an association time of 300 s and a dissociation time of 600 s. The same procedure was adopted to evaluate the binding affinity of graphene oxide (GO) and MoS 2 NSs to RBD.

Techniques: Blocking Assay, Infection, Real-time Polymerase Chain Reaction, Cell Culture, Derivative Assay, Staining

Journal: bioRxiv

Article Title: Spike Protein Targeting “Nano-Glue” that Captures and Promotes SARS-CoV-2 Elimination

doi: 10.1101/2021.04.13.439641

Figure Lengend Snippet: a) TEM images of SC2-P, CIPS and SC2-P incubated with CIPS. b) WB analysis showing SC2-P binding to CIPS (pM). c) CIPS effectively binds to SARS-CoV-2 virus. CIPS incubated with SARS-CoV-2 for 2 h and separated by centrifugation. The SARS-CoV-2 was quantitatively assessed in CIPS precipitates. Data as mean ± SEM of technical triplicates. ***, p<0.005 by Student’s t- test. d, e) Binding affinity for CIPS of RBD at different concentrations (nM) (d) and various proteins at 50 nM (e) as determined by Biolayer Interferometry (BLI). f-g) The SARS-CoV-2 inhibiting capacity in complex biological matrices. SC2-P were mixed with FBS at different ratios and incubated with 12 pM CIPS for 2 h. f) WB analysis showing the specific binding of SC2-P to CIPS in the presence of FBS. g) Anti-SARS-CoV-2 activity of CIPS in the presence of FBS. The FBS/SC2-P/CIPS were used to infect ACE2/293T cells for 2 h. Cells were lysed after 40 h incubation and the intracellular SC2-P were detected based on luciferase activity. Data in (g) are from a representative experiment out of three performed, and presented as mean ± SEM of technical triplicates. ***, p<0.005 by Student’s t- test. h-i) Inhibition of RBD binding to ACE2 (nM, on the right) by CIPS, as determined by BLI. j-k) Conformational variations in the RBD structure after CIPS interaction, as determined by CD spectra.

Article Snippet: The sensor was then immersed into the solution of different proteins, the receptor binding domain of the SARS-CoV-2 spike protein (RBD, from DIMA Biotechnology Ltd, China), human serum albumin (HSA, from Solarbio Science & Technology Co., Ltd. China), fibrinogen (FG, from Sigma-Aldrich), immunoglobulin G (IgG, from Sigma-Aldrich), transferrin (Tf, from Sigma-Aldrich), high density lipoprotein (HDL, from Yiyuan biotechnology Ltd, Guangzhou, China)) at different concentrations (12.5-200 nM) and then their binding was assessed with an association time of 300 s and a dissociation time of 600 s. The same procedure was adopted to evaluate the binding affinity of graphene oxide (GO) and MoS 2 NSs to RBD.

Techniques: Incubation, Binding Assay, Centrifugation, Activity Assay, Luciferase, Inhibition

Journal: bioRxiv

Article Title: Spike Protein Targeting “Nano-Glue” that Captures and Promotes SARS-CoV-2 Elimination

doi: 10.1101/2021.04.13.439641

Figure Lengend Snippet: a-d) CIPS promotes the phagocytosis and elimination of SC2-P (a-b) and SARS-CoV-2 (c-d) in Macrophage-like differentiated THP-1 cells. a-b) The SC2-P were pre-incubated with 12 pM CIPS for 2 h. Macrophage-like differentiated THP-1 cells were treated with SC2-P with/without CIPS pre-incubation for 24 h (Phagocytosis). SC2-P containing medium was replaced by fresh medium and cultured for another 24 h (Degradation). b) Quantitative and statistical analysis with the infected SC2-P of IF data. c) Differentiated THP-1 was challenged with SARS-CoV-2 for 4 h (Phagocytosis). The SARS-CoV-2 challenged THP-1 was continuously cultured in fresh medium for another 48 h (Degradation). Intracellular SARS-CoV-2 was evaluated by qPCR. d) SARS-CoV-2 was pre-incubated with 12 pM CIPS for 2 h, and added to differentiated THP-1 cells for 48 h in the presence or absence of Bafilomycin (BM). Intracellular SARS-CoV-2 virus was assessed by qPCR. e-f) The SC2-P (e) and SARS-CoV-2 (f) cannot infect the differentiated THP-1 cells. Differentiated THP-1 was co-cultured with SC2-P (24 h) or SARS-CoV-2 (4 h), and the cultured mediums were replaced with fresh medium for another 24 (e) or 48 h (f) incubation. The SC2-P infection was assessed with LUC activity (e), and SARS-CoV-2 infection (supernatant) were evaluated by qPCR (f). Data presented as mean ± SEM of technical triplicates. ##, **, p<0.01, ###, ***, ∇∇∇, p<0.005 by Student’s t- test. ND: not detected. g) The uptake and the degradation of CIPS by macrophages as determined by ICP-MS. Intracellular Indium was used to describe the accumulation and degradation of CIPS and related compounds resulting from CIPS degradation. Differentiated THP-1 cells were treated with 12 pM CIPS up to 24 h (Uptake) and further cultured in fresh medium for 24 and 48 h (Degradation). h) Three-dimensional tomographic images showing CIPS intracellular accumulation after 12 h uptake and 48 h degradation. Images were obtained by soft X-ray transmission microscope (Nano-CT). i-j) Cu chemical transformation and degradation from intracellular CIPS during the uptake and degradation phases, as determined by Cu K-edge XANES. (i) Chemical species of Cu in different reference samples and (j) the speciation of Cu from CIPS during the uptake and the degradation processes. The percentage of Cu forms is reported in Table S2. k-l) CD86 and HLA-DRA gene expression in SARS-CoV-2 treated macrophages in the presence or absence of CIPS. SARS-CoV-2 virus with/without CIPS were incubated with PMA-differentiated THP-1 for 48 h. HLA: HLA-DRA. GAPDH was used as housekeeping gene and the relative gene expression was normalized to the virus infection group. Data presented as mean±SEM (n=3). *, p<0.05, **, p<0.01 by Student’s t -test. (m) CIPS induced DC maturation after SC2-P incubation. SC2-P, and SC2-P with CIPS were incubated with DC for 24 h. The CD11c and CD86 positive cells were analyzed by flow cytometer. The double positive (CD11c and CD86) DC were regarded as mature DC cells. Data presented as mean±SEM (n=3). PMA used as positive control. ***, p<0.001 by One ANOVA.

Article Snippet: The sensor was then immersed into the solution of different proteins, the receptor binding domain of the SARS-CoV-2 spike protein (RBD, from DIMA Biotechnology Ltd, China), human serum albumin (HSA, from Solarbio Science & Technology Co., Ltd. China), fibrinogen (FG, from Sigma-Aldrich), immunoglobulin G (IgG, from Sigma-Aldrich), transferrin (Tf, from Sigma-Aldrich), high density lipoprotein (HDL, from Yiyuan biotechnology Ltd, Guangzhou, China)) at different concentrations (12.5-200 nM) and then their binding was assessed with an association time of 300 s and a dissociation time of 600 s. The same procedure was adopted to evaluate the binding affinity of graphene oxide (GO) and MoS 2 NSs to RBD.

Techniques: Incubation, Cell Culture, Infection, Activity Assay, Transmission Assay, Microscopy, Transformation Assay, Expressing, Flow Cytometry, Positive Control

Journal: bioRxiv

Article Title: Spike Protein Targeting “Nano-Glue” that Captures and Promotes SARS-CoV-2 Elimination

doi: 10.1101/2021.04.13.439641

Figure Lengend Snippet: The mechanism that possessed by CIPS on SARS-CoV-2 restraining and elimination. The RBD of S protein can be tightly absorbed to CIPS that induced RBD deformation. The selectively binding capacity of CIPS on RBD with a K D < 1 pM that is 1000 times higher than the reported neutralizing antibodies, causes the inhibition of SARS-CoV-2 infection. Further, the CIPS-trapped SARS-CoV-2 can be effectively phagocytized by macrophages and lead to virus elimination in lysosome, which further facilitates COVID-19 treatment.

Article Snippet: The sensor was then immersed into the solution of different proteins, the receptor binding domain of the SARS-CoV-2 spike protein (RBD, from DIMA Biotechnology Ltd, China), human serum albumin (HSA, from Solarbio Science & Technology Co., Ltd. China), fibrinogen (FG, from Sigma-Aldrich), immunoglobulin G (IgG, from Sigma-Aldrich), transferrin (Tf, from Sigma-Aldrich), high density lipoprotein (HDL, from Yiyuan biotechnology Ltd, Guangzhou, China)) at different concentrations (12.5-200 nM) and then their binding was assessed with an association time of 300 s and a dissociation time of 600 s. The same procedure was adopted to evaluate the binding affinity of graphene oxide (GO) and MoS 2 NSs to RBD.

Techniques: Binding Assay, Inhibition, Infection