nucleocapsid  (Sino Biological)


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    Name:
    SARS CoV SARS CoV 2 Nucleocapsid Antibody Rabbit MAb
    Description:
    This product is a recombinant monoclonal antibody expressed from HEK293 cells
    Catalog Number:
    40143-R001
    Price:
    None
    Category:
    Primary Antibody
    Reactivity:
    SARS
    Applications:
    WB,ELISA
    Immunogen:
    Recombinant SARS-CoV Nucleoprotein / NP Protein (Catalog#40143-V08B)
    Product Aliases:
    Anti-coronavirus NP Antibody, Anti-coronavirus Nucleocapsid Antibody, Anti-coronavirus Nucleoprotein Antibody, Anti-cov np Antibody, Anti-ncov NP Antibody, Anti-novel coronavirus NP Antibody, Anti-novel coronavirus Nucleocapsid Antibody, Anti-novel coronavirus Nucleoprotein Antibody, Anti-NP Antibody, Anti-Nucleocapsid Antibody, Anti-Nucleoprotein Antibody
    Antibody Type:
    MAb
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
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    Structured Review

    Sino Biological nucleocapsid
    This product is a recombinant monoclonal antibody expressed from HEK293 cells
    https://www.bioz.com/result/nucleocapsid/product/Sino Biological
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nucleocapsid - by Bioz Stars, 2021-05
    96/100 stars

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    Related Articles

    other:

    Article Title: Long-Term Modeling of SARS-CoV-2 Infection of In Vitro Cultured Polarized Human Airway Epithelium
    Article Snippet: Antibodies used.Primary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (clone 001) (catalog no. 40143-R001; SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6) (catalog no. T7941; MilliporeSigma, St. Louis, MO) at 1:100, mouse anti-ZO-1 (clone 1/ZO-1) (catalog no. 610966; BD Bioscience, San Jose, CA) at 1:100, and rabbit anti-Ki67 (clone SP6) (ab1666; Abcam, Cambridge, MA) at 1:50.

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium
    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Article Title: Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation
    Article Snippet: 40143-R001 (capture) and 40143-MM05 (detection) were used for N detection.

    Incubation:

    Article Title: The SARS-CoV-2 transcriptome and the dynamics of the S gene furin cleavage site in primary human airway epithelia
    Article Snippet: The fixed membrane was washed in PBS for 5 min three times and then split into several pieces for whole-mount immunostaining. .. Following permeabilization with 0.2% Triton X-100 for 15 min at room temperature, the slide was incubated with a rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (# 40143-R001; SinoBiological US, Wayne, PA) at a dilution of 1:25 in PBS with 2% fetal bovine serum for 1 h at 37°C. .. After washing, the slide was incubated with a rhodamine-conjugated secondary antibody, followed by staining of the nuclei with DAPI (4’,6-diamidino-2-phenylindole).

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies
    Article Snippet: .. 24 h post infection, cells were fixed with 4% paraformaldehyde for 30 min, followed by two PBS (pH 7.4) washes and permeabilization with 0.25% Triton X-100 in PBS for 30 min. After blocking in 5% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological, cat. 40143-R001) at a 1:2000 dilution for 1h. .. After washing and incubation with a secondary Alexa647-labeled antibody mixed with 1 ug/ml Hoechst33342 for 1 hour, plates were imaged on an automated cell-imaging reader (Cytation 5, Biotek) and nucleocapsid-positive cells were counted using the manufacturer’s supplied software.

    Infection:

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies
    Article Snippet: .. 24 h post infection, cells were fixed with 4% paraformaldehyde for 30 min, followed by two PBS (pH 7.4) washes and permeabilization with 0.25% Triton X-100 in PBS for 30 min. After blocking in 5% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological, cat. 40143-R001) at a 1:2000 dilution for 1h. .. After washing and incubation with a secondary Alexa647-labeled antibody mixed with 1 ug/ml Hoechst33342 for 1 hour, plates were imaged on an automated cell-imaging reader (Cytation 5, Biotek) and nucleocapsid-positive cells were counted using the manufacturer’s supplied software.

    Blocking Assay:

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies
    Article Snippet: .. 24 h post infection, cells were fixed with 4% paraformaldehyde for 30 min, followed by two PBS (pH 7.4) washes and permeabilization with 0.25% Triton X-100 in PBS for 30 min. After blocking in 5% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological, cat. 40143-R001) at a 1:2000 dilution for 1h. .. After washing and incubation with a secondary Alexa647-labeled antibody mixed with 1 ug/ml Hoechst33342 for 1 hour, plates were imaged on an automated cell-imaging reader (Cytation 5, Biotek) and nucleocapsid-positive cells were counted using the manufacturer’s supplied software.

    Recombinant:

    Article Title: SARS-CoV-2 RapidPlex: A Graphene-Based Multiplexed Telemedicine Platform for Rapid and Low-Cost COVID-19 Diagnosis and Monitoring
    Article Snippet: CRP polyclonal antibody labeled with horseradish peroxidase (HRP) (PA1-28329) and 3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric substrate was purchased from Invitrogen. .. Mouse NP monoclonal antibody (mAb) (40143-MM05), SARS-CoV-2 NP antigen (40588-V08B), SARS-CoV/SARS-CoV-2 nucleocapsid antibody, rabbit mAb (40143-R001), SARS-CoV NP antigen (HCoV-OC43; 40643-V07E), SARS-CoV-2 Spike S1-His recombinant protein (HPLC-verified) (40591-V08H), and SARS-CoV Spike S1 protein (S1 subunit, His tag) (40150-V08B1) were purchased from Sino Biological. ..

    High Performance Liquid Chromatography:

    Article Title: SARS-CoV-2 RapidPlex: A Graphene-Based Multiplexed Telemedicine Platform for Rapid and Low-Cost COVID-19 Diagnosis and Monitoring
    Article Snippet: CRP polyclonal antibody labeled with horseradish peroxidase (HRP) (PA1-28329) and 3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric substrate was purchased from Invitrogen. .. Mouse NP monoclonal antibody (mAb) (40143-MM05), SARS-CoV-2 NP antigen (40588-V08B), SARS-CoV/SARS-CoV-2 nucleocapsid antibody, rabbit mAb (40143-R001), SARS-CoV NP antigen (HCoV-OC43; 40643-V07E), SARS-CoV-2 Spike S1-His recombinant protein (HPLC-verified) (40591-V08H), and SARS-CoV Spike S1 protein (S1 subunit, His tag) (40150-V08B1) were purchased from Sino Biological. ..

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  • 96
    Sino Biological sars cov sars cov 2 nucleocapsid antibody rabbit mab
    Plasmablasts produce potent antibodies against <t>SARS-CoV-2</t> with diverse V genes and limited mutations. A, VH, VK and VL gene usage of antibodies from plasmablasts and memory B cells (MBCs). Up to the top four genes in each chart are shown with different colors (genes that were tied for 4 th and lower are not highlighted). B , Tukey’s plots showing heavy and light chain gene mutations of antibodies from plasmablasts and MBCs. Percent mutations were compared with the Mann-Whitney U-test. The middle line shows the median, and the box extends from the 1 st to 3 rd quartile. C , Top 21 neutralizing antibodies (IC50
    Sars Cov Sars Cov 2 Nucleocapsid Antibody Rabbit Mab, supplied by Sino Biological, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sars cov sars cov 2 nucleocapsid antibody rabbit mab/product/Sino Biological
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov sars cov 2 nucleocapsid antibody rabbit mab - by Bioz Stars, 2021-05
    96/100 stars
      Buy from Supplier

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    Plasmablasts produce potent antibodies against SARS-CoV-2 with diverse V genes and limited mutations. A, VH, VK and VL gene usage of antibodies from plasmablasts and memory B cells (MBCs). Up to the top four genes in each chart are shown with different colors (genes that were tied for 4 th and lower are not highlighted). B , Tukey’s plots showing heavy and light chain gene mutations of antibodies from plasmablasts and MBCs. Percent mutations were compared with the Mann-Whitney U-test. The middle line shows the median, and the box extends from the 1 st to 3 rd quartile. C , Top 21 neutralizing antibodies (IC50

    Journal: bioRxiv

    Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

    doi: 10.1101/2021.04.01.437942

    Figure Lengend Snippet: Plasmablasts produce potent antibodies against SARS-CoV-2 with diverse V genes and limited mutations. A, VH, VK and VL gene usage of antibodies from plasmablasts and memory B cells (MBCs). Up to the top four genes in each chart are shown with different colors (genes that were tied for 4 th and lower are not highlighted). B , Tukey’s plots showing heavy and light chain gene mutations of antibodies from plasmablasts and MBCs. Percent mutations were compared with the Mann-Whitney U-test. The middle line shows the median, and the box extends from the 1 st to 3 rd quartile. C , Top 21 neutralizing antibodies (IC50

    Article Snippet: Following fixation, the cells were permeabilized with Triton X-100 and probed with a SARS-CoV/SARS-CoV-2 nucleoprotein-specific rabbit primary antibody (Sino Biological, Wayne, PA, USA, 40143-R001) followed by an Alexa Fluor 647-conjugated secondary antibody (Life Technologies, San Diego, CA, USA, A21245).

    Techniques: MANN-WHITNEY

    Crystal structure of SARS-CoV-2 RBD in complex with CV503. A , CV503 binds to the ridge region of SARS-CoV-2 RBD. The heavy and light chains of CV503 are shown in orange and yellow, respectively. SARS-CoV-2 RBD is in white, where its ridge region (residues 471–491) is shown in blue. B, The ACE2/RBD complex structure (PDB ID: 6M0J) 52 is superimposed on the CV503/RBD complex. The heavy chain of CV503 (orange) would clash with ACE2 (green) if bound to RBD simultaneously (indicated by red circle). C-D , Epitope of CV503. Epitope residues contacting the heavy chain are in dark blue and light chain are in light blue, while residues contacting both heavy and light chains are in ocean blue. In C , CDR loops that are directly involved in RBD-binding are labeled. In D , epitope residues are labeled. Epitope residues that are also involved in ACE2 binding are labeled in red. E, ACE2-binding site on the RBD are in light pink. ACE2 is represented as semi- transparent cartoon in pale green. Epitope residues and ACE2-interacting residues are defined as those with a buried surface area (BSA) > 0 Å 2 . F , F486 at the ridge region of SARS-CoV-2 RBD (blue) is clamped in a hydrophobic pocket formed by the heavy (orange) and light chains (yellow) of CV503.

    Journal: bioRxiv

    Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

    doi: 10.1101/2021.04.01.437942

    Figure Lengend Snippet: Crystal structure of SARS-CoV-2 RBD in complex with CV503. A , CV503 binds to the ridge region of SARS-CoV-2 RBD. The heavy and light chains of CV503 are shown in orange and yellow, respectively. SARS-CoV-2 RBD is in white, where its ridge region (residues 471–491) is shown in blue. B, The ACE2/RBD complex structure (PDB ID: 6M0J) 52 is superimposed on the CV503/RBD complex. The heavy chain of CV503 (orange) would clash with ACE2 (green) if bound to RBD simultaneously (indicated by red circle). C-D , Epitope of CV503. Epitope residues contacting the heavy chain are in dark blue and light chain are in light blue, while residues contacting both heavy and light chains are in ocean blue. In C , CDR loops that are directly involved in RBD-binding are labeled. In D , epitope residues are labeled. Epitope residues that are also involved in ACE2 binding are labeled in red. E, ACE2-binding site on the RBD are in light pink. ACE2 is represented as semi- transparent cartoon in pale green. Epitope residues and ACE2-interacting residues are defined as those with a buried surface area (BSA) > 0 Å 2 . F , F486 at the ridge region of SARS-CoV-2 RBD (blue) is clamped in a hydrophobic pocket formed by the heavy (orange) and light chains (yellow) of CV503.

    Article Snippet: Following fixation, the cells were permeabilized with Triton X-100 and probed with a SARS-CoV/SARS-CoV-2 nucleoprotein-specific rabbit primary antibody (Sino Biological, Wayne, PA, USA, 40143-R001) followed by an Alexa Fluor 647-conjugated secondary antibody (Life Technologies, San Diego, CA, USA, A21245).

    Techniques: Binding Assay, Labeling

    Potent antibodies against SARS-CoV-2 target diverse epitopes on the RBD and NTD and neutralize emerging variants. A , Isoaffinity plot of antibodies targeting SARS-CoV-2 RBD (representative of n = 2 experiments). The affinity (KD) values on the top and right of the plot refer to the dotted lines crossing the plot, e.g. any antibody falling on the 10 pM dotted line has a KD value of 10 pM. B , Neutralization potency versus affinity of anti-RBD antibodies. C , Isoaffinity plot of antibodies targeting SARS-CoV-2 NTD (representative of n = 2 experiments). The affinity (KD) values on the top and right of the plot refer to the dotted lines crossing the plot, e.g. any antibody falling on the 10 pM dotted line has a KD value of 10 pM. D , Neutralization potency versus affinity of anti-NTD antibodies. E , Epitope binning of anti-RBD antibodies (representative of n = 2 experiments). ACE2 was only used as an analyte (competitor) and not as a ligand, while all other antibodies were tested as both ligands and analytes. Solid lines indicate two-way competition while dotted lines indicate one-way competition. The number and percentage of neutralizing antibodies (IC50

    Journal: bioRxiv

    Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

    doi: 10.1101/2021.04.01.437942

    Figure Lengend Snippet: Potent antibodies against SARS-CoV-2 target diverse epitopes on the RBD and NTD and neutralize emerging variants. A , Isoaffinity plot of antibodies targeting SARS-CoV-2 RBD (representative of n = 2 experiments). The affinity (KD) values on the top and right of the plot refer to the dotted lines crossing the plot, e.g. any antibody falling on the 10 pM dotted line has a KD value of 10 pM. B , Neutralization potency versus affinity of anti-RBD antibodies. C , Isoaffinity plot of antibodies targeting SARS-CoV-2 NTD (representative of n = 2 experiments). The affinity (KD) values on the top and right of the plot refer to the dotted lines crossing the plot, e.g. any antibody falling on the 10 pM dotted line has a KD value of 10 pM. D , Neutralization potency versus affinity of anti-NTD antibodies. E , Epitope binning of anti-RBD antibodies (representative of n = 2 experiments). ACE2 was only used as an analyte (competitor) and not as a ligand, while all other antibodies were tested as both ligands and analytes. Solid lines indicate two-way competition while dotted lines indicate one-way competition. The number and percentage of neutralizing antibodies (IC50

    Article Snippet: Following fixation, the cells were permeabilized with Triton X-100 and probed with a SARS-CoV/SARS-CoV-2 nucleoprotein-specific rabbit primary antibody (Sino Biological, Wayne, PA, USA, 40143-R001) followed by an Alexa Fluor 647-conjugated secondary antibody (Life Technologies, San Diego, CA, USA, A21245).

    Techniques: Neutralization

    Bispecific antibodies prevent disease mediated by WT or E484K SARS-CoV-2 in an in vivo model. A, Weight change in hamsters that were administered CV1206_521_GS IP at a dose of 2.5 or 10 mg/kg, 24 h prior to IN virus exposure at 5log10 PFU. Differences between groups that were given the antibody versus PBS were determined using a mixed-effects repeated measures analysis with Dunnett’s multiple comparisons; **P

    Journal: bioRxiv

    Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

    doi: 10.1101/2021.04.01.437942

    Figure Lengend Snippet: Bispecific antibodies prevent disease mediated by WT or E484K SARS-CoV-2 in an in vivo model. A, Weight change in hamsters that were administered CV1206_521_GS IP at a dose of 2.5 or 10 mg/kg, 24 h prior to IN virus exposure at 5log10 PFU. Differences between groups that were given the antibody versus PBS were determined using a mixed-effects repeated measures analysis with Dunnett’s multiple comparisons; **P

    Article Snippet: Following fixation, the cells were permeabilized with Triton X-100 and probed with a SARS-CoV/SARS-CoV-2 nucleoprotein-specific rabbit primary antibody (Sino Biological, Wayne, PA, USA, 40143-R001) followed by an Alexa Fluor 647-conjugated secondary antibody (Life Technologies, San Diego, CA, USA, A21245).

    Techniques: In Vivo

    Bispecific antibodies utilize both binding sites to potently neutralize SARS-CoV-2 and are effective against emerging variants. A , Scheme of DVD-Ig TM . In our bispecific antibody naming system, the first name refers to the antibody used to make the outer binding site and the second refers to the antibody at the inner binding site. GS or EL refers to the type of linker connecting the two antigen-binding sites. See Materials and Methods for details. B , Binding of individual and bispecific antibodies to various domains from SARS-CoV-1 and SARS-CoV-2 (representative of n = 2 experiments). Area under the curve (AUC) values are shown after subtraction with the negative control antigen. C , Neutralization potency of bispecific antibodies with SARS-CoV-2 authentic and pseudotyped virus (MLV). Values are averaged from two experiments done in duplicate. D , Neutralization curves of CV1206_521_GS with SARS-CoV-2 authentic and pseudotyped virus. Curves are from a representative experiment, IC50 values for authentic virus are the average from two experiments and those for the pseudovirus are from an average of two (bispecific) or three (regular antibody) experiments. E, Neutralization potency of CV1206_521_GS versus a cocktail of CV1206 and CV521, with concentrations shown in the molar scale to enable a fair comparison. For the antibody combination, the values on the x-axis refers to the concentration of each antibody in the cocktail, e.g. 10 nM refers to 10 nM of CV1206 + 10 nM of CV521. F , 3D reconstruction of CV1206_521_GS from nsEM images. Two “one RBD up” models (PDB 6VYB) in green are docked into the reconstruction. Similarly, multiple mock scFv’s in orange and purple were docked to approximate the DVD-Ig molecule. O, outer binding site; I, inner binding site. G , Binding of bispecific antibody panel to spike protein containing mutations from B.1.1.7 and B.1.351 variants (n = 1 experiment). The numbers show the percentages of mAb binding to mutants relative to D614G (which was normalized to 100). H , Neutralization potency of bispecific antibodies against D614G, B.1.1.7 and B.1.351 variants relative to wild-type (pseudotyped) SARS-CoV-2 (n = 1 experiment). Ratios are shown in brackets: numbers smaller than 1 indicate an increase in potency while numbers larger than 1 indicate a decrease in potency relative to wild-type. ND, not determined. I, Potency of CV503_664_EL versus individual component mAbs against wild-type and B.1.351 SARS-CoV-2 pseudotyped virus (lentivirus).

    Journal: bioRxiv

    Article Title: Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants

    doi: 10.1101/2021.04.01.437942

    Figure Lengend Snippet: Bispecific antibodies utilize both binding sites to potently neutralize SARS-CoV-2 and are effective against emerging variants. A , Scheme of DVD-Ig TM . In our bispecific antibody naming system, the first name refers to the antibody used to make the outer binding site and the second refers to the antibody at the inner binding site. GS or EL refers to the type of linker connecting the two antigen-binding sites. See Materials and Methods for details. B , Binding of individual and bispecific antibodies to various domains from SARS-CoV-1 and SARS-CoV-2 (representative of n = 2 experiments). Area under the curve (AUC) values are shown after subtraction with the negative control antigen. C , Neutralization potency of bispecific antibodies with SARS-CoV-2 authentic and pseudotyped virus (MLV). Values are averaged from two experiments done in duplicate. D , Neutralization curves of CV1206_521_GS with SARS-CoV-2 authentic and pseudotyped virus. Curves are from a representative experiment, IC50 values for authentic virus are the average from two experiments and those for the pseudovirus are from an average of two (bispecific) or three (regular antibody) experiments. E, Neutralization potency of CV1206_521_GS versus a cocktail of CV1206 and CV521, with concentrations shown in the molar scale to enable a fair comparison. For the antibody combination, the values on the x-axis refers to the concentration of each antibody in the cocktail, e.g. 10 nM refers to 10 nM of CV1206 + 10 nM of CV521. F , 3D reconstruction of CV1206_521_GS from nsEM images. Two “one RBD up” models (PDB 6VYB) in green are docked into the reconstruction. Similarly, multiple mock scFv’s in orange and purple were docked to approximate the DVD-Ig molecule. O, outer binding site; I, inner binding site. G , Binding of bispecific antibody panel to spike protein containing mutations from B.1.1.7 and B.1.351 variants (n = 1 experiment). The numbers show the percentages of mAb binding to mutants relative to D614G (which was normalized to 100). H , Neutralization potency of bispecific antibodies against D614G, B.1.1.7 and B.1.351 variants relative to wild-type (pseudotyped) SARS-CoV-2 (n = 1 experiment). Ratios are shown in brackets: numbers smaller than 1 indicate an increase in potency while numbers larger than 1 indicate a decrease in potency relative to wild-type. ND, not determined. I, Potency of CV503_664_EL versus individual component mAbs against wild-type and B.1.351 SARS-CoV-2 pseudotyped virus (lentivirus).

    Article Snippet: Following fixation, the cells were permeabilized with Triton X-100 and probed with a SARS-CoV/SARS-CoV-2 nucleoprotein-specific rabbit primary antibody (Sino Biological, Wayne, PA, USA, 40143-R001) followed by an Alexa Fluor 647-conjugated secondary antibody (Life Technologies, San Diego, CA, USA, A21245).

    Techniques: Binding Assay, Negative Control, Neutralization, Concentration Assay

    Antibody and T cell responses after GX-19 vaccination in macaques. Macaques ( n = 3) were immunized with 3 mg of GX-19 as described in the Methods. Serum and PBMCs (peripheral blood mononuclear cells) were collected before (week 0), during (week 4 and 5.5), and after (week 8) vaccination and were assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA ( a ) and neutralizing antibodies against SARS-CoV-2 live virus ( b ). Data represent mean SEM of individual macaques (GX-19 #1, GX-19 #2, GX-19 #3), and dashed line indicates the assay limits of detection. The number of SARS-CoV-2 S-specific IFN- γ -secreting cells in PBMCs was determined by IFN- γ ELISPOT assay after stimulation with peptide pools spanning the SARS-CoV-2 S protein. Shown are spot-forming cells (SFC) per 10 6 PBMCS in triplicate wells ( c ). The frequency of S-specific CD4 + or CD8 + T cells producing IFN- γ , TNF-α, or IL-2 was determined by intracellular cytokine staining assays stimulated with SARS-CoV-2 S peptide pools. Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle) ( d ). Data of ( a , b , d ) are represented as individual values. p -Values determined by the Wilcoxon matched-pairs signed rank test; p -values are shown.

    Journal: Vaccines

    Article Title: Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates

    doi: 10.3390/vaccines9040307

    Figure Lengend Snippet: Antibody and T cell responses after GX-19 vaccination in macaques. Macaques ( n = 3) were immunized with 3 mg of GX-19 as described in the Methods. Serum and PBMCs (peripheral blood mononuclear cells) were collected before (week 0), during (week 4 and 5.5), and after (week 8) vaccination and were assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA ( a ) and neutralizing antibodies against SARS-CoV-2 live virus ( b ). Data represent mean SEM of individual macaques (GX-19 #1, GX-19 #2, GX-19 #3), and dashed line indicates the assay limits of detection. The number of SARS-CoV-2 S-specific IFN- γ -secreting cells in PBMCs was determined by IFN- γ ELISPOT assay after stimulation with peptide pools spanning the SARS-CoV-2 S protein. Shown are spot-forming cells (SFC) per 10 6 PBMCS in triplicate wells ( c ). The frequency of S-specific CD4 + or CD8 + T cells producing IFN- γ , TNF-α, or IL-2 was determined by intracellular cytokine staining assays stimulated with SARS-CoV-2 S peptide pools. Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle) ( d ). Data of ( a , b , d ) are represented as individual values. p -Values determined by the Wilcoxon matched-pairs signed rank test; p -values are shown.

    Article Snippet: After removing the blocking buffer, 3000-fold diluted 100 µL of anti-SARS-CoV-2 NP rabbit mAb (Sino Biological, 40143-R001, Beijing, China) was added on the Vero cells and incubated at 37 °C for 1h.

    Techniques: Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Staining

    Protective efficacy of GX-19 against SARS-CoV-2 challenge. Non-vaccinated ( n = 3, blue) and GX-19-vaccinated macaques ( n = 3, red) were challenged by intratracheal, oral, conjunctival, intranasal, and intravenous administration of 2.7 × 10 7 TCID 50 SARS-CoV-2. Viral load was assessed in nasal swab ( a ) and throat swab ( b ) at multiple time points following challenge. Summary of peak viral loads and viral load area under the curve (AUC) in nasal swab ( c , e ) and throat swab ( d , f ) following challenge. Dashed line indicates the assay limit of detection. Histopathological changes in the lungs of SARS-CoV-2-challenged macaques ( g ). Interstitial pneumonia score by microscopic evaluation ( n = 6 lung lobes of each animal per group) ( h ). The lung tissue sections were stained with hematoxylin and eosin (H E). Data of ( a – f ) are represented as individual values. Data of (h) is represented as 6 lung lobes of each animal per group. p -Values determined by the Mann–Whitney test; p -values are shown * p

    Journal: Vaccines

    Article Title: Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates

    doi: 10.3390/vaccines9040307

    Figure Lengend Snippet: Protective efficacy of GX-19 against SARS-CoV-2 challenge. Non-vaccinated ( n = 3, blue) and GX-19-vaccinated macaques ( n = 3, red) were challenged by intratracheal, oral, conjunctival, intranasal, and intravenous administration of 2.7 × 10 7 TCID 50 SARS-CoV-2. Viral load was assessed in nasal swab ( a ) and throat swab ( b ) at multiple time points following challenge. Summary of peak viral loads and viral load area under the curve (AUC) in nasal swab ( c , e ) and throat swab ( d , f ) following challenge. Dashed line indicates the assay limit of detection. Histopathological changes in the lungs of SARS-CoV-2-challenged macaques ( g ). Interstitial pneumonia score by microscopic evaluation ( n = 6 lung lobes of each animal per group) ( h ). The lung tissue sections were stained with hematoxylin and eosin (H E). Data of ( a – f ) are represented as individual values. Data of (h) is represented as 6 lung lobes of each animal per group. p -Values determined by the Mann–Whitney test; p -values are shown * p

    Article Snippet: After removing the blocking buffer, 3000-fold diluted 100 µL of anti-SARS-CoV-2 NP rabbit mAb (Sino Biological, 40143-R001, Beijing, China) was added on the Vero cells and incubated at 37 °C for 1h.

    Techniques: Staining, MANN-WHITNEY

    Immunization with GX-19 elicits Th1-biased T cell responses in mice. BALB/c mice ( n = 3–7/group) were immunized at weeks 0 and 2 with indicated doses of GX-19 or pGX27 (empty control vector) as described in the Methods ( a – c ). Sera were collected at 2 weeks post-boost and assessed for SARS-CoV-2 S-specific IgG1 and IgG2a/b. Endpoint titers ( a ), and endpoint tier ratios of IgG2a/b to IgG1 ( b ) were calculated. At 2 weeks post-boost, mouse splenocytes were isolated and re-stimulated with peptide pools spanning the SARS-CoV-2 S protein ex vivo. Indicated cytokines in the supernatants of culture were quantified using a Th1/Th2 cytometric bead array kit. Mean value of the medium alone background (mean ± s.d., pg ml −1 ) was 19.17 ± 8.61 for IFN- γ , 57.12 ± 6.53 for TNF-α, 33.10 ± 6.72 for IL-2, 7.83 ± 0.45 for IL-4, and 4.66 ± 0.13 for IL-5 ( d ). T cell responses were measured by IFN- γ ELISPOT in splenocytes stimulated with peptide pools spanning the SARS-CoV-2 S protein. Shown are spot-forming cells (SFC) per 10 6 splenocytes ( c ). Cells were stained for intracellular production of IFN- γ , TNF-α, and IL-2. Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle, Sigma-Aldrich, St. Louis, MO, USA) ( e ). Data representative of two independent experiments. All data are represented as individual values. * p

    Journal: Vaccines

    Article Title: Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates

    doi: 10.3390/vaccines9040307

    Figure Lengend Snippet: Immunization with GX-19 elicits Th1-biased T cell responses in mice. BALB/c mice ( n = 3–7/group) were immunized at weeks 0 and 2 with indicated doses of GX-19 or pGX27 (empty control vector) as described in the Methods ( a – c ). Sera were collected at 2 weeks post-boost and assessed for SARS-CoV-2 S-specific IgG1 and IgG2a/b. Endpoint titers ( a ), and endpoint tier ratios of IgG2a/b to IgG1 ( b ) were calculated. At 2 weeks post-boost, mouse splenocytes were isolated and re-stimulated with peptide pools spanning the SARS-CoV-2 S protein ex vivo. Indicated cytokines in the supernatants of culture were quantified using a Th1/Th2 cytometric bead array kit. Mean value of the medium alone background (mean ± s.d., pg ml −1 ) was 19.17 ± 8.61 for IFN- γ , 57.12 ± 6.53 for TNF-α, 33.10 ± 6.72 for IL-2, 7.83 ± 0.45 for IL-4, and 4.66 ± 0.13 for IL-5 ( d ). T cell responses were measured by IFN- γ ELISPOT in splenocytes stimulated with peptide pools spanning the SARS-CoV-2 S protein. Shown are spot-forming cells (SFC) per 10 6 splenocytes ( c ). Cells were stained for intracellular production of IFN- γ , TNF-α, and IL-2. Shown are the frequency of S-specific CD4 + or CD8 + T cells after subtraction of background (DMSO vehicle, Sigma-Aldrich, St. Louis, MO, USA) ( e ). Data representative of two independent experiments. All data are represented as individual values. * p

    Article Snippet: After removing the blocking buffer, 3000-fold diluted 100 µL of anti-SARS-CoV-2 NP rabbit mAb (Sino Biological, 40143-R001, Beijing, China) was added on the Vero cells and incubated at 37 °C for 1h.

    Techniques: Mouse Assay, Plasmid Preparation, Isolation, Ex Vivo, Enzyme-linked Immunospot, Staining

    GX-19 elicits robust binding and neutralizing antibody responses in mice. BALB/c mice ( n = 4–7/group) were immunized at weeks 0 and 2 with indicated doses of GX-19 or pGX27 as described in the Methods ( a – c ). Sera were collected at 2 weeks post-prime (blue) and 2 weeks post-boost (red) and assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA ( a ), and for post-boost sera, neutralizing antibodies against SARS-CoV-2 live virus ( c ). Bronchoalveolar lavages (BALs) were collected at 2 weeks post-boost and assayed for SARS-CoV-2 S-specific IgG antibodies by ELISA ( b ). Data representative of two independent experiments. All data are represented as individual values. * p

    Journal: Vaccines

    Article Title: Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates

    doi: 10.3390/vaccines9040307

    Figure Lengend Snippet: GX-19 elicits robust binding and neutralizing antibody responses in mice. BALB/c mice ( n = 4–7/group) were immunized at weeks 0 and 2 with indicated doses of GX-19 or pGX27 as described in the Methods ( a – c ). Sera were collected at 2 weeks post-prime (blue) and 2 weeks post-boost (red) and assessed for SARS-CoV-2 S-specific IgG antibodies by ELISA ( a ), and for post-boost sera, neutralizing antibodies against SARS-CoV-2 live virus ( c ). Bronchoalveolar lavages (BALs) were collected at 2 weeks post-boost and assayed for SARS-CoV-2 S-specific IgG antibodies by ELISA ( b ). Data representative of two independent experiments. All data are represented as individual values. * p

    Article Snippet: After removing the blocking buffer, 3000-fold diluted 100 µL of anti-SARS-CoV-2 NP rabbit mAb (Sino Biological, 40143-R001, Beijing, China) was added on the Vero cells and incubated at 37 °C for 1h.

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

    Diagram and immunogenicity of SARS-CoV-2 DNA vaccines. Schematic diagram of COVID-19 DNA vaccine expressing soluble SARS-CoV-2 S protein (S ΔTM ) or full-length SARS-CoV-2 S protein (S) ( a ). BALB/c mice ( n = 4–10/group) were immunized at weeks 0 and 2 with pGX27-S ΔTM , pGX27-S, or pGX27 (empty control vector) as described in the Methods. Sera were collected at 2 weeks post-prime (blue) and 2 weeks post-boost (red) and evaluated for SARS-CoV-2 S-specific IgG antibodies ( b ). All data are represented as individual values. ** p

    Journal: Vaccines

    Article Title: Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates

    doi: 10.3390/vaccines9040307

    Figure Lengend Snippet: Diagram and immunogenicity of SARS-CoV-2 DNA vaccines. Schematic diagram of COVID-19 DNA vaccine expressing soluble SARS-CoV-2 S protein (S ΔTM ) or full-length SARS-CoV-2 S protein (S) ( a ). BALB/c mice ( n = 4–10/group) were immunized at weeks 0 and 2 with pGX27-S ΔTM , pGX27-S, or pGX27 (empty control vector) as described in the Methods. Sera were collected at 2 weeks post-prime (blue) and 2 weeks post-boost (red) and evaluated for SARS-CoV-2 S-specific IgG antibodies ( b ). All data are represented as individual values. ** p

    Article Snippet: After removing the blocking buffer, 3000-fold diluted 100 µL of anti-SARS-CoV-2 NP rabbit mAb (Sino Biological, 40143-R001, Beijing, China) was added on the Vero cells and incubated at 37 °C for 1h.

    Techniques: Expressing, Mouse Assay, Plasmid Preparation

    A diagram of HAE-ALI and model of the SARS-CoV-2 recurrent infection in HAE. ( A ) HAE-ALI model: Epithelial cells are taken from bronchia of the lung of healthy donors and plated onto Transwell ® inserts at an air-liquid interface (ALI) for four weeks. Four major types of the epithelial cells in the well differentiated polarized HAE cultures: basal, ciliated, goblet, and club cells are diagrammed in the Transwell ® insert, and their expression makers are indicated. ( B ) Basal cells in proliferation. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 9 dpi (MOI=0.2) were dissociated from the Transwell ® insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-Ki67 and together with anti-CYKT5. Confocal images were taken at a magnification of 63 ×. Nuclei were stained with DAPI (blue). ( C ) Model of airway cell regeneration model of SARS-CoV-2 recurrent infections. SARS-CoV-2 infects apical ciliated and goblet cells, in which it replicates to produce infectious progeny and causes the death of the infected cells. The destructive lesion of epithelium induces basal cell proliferation and differentiation to regenerate ciliated and goblet cells, which are readily infected by SARS-CoV-2 in the next cycle of the recurrent infections.

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: A diagram of HAE-ALI and model of the SARS-CoV-2 recurrent infection in HAE. ( A ) HAE-ALI model: Epithelial cells are taken from bronchia of the lung of healthy donors and plated onto Transwell ® inserts at an air-liquid interface (ALI) for four weeks. Four major types of the epithelial cells in the well differentiated polarized HAE cultures: basal, ciliated, goblet, and club cells are diagrammed in the Transwell ® insert, and their expression makers are indicated. ( B ) Basal cells in proliferation. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 9 dpi (MOI=0.2) were dissociated from the Transwell ® insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-Ki67 and together with anti-CYKT5. Confocal images were taken at a magnification of 63 ×. Nuclei were stained with DAPI (blue). ( C ) Model of airway cell regeneration model of SARS-CoV-2 recurrent infections. SARS-CoV-2 infects apical ciliated and goblet cells, in which it replicates to produce infectious progeny and causes the death of the infected cells. The destructive lesion of epithelium induces basal cell proliferation and differentiation to regenerate ciliated and goblet cells, which are readily infected by SARS-CoV-2 in the next cycle of the recurrent infections.

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Infection, Expressing, Staining

    Immunofluorescence analysis of SARS-CoV-2 infected primary bronchial HAE-ALI over a course of 21 days. Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( B ). Confocal images were taken at a magnification of x 40 on the indicated days post-infection (dpi). Nuclei were stained with DAPI (blue).

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: Immunofluorescence analysis of SARS-CoV-2 infected primary bronchial HAE-ALI over a course of 21 days. Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( B ). Confocal images were taken at a magnification of x 40 on the indicated days post-infection (dpi). Nuclei were stained with DAPI (blue).

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Immunofluorescence, Infection, Staining

    SARS-CoV-2 infects ciliated and goblet epithelial cells but not basal and club cells. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 4 dpi (MOI=0.2) were dissociated from the Transwell ® insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-NP and together with anti-β-tubulin IV ( A ), and anti-MUC5AC ( B ), anti-cytokeratin 5 ( C ), and anti-SCGB1A1 ( D ), respectively. Confocal images were taken at a magnification of 63 ×. Nuclei were stained with DAPI (blue).

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: SARS-CoV-2 infects ciliated and goblet epithelial cells but not basal and club cells. Epithelial cells of the mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 4 dpi (MOI=0.2) were dissociated from the Transwell ® insert and cytospun onto slides. The cells on the slides were fixed, permeabilized, and immunostained with anti-NP and together with anti-β-tubulin IV ( A ), and anti-MUC5AC ( B ), anti-cytokeratin 5 ( C ), and anti-SCGB1A1 ( D ), respectively. Confocal images were taken at a magnification of 63 ×. Nuclei were stained with DAPI (blue).

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Infection, Staining

    Immunofluorescence analysis of SARS-CoV-2 infected primary bronchial HAE at various viral loads (multiplicities of infection). HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI from 0.2 to 0.00002. At 30 dpi, both virus and mock infected HAE were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( B ). Confocal images were taken at a magnification of x 40. Nuclei were stained with DAPI (blue).

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: Immunofluorescence analysis of SARS-CoV-2 infected primary bronchial HAE at various viral loads (multiplicities of infection). HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI from 0.2 to 0.00002. At 30 dpi, both virus and mock infected HAE were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( B ). Confocal images were taken at a magnification of x 40. Nuclei were stained with DAPI (blue).

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Immunofluorescence, Infection, Staining

    Three-dimensional (z-stack) imaging of SARS-CoV-2 infected primary bronchial HAE-ALI. Mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 15 dpi were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or with anti-NP and anti-β-tubulin IV antibodies (B), or co-stained anti-NP and anti-ZO-1 antibodies ( B ). A set of confocal images were taken at a magnification of x 40 from the stained pierce of epithelium at a distance of the Z value (μm), shown in each image, from the objective (z-axis) and reconstituted as a 3-dimensional (z-stack) image as shown in each channel of fluorescence. Nuclei were stained with DAPI (blue).

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: Three-dimensional (z-stack) imaging of SARS-CoV-2 infected primary bronchial HAE-ALI. Mock- and SARS-CoV-2-infected HAE-ALI B9-20 cultures at 15 dpi were co-stained with anti-NP and anti-ZO-1 antibodies ( A ), or with anti-NP and anti-β-tubulin IV antibodies (B), or co-stained anti-NP and anti-ZO-1 antibodies ( B ). A set of confocal images were taken at a magnification of x 40 from the stained pierce of epithelium at a distance of the Z value (μm), shown in each image, from the objective (z-axis) and reconstituted as a 3-dimensional (z-stack) image as shown in each channel of fluorescence. Nuclei were stained with DAPI (blue).

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Imaging, Infection, Staining, Fluorescence

    Virus release kinetics and transepithelial electrical resistance (TEER) measurement of HAE-ALI infected with SARS-CoV-2 at various viral loads (multiplicities of infection). (A C) Virus release kinetics. HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI of 0.2 (A), 0.02 and 0.002 (C), respectively, from the apical side. At the indicated days post-infection (dpi), 100 μl of apical washes by incubation of 100 μl of D-PBS in the apical chamber and 100 μl of the basolateral media were taken for plaque assays. Plaque forming units (pfu) were plotted to the DPI. Value represents the mean +/− standard deviations. ( B D ) Transepithelial electrical resistance measurement. The TEER of mock- and SARS-CoV-2-infected HAE-ALI culture were measured using an epithelial Volt-Ohm Meter (Millipore) at the indicated dpi. The TEER values were normalized to the TEER measured on the day of infection, which is set as 1.0. Values represent the mean of relative TEER +/− standard deviations. **** P

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: Virus release kinetics and transepithelial electrical resistance (TEER) measurement of HAE-ALI infected with SARS-CoV-2 at various viral loads (multiplicities of infection). (A C) Virus release kinetics. HAE-ALI B4-20 cultures were infected with SARS-CoV-2 at an MOI of 0.2 (A), 0.02 and 0.002 (C), respectively, from the apical side. At the indicated days post-infection (dpi), 100 μl of apical washes by incubation of 100 μl of D-PBS in the apical chamber and 100 μl of the basolateral media were taken for plaque assays. Plaque forming units (pfu) were plotted to the DPI. Value represents the mean +/− standard deviations. ( B D ) Transepithelial electrical resistance measurement. The TEER of mock- and SARS-CoV-2-infected HAE-ALI culture were measured using an epithelial Volt-Ohm Meter (Millipore) at the indicated dpi. The TEER values were normalized to the TEER measured on the day of infection, which is set as 1.0. Values represent the mean of relative TEER +/− standard deviations. **** P

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Infection, Incubation

    SARS-CoV-2 infection of primary human bronchial airway epithelium (HAE) is persistent. HAE-ALI B4-20 and HAE-ALI B9-20 cultures were infected with SARS-CoV-2 at an MOI of 2 from the apical side. At the indicated days post-infection (dpi), the apical surface was washed with 100 μl of D-PBS to collect the released virus. Plaque forming units (pfu) were determined (y-axis) and plotted to the dpi. Value represents the mean +/− standard deviations.

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: SARS-CoV-2 infection of primary human bronchial airway epithelium (HAE) is persistent. HAE-ALI B4-20 and HAE-ALI B9-20 cultures were infected with SARS-CoV-2 at an MOI of 2 from the apical side. At the indicated days post-infection (dpi), the apical surface was washed with 100 μl of D-PBS to collect the released virus. Plaque forming units (pfu) were determined (y-axis) and plotted to the dpi. Value represents the mean +/− standard deviations.

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Infection

    SARS-CoV-2 does not infect HAE-ALI from the basolateral side. (A) Virus release kinetics. Both apical washes and basolateral media were collected from SARS-CoV-2 infected HAE-ALI B4-20 every day and quantified for virus titers using plaque assays. Plaque forming units (pfu) were plotted to the dpi. Value represents the mean +/− standard deviations. (B) Transepithelial electrical resistance (TEER) measurement. The TEER of infected HAE-ALI B4-20 cultures were measured using an epithelial Volt-Ohm Meter (Millipore) at the indicated dpi, and were normalized to the TEER measured on the first day, which is set as 1.0. Values represent the mean of the relative TEER +/− standard deviations. n.s. indicates statistically no significance. (C D) Immunofluorescence analysis. Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures at 23 dpi were co-stained with anti-NP and anti-ZO-1 antibodies ( C ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( D ). Confocal images were taken at a magnification of x 40. Nuclei were stained with DAPI (blue)

    Journal: bioRxiv

    Article Title: Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium

    doi: 10.1101/2020.08.27.271130

    Figure Lengend Snippet: SARS-CoV-2 does not infect HAE-ALI from the basolateral side. (A) Virus release kinetics. Both apical washes and basolateral media were collected from SARS-CoV-2 infected HAE-ALI B4-20 every day and quantified for virus titers using plaque assays. Plaque forming units (pfu) were plotted to the dpi. Value represents the mean +/− standard deviations. (B) Transepithelial electrical resistance (TEER) measurement. The TEER of infected HAE-ALI B4-20 cultures were measured using an epithelial Volt-Ohm Meter (Millipore) at the indicated dpi, and were normalized to the TEER measured on the first day, which is set as 1.0. Values represent the mean of the relative TEER +/− standard deviations. n.s. indicates statistically no significance. (C D) Immunofluorescence analysis. Mock- and SARS-CoV-2-infected HAE-ALI B4-20 cultures at 23 dpi were co-stained with anti-NP and anti-ZO-1 antibodies ( C ), or co-stained with anti-NP and anti-β-tubulin IV antibodies ( D ). Confocal images were taken at a magnification of x 40. Nuclei were stained with DAPI (blue)

    Article Snippet: Antibodies usedPrimary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (Clone 001, #40143-R001, SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6, #T7941, MilliporeSigma, St Louis, MO) at 1:100, mouse anti-ZO-1 (Clone 1/ZO-1, #610966, BD Bioscience, San Jose, CA) at 1:100, rabbit anti-Ki67 (Clone SP6, ab1666, Abcam, Cambridge, MA7) at 1:50.

    Techniques: Infection, Immunofluorescence, Staining

    ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of SARS-CoV-2 S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: ACE2 over-expression influences neutralizing activity by different classes of anti-spike mAbs. a , Surface rendering of a composite model of SARS-CoV-2 S bound to S309 (purple), S2E12 (magenta) and S2X333 (orange) 5 , 27 , 28 . The three SARS-CoV-2 S protomers are colored light blue, gold and pink whereas N-linked glycans are rendered dark blue. b-c , SARS-CoV-2 neutralization with S309, S2E12 and S2X33 on (b) Vero E6 or (c) Vero E6-TMPRSS2 cells. Cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at MOI 0.01 in the presence of the respective mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and quantified. d , Purified, fluorescently-labeled SARS-CoV-2 spike or RBD protein binding to the indicated cell lines was quantified by flow cytometry. “A”: ACE2, “T”: TMPRSS2 e , Cellular ACE2 and TMPRSS2 transcripts were quantified by RT-qPCR. f-g , A panel of 7 cell lines were infected with SARS-CoV-2-Nluc f , or VSV-SARS-CoV-2 pseudovirus (g) in the presence of S309, S2E12 or S2X333. Luciferase signal was quantified 24h post infection.

    Article Snippet: 24 h post infection, cells were fixed with 4% paraformaldehyde for 30 min, followed by two PBS (pH 7.4) washes and permeabilization with 0.25% Triton X-100 in PBS for 30 min. After blocking in 5% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological, cat. 40143-R001) at a 1:2000 dilution for 1h.

    Techniques: Over Expression, Activity Assay, Neutralization, Infection, Purification, Labeling, Protein Binding, Flow Cytometry, Quantitative RT-PCR, Luciferase

    SARS-CoV-2 live virus neutralization. HEK293T cells stably expressing ACE2, SIGLEC1, DC-SIGN or L-SIGN were infected with SARS-CoV-2 at MOI 0.02 in the presence of the indicated mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and positive cells were quantified.

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: SARS-CoV-2 live virus neutralization. HEK293T cells stably expressing ACE2, SIGLEC1, DC-SIGN or L-SIGN were infected with SARS-CoV-2 at MOI 0.02 in the presence of the indicated mAbs. Cells were fixed 24h post infection, viral nucleocapsid protein was immunostained and positive cells were quantified.

    Article Snippet: 24 h post infection, cells were fixed with 4% paraformaldehyde for 30 min, followed by two PBS (pH 7.4) washes and permeabilization with 0.25% Triton X-100 in PBS for 30 min. After blocking in 5% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological, cat. 40143-R001) at a 1:2000 dilution for 1h.

    Techniques: Neutralization, Stable Transfection, Expressing, Infection

    RBM mAbs trigger the fusogenic rearrangmement of the S protein and promote membrane fusion. a, MAbs or soluble ACE2 were incubated for 1 hour with native-like soluble prefusion S trimer of SARS-CoV-2 to track by negative stain EM imaging the fusogenic rearrangement of soluble S trimers visible as rosettes. b , Cell-cell fusion of CHO cells expressing SARS-CoV-2 S (CHO-S) on the plasma membrane in the absence (upper panel) or presence of 5 μg/ml of S2E12 mAb (lower panel) as detected by immuno-fluorescence. Nuclei stained with Hoechst dye; cytoplasm stained with CellTracker Green. ( c ), CHO-S cell-cell fusion mediated by different spike-specific mAbs quantified as described in Methods. d , Structures of 11 Fab-RBD complexes related to mAbs used in (c) (RBD orientation is fixed) and of ACE2-RBD as determined by a combination of X-ray crystallography and cryo-EM analysis (PDBs, Extended Data Table 1 ). Shown in parentheses the RBD antigenic site as defined according to Piccoli et al. 3 e , Inhibition of S2E12-induced cell-cell fusion performed as in (c) by a fixed amount (15 μg/ml) of indicated mAbs. f , Trans-fusion of S-positive CHO cells with S-negative fluorescently-labelled CHO cells. Staining as in (b).

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: RBM mAbs trigger the fusogenic rearrangmement of the S protein and promote membrane fusion. a, MAbs or soluble ACE2 were incubated for 1 hour with native-like soluble prefusion S trimer of SARS-CoV-2 to track by negative stain EM imaging the fusogenic rearrangement of soluble S trimers visible as rosettes. b , Cell-cell fusion of CHO cells expressing SARS-CoV-2 S (CHO-S) on the plasma membrane in the absence (upper panel) or presence of 5 μg/ml of S2E12 mAb (lower panel) as detected by immuno-fluorescence. Nuclei stained with Hoechst dye; cytoplasm stained with CellTracker Green. ( c ), CHO-S cell-cell fusion mediated by different spike-specific mAbs quantified as described in Methods. d , Structures of 11 Fab-RBD complexes related to mAbs used in (c) (RBD orientation is fixed) and of ACE2-RBD as determined by a combination of X-ray crystallography and cryo-EM analysis (PDBs, Extended Data Table 1 ). Shown in parentheses the RBD antigenic site as defined according to Piccoli et al. 3 e , Inhibition of S2E12-induced cell-cell fusion performed as in (c) by a fixed amount (15 μg/ml) of indicated mAbs. f , Trans-fusion of S-positive CHO cells with S-negative fluorescently-labelled CHO cells. Staining as in (b).

    Article Snippet: 24 h post infection, cells were fixed with 4% paraformaldehyde for 30 min, followed by two PBS (pH 7.4) washes and permeabilization with 0.25% Triton X-100 in PBS for 30 min. After blocking in 5% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological, cat. 40143-R001) at a 1:2000 dilution for 1h.

    Techniques: Incubation, Staining, Imaging, Expressing, Fluorescence, Cryo-EM Sample Prep, Inhibition

    S309 or a cocktail of S309 and S2E12 provide robust in vivo protection against SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount of mAb(s) 48 hours before intra-nasal challenge with SARS-CoV-2. ( a-b ) Quantification of viral RNA in the lungs 4 days post-infection. ( c-d ) Quantification of replicating virus in lung homogenates harvested 4 days post infection using a TCID50 assay. ( e-f ) Histopathological score of the lung tissue was assessed 4 days post infection. ( g-h ) Efficacy plots based on the correlation between the level of serum antibody measured at the time of infection and the level of SARS-CoV2 (viral RNA) measured in lungs on day 4 after infection. The dotted lines represents EC50 and EC90 for viral reduction (EC90 of S309 alone vs S309+S2E12: 9 vs 11 μg/ml, respectively).

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: S309 or a cocktail of S309 and S2E12 provide robust in vivo protection against SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount of mAb(s) 48 hours before intra-nasal challenge with SARS-CoV-2. ( a-b ) Quantification of viral RNA in the lungs 4 days post-infection. ( c-d ) Quantification of replicating virus in lung homogenates harvested 4 days post infection using a TCID50 assay. ( e-f ) Histopathological score of the lung tissue was assessed 4 days post infection. ( g-h ) Efficacy plots based on the correlation between the level of serum antibody measured at the time of infection and the level of SARS-CoV2 (viral RNA) measured in lungs on day 4 after infection. The dotted lines represents EC50 and EC90 for viral reduction (EC90 of S309 alone vs S309+S2E12: 9 vs 11 μg/ml, respectively).

    Article Snippet: 24 h post infection, cells were fixed with 4% paraformaldehyde for 30 min, followed by two PBS (pH 7.4) washes and permeabilization with 0.25% Triton X-100 in PBS for 30 min. After blocking in 5% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological, cat. 40143-R001) at a 1:2000 dilution for 1h.

    Techniques: In Vivo, Injection, Infection, TCID50 Assay

    SIGLEC1, DC-SIGN and L-SIGN modulate neutralizing activity by different classes of antibodies. a-d , Neutralization of infection by authentic SARS-CoV-2 pre-incubated with indicated mAbs of HEK293T cell lines stably overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2. Infection was measured by immunostaining at 24 hours for the SARS-CoV-2 nucleoprotein. e , Summary of the mechanisms of action of different classes of spike-specific mAbs based on this and previous studies. *, mAb-mediated inhibition of fusion between CHO-spike cells and ACE2+ Vero-E6 cells; **, based on mAb-dependent activation of human FcγRs performed with a bioluminescent reporter assay as in 27 . æ , S2X58 binds to open RDB due to a confomational clash with neighboring NTD

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: SIGLEC1, DC-SIGN and L-SIGN modulate neutralizing activity by different classes of antibodies. a-d , Neutralization of infection by authentic SARS-CoV-2 pre-incubated with indicated mAbs of HEK293T cell lines stably overexpressing DC-SIGN, L-SIGN, SIGLEC1 or ACE2. Infection was measured by immunostaining at 24 hours for the SARS-CoV-2 nucleoprotein. e , Summary of the mechanisms of action of different classes of spike-specific mAbs based on this and previous studies. *, mAb-mediated inhibition of fusion between CHO-spike cells and ACE2+ Vero-E6 cells; **, based on mAb-dependent activation of human FcγRs performed with a bioluminescent reporter assay as in 27 . æ , S2X58 binds to open RDB due to a confomational clash with neighboring NTD

    Article Snippet: 24 h post infection, cells were fixed with 4% paraformaldehyde for 30 min, followed by two PBS (pH 7.4) washes and permeabilization with 0.25% Triton X-100 in PBS for 30 min. After blocking in 5% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological, cat. 40143-R001) at a 1:2000 dilution for 1h.

    Techniques: Activity Assay, Neutralization, Infection, Incubation, Stable Transfection, Immunostaining, Inhibition, Activation Assay, Reporter Assay

    Expression of auxiliary receptors in infected tissues and their role in mediating trans-infection in vitro a, Distribution and expression of ACE2, DC-SIGN, L-SIGN, and SIGLEC1 in the human lung cell atlas. b, Major cell types with detectable SARS-CoV-2 genome in bronchoalverolar lavage fluid and sputum of severe COVID-19 patients. Left panel shows a t-SNE embedding of single-cell gene expression profiles coloured by cell type and sized by viral load (logCPM); right panel, distribution plots by annotated cell type denote the cumulative fraction of cells (y-axis) with detected viral RNA per cell up to the corresponding logCPM value (x-axis). c, Left panel shows a heatmap matrix of counts for cells with detected transcripts for receptor gene(s) on x-axis by SARS-CoV-2 + cell type on y-axis (total n=3,085 cells from 8 subjects in Ren et al. 20 ); right panel, correlation of receptor transcript counts with SARS-CoV-2 RNA counts in macrophages and in secretory cells. Correlation is based on counts (before log transformation), from Ren et al. 22 . d, Trans-infection: HeLa cells transduced with DC-SIGN, L-SIGN or SIGLEC1 were incubated with VSV-SARS-CoV-2, extensively washed and co-cultured with Vero-E6-TMPRSS2 susceptible target cells. Shown is RLU in the presence or absence of target cells. e, Trans-infection performed as in (d). VSV-SARS-CoV-2 viral adsorption was performed in the presence or absence of an anti-SIGLEC1 blocking antibody.

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: Expression of auxiliary receptors in infected tissues and their role in mediating trans-infection in vitro a, Distribution and expression of ACE2, DC-SIGN, L-SIGN, and SIGLEC1 in the human lung cell atlas. b, Major cell types with detectable SARS-CoV-2 genome in bronchoalverolar lavage fluid and sputum of severe COVID-19 patients. Left panel shows a t-SNE embedding of single-cell gene expression profiles coloured by cell type and sized by viral load (logCPM); right panel, distribution plots by annotated cell type denote the cumulative fraction of cells (y-axis) with detected viral RNA per cell up to the corresponding logCPM value (x-axis). c, Left panel shows a heatmap matrix of counts for cells with detected transcripts for receptor gene(s) on x-axis by SARS-CoV-2 + cell type on y-axis (total n=3,085 cells from 8 subjects in Ren et al. 20 ); right panel, correlation of receptor transcript counts with SARS-CoV-2 RNA counts in macrophages and in secretory cells. Correlation is based on counts (before log transformation), from Ren et al. 22 . d, Trans-infection: HeLa cells transduced with DC-SIGN, L-SIGN or SIGLEC1 were incubated with VSV-SARS-CoV-2, extensively washed and co-cultured with Vero-E6-TMPRSS2 susceptible target cells. Shown is RLU in the presence or absence of target cells. e, Trans-infection performed as in (d). VSV-SARS-CoV-2 viral adsorption was performed in the presence or absence of an anti-SIGLEC1 blocking antibody.

    Article Snippet: 24 h post infection, cells were fixed with 4% paraformaldehyde for 30 min, followed by two PBS (pH 7.4) washes and permeabilization with 0.25% Triton X-100 in PBS for 30 min. After blocking in 5% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological, cat. 40143-R001) at a 1:2000 dilution for 1h.

    Techniques: Expressing, Infection, In Vitro, Transformation Assay, Transduction, Incubation, Cell Culture, Adsorption, Blocking Assay

    Role of host effector function in SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount (mg/kg) of hamster IgG2a S309 either wt or Fc silenced (S309-N297A). a , Quantification of viral RNA in the lung 4 days post infection. b, Quantification of replicating virus in the lung 4 days post infection. c, Histopathological score in the lung 4 days post infection. Control animals (white symbols) were injected with 4 mg/kg unrelated control isotype mAb. *, **, ***, **** p

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: Role of host effector function in SARS-CoV-2 challenge. Syrian hamsters were injected with the indicated amount (mg/kg) of hamster IgG2a S309 either wt or Fc silenced (S309-N297A). a , Quantification of viral RNA in the lung 4 days post infection. b, Quantification of replicating virus in the lung 4 days post infection. c, Histopathological score in the lung 4 days post infection. Control animals (white symbols) were injected with 4 mg/kg unrelated control isotype mAb. *, **, ***, **** p

    Article Snippet: 24 h post infection, cells were fixed with 4% paraformaldehyde for 30 min, followed by two PBS (pH 7.4) washes and permeabilization with 0.25% Triton X-100 in PBS for 30 min. After blocking in 5% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological, cat. 40143-R001) at a 1:2000 dilution for 1h.

    Techniques: Injection, Infection

    HeLa cells expressing DC-SIGN are refractory to SARS-CoV-2 infection. 293T or HeLa cells stably expressing DC-SIGN were infected with SARS-CoV-2-Nluc at MOI0.04 in the presence of the indicated antibodies. Infection was analyzed by quantification of luminescent signal at 24 h post infection.

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: HeLa cells expressing DC-SIGN are refractory to SARS-CoV-2 infection. 293T or HeLa cells stably expressing DC-SIGN were infected with SARS-CoV-2-Nluc at MOI0.04 in the presence of the indicated antibodies. Infection was analyzed by quantification of luminescent signal at 24 h post infection.

    Article Snippet: 24 h post infection, cells were fixed with 4% paraformaldehyde for 30 min, followed by two PBS (pH 7.4) washes and permeabilization with 0.25% Triton X-100 in PBS for 30 min. After blocking in 5% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological, cat. 40143-R001) at a 1:2000 dilution for 1h.

    Techniques: Expressing, Infection, Stable Transfection

    Characterization of DC-SIGN, L-SIGN and SIGLEC-1 as SARS-CoV-2 attachment factors. a-b, Binding of antibodies targeting DC/-L-SIGN, DC-SIGN, SIGLEC1 or ACE2 on HEK293T stably over-expressing the respective attachment receptors was analyzed by flow cytometry (a) and immunofluorescence analysis (b). c, HEK293T cells over-expressing the respective attachment receptors were infected with VSV-SARS-COV-2 wildtype spike (grey bars) or spike bearing mutations of the B.1.1.7 variant (red bars). Luminescence was analyzed one day post infection.

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: Characterization of DC-SIGN, L-SIGN and SIGLEC-1 as SARS-CoV-2 attachment factors. a-b, Binding of antibodies targeting DC/-L-SIGN, DC-SIGN, SIGLEC1 or ACE2 on HEK293T stably over-expressing the respective attachment receptors was analyzed by flow cytometry (a) and immunofluorescence analysis (b). c, HEK293T cells over-expressing the respective attachment receptors were infected with VSV-SARS-COV-2 wildtype spike (grey bars) or spike bearing mutations of the B.1.1.7 variant (red bars). Luminescence was analyzed one day post infection.

    Article Snippet: 24 h post infection, cells were fixed with 4% paraformaldehyde for 30 min, followed by two PBS (pH 7.4) washes and permeabilization with 0.25% Triton X-100 in PBS for 30 min. After blocking in 5% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological, cat. 40143-R001) at a 1:2000 dilution for 1h.

    Techniques: Binding Assay, Stable Transfection, Expressing, Flow Cytometry, Immunofluorescence, Infection, Variant Assay

    Data collection and processing of the S/S2X58 complex cryoEM datasets. a,b , Representative electron micrograph and 2D class averages of SARS-CoV-2 S in complex with the S2X58 Fab embedded in vitreous ice. Scale bar: 400 Å. c , Gold-standard Fourier shell correlation curves for the S2X58-bound SARS-CoV-2 S trimer in one RBD closed (black line) and three RBDs open conformations (gray line). The 0.143 cutoff is indicated by a horizontal dashed line. d, Local resolution maps calculated using cryoSPARC for the SARS-CoV-2 S/S2X58 Fab complex structure with one RBD closed and three RBDs open shown in two orthogonal orientations.

    Journal: bioRxiv

    Article Title: Membrane lectins enhance SARS-CoV-2 infection and influence the neutralizing activity of different classes of antibodies

    doi: 10.1101/2021.04.03.438258

    Figure Lengend Snippet: Data collection and processing of the S/S2X58 complex cryoEM datasets. a,b , Representative electron micrograph and 2D class averages of SARS-CoV-2 S in complex with the S2X58 Fab embedded in vitreous ice. Scale bar: 400 Å. c , Gold-standard Fourier shell correlation curves for the S2X58-bound SARS-CoV-2 S trimer in one RBD closed (black line) and three RBDs open conformations (gray line). The 0.143 cutoff is indicated by a horizontal dashed line. d, Local resolution maps calculated using cryoSPARC for the SARS-CoV-2 S/S2X58 Fab complex structure with one RBD closed and three RBDs open shown in two orthogonal orientations.

    Article Snippet: 24 h post infection, cells were fixed with 4% paraformaldehyde for 30 min, followed by two PBS (pH 7.4) washes and permeabilization with 0.25% Triton X-100 in PBS for 30 min. After blocking in 5% milk powder/PBS for 30 min, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological, cat. 40143-R001) at a 1:2000 dilution for 1h.

    Techniques: