sars cov 2 spike protein  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike RBD R408I His Recombinant Protein
    Description:
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike RBD R408I His Recombinant Protein YP 009724390 1 Arg319 Phe541 R408I was expressed with a polyhistidine tag at the C terminus
    Catalog Number:
    40592-V08H10
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus spike Protein 2019-nCoV, cov spike Protein 2019-nCoV, ncov RBD Protein 2019-nCoV, ncov s1 Protein 2019-nCoV, ncov s2 Protein 2019-nCoV, ncov spike Protein 2019-nCoV, NCP-CoV RBD Protein 2019-nCoV, NCP-CoV s1 Protein 2019-nCoV, NCP-CoV s2 Protein 2019-nCoV, NCP-CoV Spike Protein 2019-nCoV, novel coronavirus RBD Protein 2019-nCoV, novel coronavirus s1 Protein 2019-nCoV, novel coronavirus s2 Protein 2019-nCoV, novel coronavirus spike Protein 2019-nCoV, RBD Protein 2019-nCoV, S1 Protein 2019-nCoV, S2 Protein 2019-nCoV, Spike RBD Protein 2019-nCoV
    Host:
    HEK293 Cells
    Buy from Supplier


    Structured Review

    Sino Biological sars cov 2 spike protein
    ACE2 receptor competition assay development. (A) Competition ELISA schematic displaying immobilized anti-His pAb (red) capturing His6×-tagged <t>SARS-CoV-2</t> spike protein (rainbow). Premixed ACE2-IgHu (green, blue) at a constant concentration with a dilution series of competitors (green, red) is added, and anti-human HRP (green) determines the amount of ACE2-IgHu remaining in the presence of competitors through a colorimetric readout. (B) Four constant concentrations of ACE2-IgHu were tested with various concentrations of the ACE2-IgMu competitor to establish an optimal ACE2-IgHu concentration which displays a full blocking curve (red, 0.10 μg/ml) from the competitor dilution series while retaining a wide range in signal. (C) Pseudovirus neutralization curves for a control antibody (non-SARS-CoV-2) in red and for ACE2-IgHu in blue.
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike RBD R408I His Recombinant Protein YP 009724390 1 Arg319 Phe541 R408I was expressed with a polyhistidine tag at the C terminus
    https://www.bioz.com/result/sars cov 2 spike protein/product/Sino Biological
    Average 95 stars, based on 1 article reviews
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    sars cov 2 spike protein - by Bioz Stars, 2021-04
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    Images

    1) Product Images from "SARS-CoV-2 Assays To Detect Functional Antibody Responses That Block ACE2 Recognition in Vaccinated Animals and Infected Patients"

    Article Title: SARS-CoV-2 Assays To Detect Functional Antibody Responses That Block ACE2 Recognition in Vaccinated Animals and Infected Patients

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.01533-20

    ACE2 receptor competition assay development. (A) Competition ELISA schematic displaying immobilized anti-His pAb (red) capturing His6×-tagged SARS-CoV-2 spike protein (rainbow). Premixed ACE2-IgHu (green, blue) at a constant concentration with a dilution series of competitors (green, red) is added, and anti-human HRP (green) determines the amount of ACE2-IgHu remaining in the presence of competitors through a colorimetric readout. (B) Four constant concentrations of ACE2-IgHu were tested with various concentrations of the ACE2-IgMu competitor to establish an optimal ACE2-IgHu concentration which displays a full blocking curve (red, 0.10 μg/ml) from the competitor dilution series while retaining a wide range in signal. (C) Pseudovirus neutralization curves for a control antibody (non-SARS-CoV-2) in red and for ACE2-IgHu in blue.
    Figure Legend Snippet: ACE2 receptor competition assay development. (A) Competition ELISA schematic displaying immobilized anti-His pAb (red) capturing His6×-tagged SARS-CoV-2 spike protein (rainbow). Premixed ACE2-IgHu (green, blue) at a constant concentration with a dilution series of competitors (green, red) is added, and anti-human HRP (green) determines the amount of ACE2-IgHu remaining in the presence of competitors through a colorimetric readout. (B) Four constant concentrations of ACE2-IgHu were tested with various concentrations of the ACE2-IgMu competitor to establish an optimal ACE2-IgHu concentration which displays a full blocking curve (red, 0.10 μg/ml) from the competitor dilution series while retaining a wide range in signal. (C) Pseudovirus neutralization curves for a control antibody (non-SARS-CoV-2) in red and for ACE2-IgHu in blue.

    Techniques Used: Competitive Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Blocking Assay, Neutralization

    Animal IgG and serological competition. (A) IgG and serological competition schematic. Anti-His pAb captures SARS-CoV-2 spike protein. Immunized sera or IgG from small animals are used as competitors to block ACE2-IgHu receptor binding when premixed. ACE2-IgHu remaining is determined from an anti-human-HRP colorimetric readout. (B) IgGs present in a vaccinated BALB/c mouse block ACE2-IgHu binding with greater effect when the full-length SARS-CoV-2 S1-S2 spike protein is immobilized versus the S1 subunit by itself. (C) Area under the concentration-time curve (AUC) schematic displaying the larger area for uninhibited ACE2 binding versus the area from curves showing competition with ACE2. (D) AUC of IgGs purified from immunized rabbit sera (IgGr low dose, blue; IgGr high dose, red) versus naive IgGr or day 0 IgGr. (E) AUC of sera from immunized rabbits (low dose rabbit sera, blue; high dose rabbit sera, red) versus naive rabbit sera or day 0 rabbit sera. (F) AUC of sera from immunized guinea pigs at week 2 (dark blue) and individual animals (blue), naive sera (gray), and pooled day 0 sera from all animals (black). The pooled immunized curve displayed a comparable AUC to the average AUC from all individual immunized animals.
    Figure Legend Snippet: Animal IgG and serological competition. (A) IgG and serological competition schematic. Anti-His pAb captures SARS-CoV-2 spike protein. Immunized sera or IgG from small animals are used as competitors to block ACE2-IgHu receptor binding when premixed. ACE2-IgHu remaining is determined from an anti-human-HRP colorimetric readout. (B) IgGs present in a vaccinated BALB/c mouse block ACE2-IgHu binding with greater effect when the full-length SARS-CoV-2 S1-S2 spike protein is immobilized versus the S1 subunit by itself. (C) Area under the concentration-time curve (AUC) schematic displaying the larger area for uninhibited ACE2 binding versus the area from curves showing competition with ACE2. (D) AUC of IgGs purified from immunized rabbit sera (IgGr low dose, blue; IgGr high dose, red) versus naive IgGr or day 0 IgGr. (E) AUC of sera from immunized rabbits (low dose rabbit sera, blue; high dose rabbit sera, red) versus naive rabbit sera or day 0 rabbit sera. (F) AUC of sera from immunized guinea pigs at week 2 (dark blue) and individual animals (blue), naive sera (gray), and pooled day 0 sera from all animals (black). The pooled immunized curve displayed a comparable AUC to the average AUC from all individual immunized animals.

    Techniques Used: Blocking Assay, Binding Assay, Concentration Assay, Purification

    ACE2 receptor competition assay development. (A) Overview of SPR experiment depicting SARS-CoV-2 RBD capture by streptavidin-biotin interaction, sera injected as analyte, and ACE2 injected as second analyte. (B) Sensorgram for ACE2 blocking SPR assay with ACE2-IgHu injected as sample (ACE2 sample ) as indicated and ACE-IgHu injected as receptor (ACE2 receptor ) as indicated. Sample responses were referenced to blank injections. Each curve corresponds to a 3-fold dilution of ACE2 sample starting at 1,500 nM as indicated on the right, and the ACE2 receptor was injected at a constant concentration of 100 nM to all curves. (C) Response in RUs measured at the end of sample (ACE2 sample ) injection (blue) and receptor (ACE2 receptor ) injection (red) at each concentration of sample. (D) ACE2 inhibition curve derived from RUs at each concentration.
    Figure Legend Snippet: ACE2 receptor competition assay development. (A) Overview of SPR experiment depicting SARS-CoV-2 RBD capture by streptavidin-biotin interaction, sera injected as analyte, and ACE2 injected as second analyte. (B) Sensorgram for ACE2 blocking SPR assay with ACE2-IgHu injected as sample (ACE2 sample ) as indicated and ACE-IgHu injected as receptor (ACE2 receptor ) as indicated. Sample responses were referenced to blank injections. Each curve corresponds to a 3-fold dilution of ACE2 sample starting at 1,500 nM as indicated on the right, and the ACE2 receptor was injected at a constant concentration of 100 nM to all curves. (C) Response in RUs measured at the end of sample (ACE2 sample ) injection (blue) and receptor (ACE2 receptor ) injection (red) at each concentration of sample. (D) ACE2 inhibition curve derived from RUs at each concentration.

    Techniques Used: Competitive Binding Assay, SPR Assay, Injection, Blocking Assay, Concentration Assay, Inhibition, Derivative Assay

    Primate serological competition. (A) Competition ELISA schematic displaying immobilized His6×-tagged SARS-CoV-2 spike protein (rainbow). Preblocking of the spike protein with primate sera (blue) at various concentrations was added followed by ACE2-IgMu (green, blue) at a constant concentration. Anti-mouse HRP (green) determines the amount of ACE2-IgMu remaining in the presence of competitors through a colorimetric readout. (B) Affinity of ACE2-IgMu for immobilized SARS-CoV-2 S1+S2 full-length spike protein assessed by ELISA. Optimal concentration of ACE2-IgMu for competition assays (red arrow, 0.4 μg/ml) requires high signal without excess receptor present. (C) Optimal ACE2-IgMu concentration which displays a full blocking curve (0.40 μg/ml) from the competitor dilution series (ACE2-IgHu) while retaining a wide range in signal. (D) NHP sera pooled from five vaccinated animals were used as competitors in the primate competition assay. The AUC from vaccinated NHP sera (blue) versus day 0 NHP sera (black). (E) Human sera from nine SARS-CoV-2-positive COVID-19 patients were tested in the primate competition assay and compared with 16 naive human sera collected prepandemic. The AUC of the COVID-19 patient serum (purple) is significantly decreased compared to the prepandemic human serum (gray). The median is shown as a solid black line, and quartiles are shown as dashed black lines. (F) Human sera were analyzed by a pseudovirus neutralization assay. The samples and the coloring are the same as in (E). Statistics include a two-tailed t test with P values indicated.
    Figure Legend Snippet: Primate serological competition. (A) Competition ELISA schematic displaying immobilized His6×-tagged SARS-CoV-2 spike protein (rainbow). Preblocking of the spike protein with primate sera (blue) at various concentrations was added followed by ACE2-IgMu (green, blue) at a constant concentration. Anti-mouse HRP (green) determines the amount of ACE2-IgMu remaining in the presence of competitors through a colorimetric readout. (B) Affinity of ACE2-IgMu for immobilized SARS-CoV-2 S1+S2 full-length spike protein assessed by ELISA. Optimal concentration of ACE2-IgMu for competition assays (red arrow, 0.4 μg/ml) requires high signal without excess receptor present. (C) Optimal ACE2-IgMu concentration which displays a full blocking curve (0.40 μg/ml) from the competitor dilution series (ACE2-IgHu) while retaining a wide range in signal. (D) NHP sera pooled from five vaccinated animals were used as competitors in the primate competition assay. The AUC from vaccinated NHP sera (blue) versus day 0 NHP sera (black). (E) Human sera from nine SARS-CoV-2-positive COVID-19 patients were tested in the primate competition assay and compared with 16 naive human sera collected prepandemic. The AUC of the COVID-19 patient serum (purple) is significantly decreased compared to the prepandemic human serum (gray). The median is shown as a solid black line, and quartiles are shown as dashed black lines. (F) Human sera were analyzed by a pseudovirus neutralization assay. The samples and the coloring are the same as in (E). Statistics include a two-tailed t test with P values indicated.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay, Blocking Assay, Competitive Binding Assay, Neutralization, Two Tailed Test

    ACE2 receptor expression and affinity. (A) Overview of soluble ACE2 receptor design (ACE2-IgHu). (B) Affinity of SARS CoV-2 receptor binding domain for immobilized ACE2-IgHu assessed by SPR (27 nM) curves are concentrations of RBD X, Y, and Z. (C) Affinity of ACE2-IgHu for immobilized SARS CoV-2 full-length spike protein assessed by ELISA. Optimal concentration of ACE2-IgHu for competition assays (∼EC 90 , red arrow) requires high signal without excess receptor present.
    Figure Legend Snippet: ACE2 receptor expression and affinity. (A) Overview of soluble ACE2 receptor design (ACE2-IgHu). (B) Affinity of SARS CoV-2 receptor binding domain for immobilized ACE2-IgHu assessed by SPR (27 nM) curves are concentrations of RBD X, Y, and Z. (C) Affinity of ACE2-IgHu for immobilized SARS CoV-2 full-length spike protein assessed by ELISA. Optimal concentration of ACE2-IgHu for competition assays (∼EC 90 , red arrow) requires high signal without excess receptor present.

    Techniques Used: Expressing, Binding Assay, SPR Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

    ACE2 receptor blocking correlates with pseudovirus neuralization. A symbol represents each of the individual datapoints where we had a paired AUC blocking and pseudovirus ID 50 values. The human samples are in triangles, the mice in circles, individual Guinea pigs in squares, Guinea pig pools in diamonds, and rabbit pools in hexagons. SARS-CoV-2 spike-experienced samples are shown in color. Naïve samples and healthy donors are shown in gray. Least-squares fit line is shown with P value and R squared from Prism.
    Figure Legend Snippet: ACE2 receptor blocking correlates with pseudovirus neuralization. A symbol represents each of the individual datapoints where we had a paired AUC blocking and pseudovirus ID 50 values. The human samples are in triangles, the mice in circles, individual Guinea pigs in squares, Guinea pig pools in diamonds, and rabbit pools in hexagons. SARS-CoV-2 spike-experienced samples are shown in color. Naïve samples and healthy donors are shown in gray. Least-squares fit line is shown with P value and R squared from Prism.

    Techniques Used: Blocking Assay, Mouse Assay

    2) Product Images from "A Single Dose of Self-Transcribing and Replicating RNA Based SARS-CoV-2 Vaccine Produces Protective Adaptive Immunity In Mice"

    Article Title: A Single Dose of Self-Transcribing and Replicating RNA Based SARS-CoV-2 Vaccine Produces Protective Adaptive Immunity In Mice

    Journal: bioRxiv

    doi: 10.1101/2020.09.03.280446

    Cellular immune responses following vaccination with LUNAR-COV19 and conventional mRNA. C57BL/6 mice ( n =5 per group) were immunized with 0.2 μg, 2.0 μg, or 10.0 μg of LUNAR-COV19 or conventional mRNA via IM, sacrificed at day 7 post-vaccination and spleens analyzed for cellular T cell responses by flow-cytometry and ELISPOT. A-B ) CD8 + and C ) CD4 + T effector cells were assessed in vaccinated animals using surface staining for T cell markers and flow-cytometry. D-E ) IFNγ + CD8 + T cells and F ) Ratio of IFNγ + / IL4 + CD4 + T cells in spleens of immunized mice were assessed following ex vivo stimulation with PMA/IO and intracellular staining. G-I ) SARS-CoV-2 S protein-specific responses to pooled S protein peptides were assessed using IFNγ ELISPOT assays following vaccination with mRNA ( H ) or LUNAR-COV19 ( I ). Percentage of CD8+ cells, CD4+ cells, IFNγ and IL4 producing T cells were compared between groups using two-tailed Mann-Whitney U test with * denoting 0.05
    Figure Legend Snippet: Cellular immune responses following vaccination with LUNAR-COV19 and conventional mRNA. C57BL/6 mice ( n =5 per group) were immunized with 0.2 μg, 2.0 μg, or 10.0 μg of LUNAR-COV19 or conventional mRNA via IM, sacrificed at day 7 post-vaccination and spleens analyzed for cellular T cell responses by flow-cytometry and ELISPOT. A-B ) CD8 + and C ) CD4 + T effector cells were assessed in vaccinated animals using surface staining for T cell markers and flow-cytometry. D-E ) IFNγ + CD8 + T cells and F ) Ratio of IFNγ + / IL4 + CD4 + T cells in spleens of immunized mice were assessed following ex vivo stimulation with PMA/IO and intracellular staining. G-I ) SARS-CoV-2 S protein-specific responses to pooled S protein peptides were assessed using IFNγ ELISPOT assays following vaccination with mRNA ( H ) or LUNAR-COV19 ( I ). Percentage of CD8+ cells, CD4+ cells, IFNγ and IL4 producing T cells were compared between groups using two-tailed Mann-Whitney U test with * denoting 0.05

    Techniques Used: Mouse Assay, Flow Cytometry, Enzyme-linked Immunospot, Staining, Ex Vivo, Two Tailed Test, MANN-WHITNEY

    LUNAR-COV19 elicits Th1 biased immune responses. SARS-CoV-2 spike-specific IgG subclasses and the ratio of IgG2a/c/IgG1 at 30 days post-vaccination with LUNAR-COV19 and conventional mRNA in A ) BALB/c and B ) C57BL/6J mice. Th2 cytokine and Th1/Th2 skew in CD4 T cells at day 7 post-vaccination in C57BL/6J mice measured by ICS as C ) percentage of IL4+ CD4 T cells and D ) ratio of IFNγ + /IL4 + CD4 + T cells. Antibody titers and T cell data were compared between groups using a two-tailed Mann-Whitney U test with * denoting 0.05
    Figure Legend Snippet: LUNAR-COV19 elicits Th1 biased immune responses. SARS-CoV-2 spike-specific IgG subclasses and the ratio of IgG2a/c/IgG1 at 30 days post-vaccination with LUNAR-COV19 and conventional mRNA in A ) BALB/c and B ) C57BL/6J mice. Th2 cytokine and Th1/Th2 skew in CD4 T cells at day 7 post-vaccination in C57BL/6J mice measured by ICS as C ) percentage of IL4+ CD4 T cells and D ) ratio of IFNγ + /IL4 + CD4 + T cells. Antibody titers and T cell data were compared between groups using a two-tailed Mann-Whitney U test with * denoting 0.05

    Techniques Used: Mouse Assay, Two Tailed Test, MANN-WHITNEY

    LUNAR-COV19 elicits a higher quality humoral response than conventional mRNA platform. A) BALB/c and C57BL/6J mice were immunized via IM with 0.2 μg, 2μg, or 10 μg of LUNAR-COV19 or conventional mRNA ( n =5/group). Blood sampling was conducted at baseline, and days 10, 19, 30, 40, 50 and 60 post-vaccination for BALB/c and days 10, 20 and 30 for C57BL/6J. B - C ) IgM and D - E ) IgG against the SARS-CoV-2 S protein over time, assessed using insect cell-derived whole S protein in a Luminex immuno-assay (measured as MFI). IgG endpoint titers to mammalian-derived whole S protein, S1, S2 and RBD proteins to mammalian-derived whole S protein at day 30 post-vaccination were assessed in F) BALB/c and G) C57BL/6J. H ) Avidity of SARS-CoV-2 S protein-specific IgG at day 30 post-immunization was measured using 8M urea washes. I ) Neutralizing antibody (PRNT 50 titers) at day 30 post-vaccination against a clinically isolated live SARS-CoV-2 virus measured in both BALB/c and C57BL/6J. Gray dashed lines depict the serum dilution range (i.e. from 1:20 to 1:320) tested by PRNT. J ) PRNT50 and K ) PRNT70 of SARS-CoV-2 neutralization at day 30 and day 60 post-vaccination in BALB/c and convalescent sera from COVID-19 patients. L ) Correlation analysis of Spike-specific IgG endpoint titers against SARS-CoV-2 neutralization (PRNT50). Antibody data were compared between groups using a two-tailed Mann-Whitney U test with * denoting 0.05
    Figure Legend Snippet: LUNAR-COV19 elicits a higher quality humoral response than conventional mRNA platform. A) BALB/c and C57BL/6J mice were immunized via IM with 0.2 μg, 2μg, or 10 μg of LUNAR-COV19 or conventional mRNA ( n =5/group). Blood sampling was conducted at baseline, and days 10, 19, 30, 40, 50 and 60 post-vaccination for BALB/c and days 10, 20 and 30 for C57BL/6J. B - C ) IgM and D - E ) IgG against the SARS-CoV-2 S protein over time, assessed using insect cell-derived whole S protein in a Luminex immuno-assay (measured as MFI). IgG endpoint titers to mammalian-derived whole S protein, S1, S2 and RBD proteins to mammalian-derived whole S protein at day 30 post-vaccination were assessed in F) BALB/c and G) C57BL/6J. H ) Avidity of SARS-CoV-2 S protein-specific IgG at day 30 post-immunization was measured using 8M urea washes. I ) Neutralizing antibody (PRNT 50 titers) at day 30 post-vaccination against a clinically isolated live SARS-CoV-2 virus measured in both BALB/c and C57BL/6J. Gray dashed lines depict the serum dilution range (i.e. from 1:20 to 1:320) tested by PRNT. J ) PRNT50 and K ) PRNT70 of SARS-CoV-2 neutralization at day 30 and day 60 post-vaccination in BALB/c and convalescent sera from COVID-19 patients. L ) Correlation analysis of Spike-specific IgG endpoint titers against SARS-CoV-2 neutralization (PRNT50). Antibody data were compared between groups using a two-tailed Mann-Whitney U test with * denoting 0.05

    Techniques Used: Mouse Assay, Sampling, Derivative Assay, Luminex, Immuno Assay, Plaque Reduction Neutralization Test, Isolation, Neutralization, Two Tailed Test, MANN-WHITNEY

    Design and Expression of a SARS-COV-2 vaccine with conventional mRNA and self-transcribing and replicating RNA (STARR ® ) platforms. A) Schematic diagram of the SARS-CoV-2 self-replicating STARR RNA (LUNAR ® -COV19) and conventional mRNA vaccine constructs. The STARR construct encodes for the four non-structural proteins, ns1-ns4, from Venezuelan equine encephalitis virus (VEEV) and the unmodified full-length pre-fusion spike (S) protein of SARS-CoV-2. The mRNA construct also codes for the same SARS-CoV-2 full length spike S protein. B) Physical characteristics and RNA trapping efficiency of the LNP encapsulating conventional mRNA and LUNAR-COV19 vaccines. C) Western blot detection of SARS-CoV-2 S protein following transfection of HEK293 cells with LUNAR-COV19 and conventional mRNA. D) In vivo comparison of protein expression following IM administration of LNP containing luciferase-expressing STARR RNA or conventional mRNA. Balb/c mice ( n =3/group) were injected IM with 0.2 μg, 2.0 μg and 10.0 μg of STARR RNA or conventional mRNA formulated with the same lipid nanoparticle. Luciferase expression was measured by in vivo bioluminescence on days 1, 3 and 7 post-IM administration. S domain 1 = S1, S domain 2 = S2, transmembrane domain = TM, cytoplasmic domain = CP; aka = also known as.
    Figure Legend Snippet: Design and Expression of a SARS-COV-2 vaccine with conventional mRNA and self-transcribing and replicating RNA (STARR ® ) platforms. A) Schematic diagram of the SARS-CoV-2 self-replicating STARR RNA (LUNAR ® -COV19) and conventional mRNA vaccine constructs. The STARR construct encodes for the four non-structural proteins, ns1-ns4, from Venezuelan equine encephalitis virus (VEEV) and the unmodified full-length pre-fusion spike (S) protein of SARS-CoV-2. The mRNA construct also codes for the same SARS-CoV-2 full length spike S protein. B) Physical characteristics and RNA trapping efficiency of the LNP encapsulating conventional mRNA and LUNAR-COV19 vaccines. C) Western blot detection of SARS-CoV-2 S protein following transfection of HEK293 cells with LUNAR-COV19 and conventional mRNA. D) In vivo comparison of protein expression following IM administration of LNP containing luciferase-expressing STARR RNA or conventional mRNA. Balb/c mice ( n =3/group) were injected IM with 0.2 μg, 2.0 μg and 10.0 μg of STARR RNA or conventional mRNA formulated with the same lipid nanoparticle. Luciferase expression was measured by in vivo bioluminescence on days 1, 3 and 7 post-IM administration. S domain 1 = S1, S domain 2 = S2, transmembrane domain = TM, cytoplasmic domain = CP; aka = also known as.

    Techniques Used: Expressing, Construct, Western Blot, Transfection, In Vivo, Luciferase, Mouse Assay, Injection

    Correlation analysis of live SARS-CoV-2 neutralization against binding IgG and IgG subclasses in BALB/c and C57BL/6J mouse strains. A ) Spearman correlation analysis of SARS-CoV-2 neutralization (PRNT50) against total IgG specific to several SARS-CoV2 antigens, including S, S1, and RBD recombinant proteins. B ) Spearman correlation analysis of SARS-CoV-2 neutralization (PRNT50) against SARS-CoV2 S-specific IgG subclasses (IgG1 and IgG2a or IgG2c).
    Figure Legend Snippet: Correlation analysis of live SARS-CoV-2 neutralization against binding IgG and IgG subclasses in BALB/c and C57BL/6J mouse strains. A ) Spearman correlation analysis of SARS-CoV-2 neutralization (PRNT50) against total IgG specific to several SARS-CoV2 antigens, including S, S1, and RBD recombinant proteins. B ) Spearman correlation analysis of SARS-CoV-2 neutralization (PRNT50) against SARS-CoV2 S-specific IgG subclasses (IgG1 and IgG2a or IgG2c).

    Techniques Used: Neutralization, Binding Assay, Recombinant

    Single dose of LUNAR-COV19 protects hACE2 mice against a lethal challenge of SARS-CoV-2 virus. A ) hACE2 transgenic mice were immunized with a single dose of either PBS or 2 μg or 10 μg of LUNAR-COV19 ( n =5 per group), then challenged with live SARS-CoV-2 at 30 days post-vaccination, and assessed for either survival (with daily weights and clinical scores) or sacrificed at day 5 post-challenge and measured lung and brain tissue viral loads. Study design schematic diagram was created with BioRender.com B ) Live SARS-CoV-2 neutralizing antibody titers (PRNT70) measured at 28 days post-vaccination. C ) Weight, D ) clinical score and E ) survival was estimated following challenge with a lethal dose (5×10^5 TCID 50 ) of live SARS-CoV-2 virus. F ) Viral RNA and G ) infectious virus in the lungs and brain of challenged mice were measured with qRT-PCR or plaque assay, respectively. PRNT 70 and viral titers (RNA and plaque titers) were compared across groups using the non-parametric Mann-Whitney U test. Weights and clinical scores at different timepoints were compared between PBS and 10ug LUNAR-CoV19 immunized mice using multiple t -tests. P -values are denoted by * for 0.05
    Figure Legend Snippet: Single dose of LUNAR-COV19 protects hACE2 mice against a lethal challenge of SARS-CoV-2 virus. A ) hACE2 transgenic mice were immunized with a single dose of either PBS or 2 μg or 10 μg of LUNAR-COV19 ( n =5 per group), then challenged with live SARS-CoV-2 at 30 days post-vaccination, and assessed for either survival (with daily weights and clinical scores) or sacrificed at day 5 post-challenge and measured lung and brain tissue viral loads. Study design schematic diagram was created with BioRender.com B ) Live SARS-CoV-2 neutralizing antibody titers (PRNT70) measured at 28 days post-vaccination. C ) Weight, D ) clinical score and E ) survival was estimated following challenge with a lethal dose (5×10^5 TCID 50 ) of live SARS-CoV-2 virus. F ) Viral RNA and G ) infectious virus in the lungs and brain of challenged mice were measured with qRT-PCR or plaque assay, respectively. PRNT 70 and viral titers (RNA and plaque titers) were compared across groups using the non-parametric Mann-Whitney U test. Weights and clinical scores at different timepoints were compared between PBS and 10ug LUNAR-CoV19 immunized mice using multiple t -tests. P -values are denoted by * for 0.05

    Techniques Used: Mouse Assay, Transgenic Assay, Quantitative RT-PCR, Plaque Assay, Plaque Reduction Neutralization Test, MANN-WHITNEY

    Clinical Scores, mouse weights and transcriptomic analysis of immune genes following vaccination with LUNAR-COV19 or conventional mRNA SARS-CoV-2 vaccine candidates. A) C57BL/6 mice ( n =5/group) were immunized with either PBS, mRNA or LUNAR-COV19 (doses 0.2 μg, 2 μg or 10 μg), weight and clinical scores assessed every day, bled at day 1 post-immunization, sacrificed at 7 days post-vaccination and lymph nodes harvested. Gene expression of inflammatory genes and immune genes were measured in whole blood (at day 1) and lymph nodes (at day 7), respectively. B ) Expression of IFN and inflammatory response genes in whole blood presented as heatmap of z scores. C) Lymph node weights at 7 days post-vaccination. Principal component analysis (PCA) of immune gene expression following vaccination with conventional mRNA or LUNAR-COV19 at doses D) 0.2 μg, E) 2 μg and F) 10 μg. Volcano plots of fold change of LUNAR-COV19 versus conventional mRNA (x-axis) and Log 10 P -value of LUNAR-COV19 versus conventional mRNA (y-axis) for doses G) 0.2 μg, H) 2 μg and I) 10 μg. Study design schematic diagram created with BioRender.com. Weights of lymph nodes were compared between groups using a two-tailed Mann-Whitney U test with * denoting 0.05
    Figure Legend Snippet: Clinical Scores, mouse weights and transcriptomic analysis of immune genes following vaccination with LUNAR-COV19 or conventional mRNA SARS-CoV-2 vaccine candidates. A) C57BL/6 mice ( n =5/group) were immunized with either PBS, mRNA or LUNAR-COV19 (doses 0.2 μg, 2 μg or 10 μg), weight and clinical scores assessed every day, bled at day 1 post-immunization, sacrificed at 7 days post-vaccination and lymph nodes harvested. Gene expression of inflammatory genes and immune genes were measured in whole blood (at day 1) and lymph nodes (at day 7), respectively. B ) Expression of IFN and inflammatory response genes in whole blood presented as heatmap of z scores. C) Lymph node weights at 7 days post-vaccination. Principal component analysis (PCA) of immune gene expression following vaccination with conventional mRNA or LUNAR-COV19 at doses D) 0.2 μg, E) 2 μg and F) 10 μg. Volcano plots of fold change of LUNAR-COV19 versus conventional mRNA (x-axis) and Log 10 P -value of LUNAR-COV19 versus conventional mRNA (y-axis) for doses G) 0.2 μg, H) 2 μg and I) 10 μg. Study design schematic diagram created with BioRender.com. Weights of lymph nodes were compared between groups using a two-tailed Mann-Whitney U test with * denoting 0.05

    Techniques Used: Mouse Assay, Expressing, Two Tailed Test, MANN-WHITNEY

    3) Product Images from "SARS-CoV-2 proteins and anti-COVID-19 drugs induce lytic reactivation of an oncogenic virus"

    Article Title: SARS-CoV-2 proteins and anti-COVID-19 drugs induce lytic reactivation of an oncogenic virus

    Journal: bioRxiv

    doi: 10.1101/2020.10.02.324228

    Ectopic expression of SARS-CoV-2 proteins induces KSHV lytic gene expression from latently infected cells. ( A ) The iSLK.219 cells were transfected with vector control or vectors encoding SARS-CoV-2 spike protein (S), nucleocapsid protein (N) and KSHV RTA (as a positive control) with or without low dose of doxycycline (Dox, 0.1 μg/mL) induction for 72 h. The expression of RFP (representing viral lytic reactivation) and GFP (representing infected cells) were detected using fluorescence microscopy. ( B ) BCP-1 cells were transfected as above with or without low dose of 12- O -tetradecanoyl-phorbol-13-acetate (TPA, 1.0 ng/mL) induction for 72 h, then the transcripts of representative lytic genes were quantified by using qRT-PCR. Error bars represent S.D. for 3 independent experiments, ** = p
    Figure Legend Snippet: Ectopic expression of SARS-CoV-2 proteins induces KSHV lytic gene expression from latently infected cells. ( A ) The iSLK.219 cells were transfected with vector control or vectors encoding SARS-CoV-2 spike protein (S), nucleocapsid protein (N) and KSHV RTA (as a positive control) with or without low dose of doxycycline (Dox, 0.1 μg/mL) induction for 72 h. The expression of RFP (representing viral lytic reactivation) and GFP (representing infected cells) were detected using fluorescence microscopy. ( B ) BCP-1 cells were transfected as above with or without low dose of 12- O -tetradecanoyl-phorbol-13-acetate (TPA, 1.0 ng/mL) induction for 72 h, then the transcripts of representative lytic genes were quantified by using qRT-PCR. Error bars represent S.D. for 3 independent experiments, ** = p

    Techniques Used: Expressing, Infection, Transfection, Plasmid Preparation, Positive Control, Fluorescence, Microscopy, Quantitative RT-PCR

    4) Product Images from "SARS-CoV-2 Entry Receptor ACE2 Is Expressed on Very Small CD45− Precursors of Hematopoietic and Endothelial Cells and in Response to Virus Spike Protein Activates the Nlrp3 Inflammasome"

    Article Title: SARS-CoV-2 Entry Receptor ACE2 Is Expressed on Very Small CD45− Precursors of Hematopoietic and Endothelial Cells and in Response to Virus Spike Protein Activates the Nlrp3 Inflammasome

    Journal: Stem Cell Reviews and Reports

    doi: 10.1007/s12015-020-10010-z

    Human CD34 + HSC activate Nlrp3 inflammasome in response to SARS-Cov-2 spike protein. Effect of NCP-CoV (2019-nCoV) Spike protein (S1 + S2 ECD, His tag) and Angiotensin 1–7 on the expression of inflammasome related genes. Real-time PCR quantitation of FACS sorted human UCB derived HSCs in comparison to mononuclear cells; * P
    Figure Legend Snippet: Human CD34 + HSC activate Nlrp3 inflammasome in response to SARS-Cov-2 spike protein. Effect of NCP-CoV (2019-nCoV) Spike protein (S1 + S2 ECD, His tag) and Angiotensin 1–7 on the expression of inflammasome related genes. Real-time PCR quantitation of FACS sorted human UCB derived HSCs in comparison to mononuclear cells; * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitation Assay, FACS, Derivative Assay

    Human CD34 + VSELs activate Nlrp3 inflammasome in response to SARS-Cov-2 spike protein. Effect of NCP-CoV (2019-nCoV) Spike protein (S1 + S2 ECD, His tag) on the expression of inflammasome related genes. Real-time PCR quantitation of FACS sorted human UCB derived VSELs in comparison to mononuclear cells; *P
    Figure Legend Snippet: Human CD34 + VSELs activate Nlrp3 inflammasome in response to SARS-Cov-2 spike protein. Effect of NCP-CoV (2019-nCoV) Spike protein (S1 + S2 ECD, His tag) on the expression of inflammasome related genes. Real-time PCR quantitation of FACS sorted human UCB derived VSELs in comparison to mononuclear cells; *P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Quantitation Assay, FACS, Derivative Assay

    Expression of SARS-CoV-2 entry receptors and selected RAAS genes in purified human VSELs and HSCs. Expression of ACE2, AGTR1, AGTR2, MAS1, CMA1, RENIN and TMPRSS2 mRNAs in UCB MNC and UCB purified VSELs and HSCs as measured by RT-PCR. To evaluate relative expression, comparative ΔCT method was employed. Results are combined from three independent purification of UCB VSELs and HSCs. Results are presented as mean ± SEM. * P
    Figure Legend Snippet: Expression of SARS-CoV-2 entry receptors and selected RAAS genes in purified human VSELs and HSCs. Expression of ACE2, AGTR1, AGTR2, MAS1, CMA1, RENIN and TMPRSS2 mRNAs in UCB MNC and UCB purified VSELs and HSCs as measured by RT-PCR. To evaluate relative expression, comparative ΔCT method was employed. Results are combined from three independent purification of UCB VSELs and HSCs. Results are presented as mean ± SEM. * P

    Techniques Used: Expressing, Purification, Reverse Transcription Polymerase Chain Reaction

    5) Product Images from "The structure-activity relationship of the interactions of SARS-CoV-2 spike glycoproteins with glucuronomannan and sulfated galactofucan from Saccharina japonica"

    Article Title: The structure-activity relationship of the interactions of SARS-CoV-2 spike glycoproteins with glucuronomannan and sulfated galactofucan from Saccharina japonica

    Journal: International Journal of Biological Macromolecules

    doi: 10.1016/j.ijbiomac.2020.09.184

    Bar graphs of normalized ACE2 binding preference to surface SARS-CoV-2 spike protein by competing with different polysaccharides. Concentration of ACE2 was 500 nM and concentrations of different polysaccharides were 1000 nM. All bar graphs with standard deviations were based on triplicate experiments.
    Figure Legend Snippet: Bar graphs of normalized ACE2 binding preference to surface SARS-CoV-2 spike protein by competing with different polysaccharides. Concentration of ACE2 was 500 nM and concentrations of different polysaccharides were 1000 nM. All bar graphs with standard deviations were based on triplicate experiments.

    Techniques Used: Binding Assay, Concentration Assay

    6) Product Images from "Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies"

    Article Title: Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies

    Journal: bioRxiv

    doi: 10.1101/2020.12.13.422550

    The predicted linear B-cell epitopes in the Spike protein of SARS-CoV-2. a , The number of linear B-cell epitopes shared among the distinct methods and literature mining. The pink, green and light blue represent epitopes with antigenicity scores > 0.9, 0.4 and 0.9, and
    Figure Legend Snippet: The predicted linear B-cell epitopes in the Spike protein of SARS-CoV-2. a , The number of linear B-cell epitopes shared among the distinct methods and literature mining. The pink, green and light blue represent epitopes with antigenicity scores > 0.9, 0.4 and 0.9, and

    Techniques Used:

    Measurements of the selected Linear B cell epitope binding to antibody and neutralization efficiency of selected epitopes against SARS-CoV-2. a-d, The binding affinity assessed by ELISA between linear B-cell epitopes and serum antibodies from immunized horse with S1-based vaccines (a), immunized mouse with RBD-based vaccines (b), immunized monkey with RBD-based vaccines (c), and a patient recovering from COVID-19 (d). e, The binding affinity assessed by ELISA between the linear B-cell epitopes and serum antibodies from immunized mice with corresponding epitopes of ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. f, Neutralization assay against SARS-CoV-2 pseudovirus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of EC 50 . g, Neutralization assay against SARS-CoV-2 live virus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of NT 50 .
    Figure Legend Snippet: Measurements of the selected Linear B cell epitope binding to antibody and neutralization efficiency of selected epitopes against SARS-CoV-2. a-d, The binding affinity assessed by ELISA between linear B-cell epitopes and serum antibodies from immunized horse with S1-based vaccines (a), immunized mouse with RBD-based vaccines (b), immunized monkey with RBD-based vaccines (c), and a patient recovering from COVID-19 (d). e, The binding affinity assessed by ELISA between the linear B-cell epitopes and serum antibodies from immunized mice with corresponding epitopes of ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. f, Neutralization assay against SARS-CoV-2 pseudovirus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of EC 50 . g, Neutralization assay against SARS-CoV-2 live virus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of NT 50 .

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

    The characteristics of the 18 selected linear B cell epitopes. a , The sequences of 18 selected linear B-cell epitopes. The bold is the mutated site in less than ten of 118,694 virus strains; The red is the predicted discontinuous residues. The bars on the right side are the Wilcoxon test p value for the comparisons of IgG or IgA antibody enrichment scores associated with each linear B-cell epitope between COVID-19 patients and negative controls. b , The digesting enzymes profile of the epitope sequence. Red indicated not digest, blue indicated digest. c-d , The localization of the 18 selected epitopes mapped on SARS-CoV-2 S (PDB: 6VSB) protein (c) and ACE-RBD complex (d). e-f , The localizations of B cell discontinuous epitopes on SARS-CoV-2 S (PDB: 6VSB) protein (e) and ACE-RBD complex (f). The spike protein is grey, the RBD region is wheat color, the selected epitopes are green, the mutation sites are red, the human ACE domain is blue, and the discontinuous B-cell epitopes are purple.
    Figure Legend Snippet: The characteristics of the 18 selected linear B cell epitopes. a , The sequences of 18 selected linear B-cell epitopes. The bold is the mutated site in less than ten of 118,694 virus strains; The red is the predicted discontinuous residues. The bars on the right side are the Wilcoxon test p value for the comparisons of IgG or IgA antibody enrichment scores associated with each linear B-cell epitope between COVID-19 patients and negative controls. b , The digesting enzymes profile of the epitope sequence. Red indicated not digest, blue indicated digest. c-d , The localization of the 18 selected epitopes mapped on SARS-CoV-2 S (PDB: 6VSB) protein (c) and ACE-RBD complex (d). e-f , The localizations of B cell discontinuous epitopes on SARS-CoV-2 S (PDB: 6VSB) protein (e) and ACE-RBD complex (f). The spike protein is grey, the RBD region is wheat color, the selected epitopes are green, the mutation sites are red, the human ACE domain is blue, and the discontinuous B-cell epitopes are purple.

    Techniques Used: Sequencing, Mutagenesis

    7) Product Images from "Presence of antibodies against SARS-CoV-2 spike protein in bovine whey IgG enriched fraction"

    Article Title: Presence of antibodies against SARS-CoV-2 spike protein in bovine whey IgG enriched fraction

    Journal: International Dairy Journal

    doi: 10.1016/j.idairyj.2021.105002

    Bovine IgG enriched fraction containing IgG against SARS-CoV-2 assessed by direct enzyme-linked immunosorbent assays (ELISA) using a partial-length of recombinant SARS-CoV-2 S (aa 177–512, 288–512, 348–578, 387–516 and 408–664), full-recombinant SARS-CoV-2 N (aa 1-419) and partial-length of recombinant SARS-CoV-2 N (aa 1–120, 111–220, 1–220 and 210–419) ( Fig.1 ) as coating antigens. Two different lots of bovine IgG enriched fraction prepared in 2019 and 2018 were used (2a and 2b, respectively): Image 1 , 0.003 μg mL -1 ; Image 2 , 0.03 μg mL -1 ; Image 3 , 0.3 μg mL -1 ; Image 4 , 3 μg mL -1 ; Image 5 , 30 μg mL -1 . A picture of a representative ELISA result is shown in 2c.
    Figure Legend Snippet: Bovine IgG enriched fraction containing IgG against SARS-CoV-2 assessed by direct enzyme-linked immunosorbent assays (ELISA) using a partial-length of recombinant SARS-CoV-2 S (aa 177–512, 288–512, 348–578, 387–516 and 408–664), full-recombinant SARS-CoV-2 N (aa 1-419) and partial-length of recombinant SARS-CoV-2 N (aa 1–120, 111–220, 1–220 and 210–419) ( Fig.1 ) as coating antigens. Two different lots of bovine IgG enriched fraction prepared in 2019 and 2018 were used (2a and 2b, respectively): Image 1 , 0.003 μg mL -1 ; Image 2 , 0.03 μg mL -1 ; Image 3 , 0.3 μg mL -1 ; Image 4 , 3 μg mL -1 ; Image 5 , 30 μg mL -1 . A picture of a representative ELISA result is shown in 2c.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Recombinant

    Competitive inhibition ELISA of the bovine IgG enriched fraction (IgG 0.3 μg mL -1 ), incubated with one of three peptides of S protein of SARS-CoV-2 ( Image 7 , aa 382–401; Image 8 , aa 427–446; Image 9 , aa 502–520) at concentrations 0.001, 0.01, 0,1, 1, and 10 μg mL -1 , the remaining free IgG against the S protein of SARS-CoV-2 was assayed by direct ELISA, using plates coated with the peptide corresponding to aa 288–512 of S protein of SARS-CoV-2. Two lots of bovine IgG enriched fraction prepared in 2019 ( Image 10 ) and 2018 ( Image 11 ) were tested.
    Figure Legend Snippet: Competitive inhibition ELISA of the bovine IgG enriched fraction (IgG 0.3 μg mL -1 ), incubated with one of three peptides of S protein of SARS-CoV-2 ( Image 7 , aa 382–401; Image 8 , aa 427–446; Image 9 , aa 502–520) at concentrations 0.001, 0.01, 0,1, 1, and 10 μg mL -1 , the remaining free IgG against the S protein of SARS-CoV-2 was assayed by direct ELISA, using plates coated with the peptide corresponding to aa 288–512 of S protein of SARS-CoV-2. Two lots of bovine IgG enriched fraction prepared in 2019 ( Image 10 ) and 2018 ( Image 11 ) were tested.

    Techniques Used: Inhibition, Enzyme-linked Immunosorbent Assay, Incubation, Direct ELISA

    Determination of epitopes by direct ELISA using nine peptides of SARS-CoV-2 S protein, corresponding to aa 382–401, 397–416, 427–446, 442–461, 457–476, 472–491, 487–506 and 502–520, with plates coated with a recombinant protein covering the RBD of SARS-CoV-2 S protein. Two lots of bovine IgG enriched fraction prepared in 2019 and 2018 (3a and 3b, respectively), were tested: Image 6 , 0.3 μg mL -1 ; Image 2 , 3 μg mL -1 ; Image 3 , 30 μg mL -1 .
    Figure Legend Snippet: Determination of epitopes by direct ELISA using nine peptides of SARS-CoV-2 S protein, corresponding to aa 382–401, 397–416, 427–446, 442–461, 457–476, 472–491, 487–506 and 502–520, with plates coated with a recombinant protein covering the RBD of SARS-CoV-2 S protein. Two lots of bovine IgG enriched fraction prepared in 2019 and 2018 (3a and 3b, respectively), were tested: Image 6 , 0.3 μg mL -1 ; Image 2 , 3 μg mL -1 ; Image 3 , 30 μg mL -1 .

    Techniques Used: Direct ELISA, Recombinant

    Overall topology of (a) SARS-CoV-2 spike protein (S) and five regions of recombinant SARS-CoV-2 S and (b) SARS-CoV-2 nucleocapsid protein (N) and regions of recombinant SARS-CoV-2 N. NTD, N-terminal domain; CTD, C-terminal domain; RBD, receptor binding domain; RDM, receptor binding motif; SD1, subdomain 1; SD2, subdomain 2; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane region; IC, intracellular domain.
    Figure Legend Snippet: Overall topology of (a) SARS-CoV-2 spike protein (S) and five regions of recombinant SARS-CoV-2 S and (b) SARS-CoV-2 nucleocapsid protein (N) and regions of recombinant SARS-CoV-2 N. NTD, N-terminal domain; CTD, C-terminal domain; RBD, receptor binding domain; RDM, receptor binding motif; SD1, subdomain 1; SD2, subdomain 2; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane region; IC, intracellular domain.

    Techniques Used: Recombinant, Binding Assay

    8) Product Images from "AR12 (OSU-03012) suppresses GRP78 expression and inhibits SARS-CoV-2 replication"

    Article Title: AR12 (OSU-03012) suppresses GRP78 expression and inhibits SARS-CoV-2 replication

    Journal: Biochemical Pharmacology

    doi: 10.1016/j.bcp.2020.114227

    HCT116 ATG16L1 T300 cells express greater amounts of GRP78 and ACE2 compared to cells expressing ATG16L1 A300 A. Vero, ADOR and HCT116 ATG16L1 T300 cells were plated and 24h after plating, fixed and stained. Fixed cells were subjected to in-cell western blotting as described in the Methods to determine the expression of the indicated proteins (n = 3 independent studies +/-SD). B. ADOR and Vero cells were treated with vehicle control or with AR12 (2 μM) and 6h later infected with SARS-CoV-2 (0.1 MOI, 0.01 MOI). Cells were fixed in place 24h after infection. Fixed cells were subjected to in-cell western blotting as described in the Methods to determine the expression of the indicated proteins (n = 3 independent studies +/-SD). * p
    Figure Legend Snippet: HCT116 ATG16L1 T300 cells express greater amounts of GRP78 and ACE2 compared to cells expressing ATG16L1 A300 A. Vero, ADOR and HCT116 ATG16L1 T300 cells were plated and 24h after plating, fixed and stained. Fixed cells were subjected to in-cell western blotting as described in the Methods to determine the expression of the indicated proteins (n = 3 independent studies +/-SD). B. ADOR and Vero cells were treated with vehicle control or with AR12 (2 μM) and 6h later infected with SARS-CoV-2 (0.1 MOI, 0.01 MOI). Cells were fixed in place 24h after infection. Fixed cells were subjected to in-cell western blotting as described in the Methods to determine the expression of the indicated proteins (n = 3 independent studies +/-SD). * p

    Techniques Used: Expressing, Staining, In-Cell ELISA, Infection

    AR12 suppresses the synthesis of the SARS-CoV-2 spike protein. A. Vero cells were treated with vehicle control or AR12 and 6h later infected with SARS-CoV-2 as described in the Methods and Figure. Cells were fixed after 24h or 48h and the expression of the spike protein determined (n = 2 independent experiments each with 4 independent assessments +/-SD) *p
    Figure Legend Snippet: AR12 suppresses the synthesis of the SARS-CoV-2 spike protein. A. Vero cells were treated with vehicle control or AR12 and 6h later infected with SARS-CoV-2 as described in the Methods and Figure. Cells were fixed after 24h or 48h and the expression of the spike protein determined (n = 2 independent experiments each with 4 independent assessments +/-SD) *p

    Techniques Used: Infection, Expressing

    AR-12 inactivates eIF2α regardless of viral infection. A. Vero cells were treated with vehicle control or AR12 (2 μM) and 6h later infected with SARS-CoV-2 as described in the Methods. Cells were fixed after 24h and the expression of eIF2α, phospho-eIF2α serine 51 and ERK2 determined (n = 2 independent experiments each with 4 independent assessments +/-SD) # p
    Figure Legend Snippet: AR-12 inactivates eIF2α regardless of viral infection. A. Vero cells were treated with vehicle control or AR12 (2 μM) and 6h later infected with SARS-CoV-2 as described in the Methods. Cells were fixed after 24h and the expression of eIF2α, phospho-eIF2α serine 51 and ERK2 determined (n = 2 independent experiments each with 4 independent assessments +/-SD) # p

    Techniques Used: Infection, Expressing

    Sorafenib and pazopanib modestly reduce spike protein and GRP78 expression in SARS-CoV-2 infected cells. Vero cells were infected with the indicated multiplicities of infection with SARS-CoV-2. One hour after infection, cells were treated with vehicle control, sorafenib tosylate (1.0 μM) or pazopanib (0.5 μM). Twenty-four h after infection, cells were fixed in place. Fixed cells were subjected to in-cell western blotting as described in the Methods to determine the expression of the indicated proteins (n = 3 independent studies +/-SD).
    Figure Legend Snippet: Sorafenib and pazopanib modestly reduce spike protein and GRP78 expression in SARS-CoV-2 infected cells. Vero cells were infected with the indicated multiplicities of infection with SARS-CoV-2. One hour after infection, cells were treated with vehicle control, sorafenib tosylate (1.0 μM) or pazopanib (0.5 μM). Twenty-four h after infection, cells were fixed in place. Fixed cells were subjected to in-cell western blotting as described in the Methods to determine the expression of the indicated proteins (n = 3 independent studies +/-SD).

    Techniques Used: Expressing, Infection, In-Cell ELISA

    AR12 reduces the expression of SARS-CoV-2 spike protein, GRP78, HSP90 and HSP70 via autophagy. A. HCT116 cells (ATG16L1 A300 and T300) were transfected with a plasmid to express LC3-GFP-RFP. Twenty-four h after transfection, cells were treated with vehicle control or with AR12 (2 μM). Cells were imaged 4h and 8h after drug exposure and the mean number of GFP+ and RFP+ punctae per cell determined from at least 40 cells per condition (n = 3 +/-SD) # p
    Figure Legend Snippet: AR12 reduces the expression of SARS-CoV-2 spike protein, GRP78, HSP90 and HSP70 via autophagy. A. HCT116 cells (ATG16L1 A300 and T300) were transfected with a plasmid to express LC3-GFP-RFP. Twenty-four h after transfection, cells were treated with vehicle control or with AR12 (2 μM). Cells were imaged 4h and 8h after drug exposure and the mean number of GFP+ and RFP+ punctae per cell determined from at least 40 cells per condition (n = 3 +/-SD) # p

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    The weak infectivity of ADOR cells is associated with reduced TMPRSS2 and TMPRSS11D expression. A. and B. Vero, ADOR and HCT116 cells were infected with SARS-CoV-2 (1.0 MOI, 0.01 MOI) and the media collected 24h, 48h and 96h after infection. The media was diluted 1:10, with repeated 1:10 dilutions using the Reed-Muench method. Vero cells were afterwards infected with the media, and 72h later the media was removed from the cells and TCID50/ml values determined. C. ADOR cells were infected with SARS-CoV-2 and fixed 24h after infection. Staining was performed to determine the localization of virus spike protein, GRP78 and nuclear DNA. A representative image is presented. D. Vero and ADOR were plated and 24h after plating, fixed and stained. Fixed cells were subjected to in-cell western blotting as described in the Methods to determine the expression of the indicated proteins (n = 3 independent studies +/-SD). * p
    Figure Legend Snippet: The weak infectivity of ADOR cells is associated with reduced TMPRSS2 and TMPRSS11D expression. A. and B. Vero, ADOR and HCT116 cells were infected with SARS-CoV-2 (1.0 MOI, 0.01 MOI) and the media collected 24h, 48h and 96h after infection. The media was diluted 1:10, with repeated 1:10 dilutions using the Reed-Muench method. Vero cells were afterwards infected with the media, and 72h later the media was removed from the cells and TCID50/ml values determined. C. ADOR cells were infected with SARS-CoV-2 and fixed 24h after infection. Staining was performed to determine the localization of virus spike protein, GRP78 and nuclear DNA. A representative image is presented. D. Vero and ADOR were plated and 24h after plating, fixed and stained. Fixed cells were subjected to in-cell western blotting as described in the Methods to determine the expression of the indicated proteins (n = 3 independent studies +/-SD). * p

    Techniques Used: Infection, Expressing, Endpoint Dilution Assay, Staining, In-Cell ELISA

    AR12 reduces SARS-CoV-2 spike protein expression in a dose-dependent fashion. HCT116 ATG16L1 T300 cells were transfected with a plasmid to express the SARS-CoV-2 spike protein. Twenty-four h later, cells were treated with vehicle control or with AR12 (100-2,000 nM) for 6h and fixed. Cells were stained to determine the expression of the SARS-CoV-2 spike protein and for ERK2 as a loading control. (n = 3 +/-SD).
    Figure Legend Snippet: AR12 reduces SARS-CoV-2 spike protein expression in a dose-dependent fashion. HCT116 ATG16L1 T300 cells were transfected with a plasmid to express the SARS-CoV-2 spike protein. Twenty-four h later, cells were treated with vehicle control or with AR12 (100-2,000 nM) for 6h and fixed. Cells were stained to determine the expression of the SARS-CoV-2 spike protein and for ERK2 as a loading control. (n = 3 +/-SD).

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Staining

    Representative images of GRP78 and SARS-CoV-2 spike protein expression and co-localization. A. and B. Vero cells were treated with vehicle control or AR12 (1 μM, 2 μM) and 6h later infected with SARS-CoV-2 as described in the Methods. Cells were fixed after 24h and the expression of GRP78 and ERK2 determined (n = 2 independent experiments each with 4 independent assessments +/-SD). Representative 60X images at the 24h timepoint of the colocalization of the virus spike protein (green) and GRP78 (red). DAPI (blue) staining is the nucleus.
    Figure Legend Snippet: Representative images of GRP78 and SARS-CoV-2 spike protein expression and co-localization. A. and B. Vero cells were treated with vehicle control or AR12 (1 μM, 2 μM) and 6h later infected with SARS-CoV-2 as described in the Methods. Cells were fixed after 24h and the expression of GRP78 and ERK2 determined (n = 2 independent experiments each with 4 independent assessments +/-SD). Representative 60X images at the 24h timepoint of the colocalization of the virus spike protein (green) and GRP78 (red). DAPI (blue) staining is the nucleus.

    Techniques Used: Expressing, Infection, Staining

    AR12 suppresses the production of infectious SARS-CoV-2 virions. A. Four independent TCID 50 / ml studies are presented, each study in independent quadruplicate. Vero cells were pre-treated with AR12 and 6h afterwards infected, and 24h later the media was removed from the cells. The media was diluted 1:10, with repeated 1:10 dilutions using the Reed-Muench method. B. Four independent TCID 50 / ml studies are presented, each study in independent quadruplicate. Vero cells were infected then after 6h treated with AR12, and 24h later the media was removed from the cells. The media was diluted 1:10, with repeated 1:10 dilutions using the Reed-Muench method.
    Figure Legend Snippet: AR12 suppresses the production of infectious SARS-CoV-2 virions. A. Four independent TCID 50 / ml studies are presented, each study in independent quadruplicate. Vero cells were pre-treated with AR12 and 6h afterwards infected, and 24h later the media was removed from the cells. The media was diluted 1:10, with repeated 1:10 dilutions using the Reed-Muench method. B. Four independent TCID 50 / ml studies are presented, each study in independent quadruplicate. Vero cells were infected then after 6h treated with AR12, and 24h later the media was removed from the cells. The media was diluted 1:10, with repeated 1:10 dilutions using the Reed-Muench method.

    Techniques Used: Infection, Endpoint Dilution Assay

    AR12 suppresses the expression of GRP78 and reduces the increase in GRP78 expression caused by SARS-CoV-2. A. Vero cells were pre-treated with vehicle control or with AR12 for 6h. Cells were infected with SARS-CoV-2 (10 MOI). Forty minutes after infection, the media was removed, and the cells washed with PBS three times. The cells were fixed in place without permeabilization and cells stained to determine the levels of spike protein on the outer leaflet of the plasma membrane, with DAPI and ERK2 as loading controls. B. Vero cells were pre-treated with vehicle control or with AR12 for 6h. Cells were then infected (10 MOI). The cells were fixed in place with permeabilization 10 min and 40 min after infection and the total expression of ACE2, GRP78, HSP90, HSP70, eIF2α, P-eIF2α S51 and ERK2 (not shown) determined. (n = 2 independent experiments each with 4 independent assessments +/-SD) *p
    Figure Legend Snippet: AR12 suppresses the expression of GRP78 and reduces the increase in GRP78 expression caused by SARS-CoV-2. A. Vero cells were pre-treated with vehicle control or with AR12 for 6h. Cells were infected with SARS-CoV-2 (10 MOI). Forty minutes after infection, the media was removed, and the cells washed with PBS three times. The cells were fixed in place without permeabilization and cells stained to determine the levels of spike protein on the outer leaflet of the plasma membrane, with DAPI and ERK2 as loading controls. B. Vero cells were pre-treated with vehicle control or with AR12 for 6h. Cells were then infected (10 MOI). The cells were fixed in place with permeabilization 10 min and 40 min after infection and the total expression of ACE2, GRP78, HSP90, HSP70, eIF2α, P-eIF2α S51 and ERK2 (not shown) determined. (n = 2 independent experiments each with 4 independent assessments +/-SD) *p

    Techniques Used: Expressing, Infection, Staining

    9) Product Images from "Adopting STING agonist cyclic dinucleotides as a potential adjuvant for SARS-CoV-2 vaccine"

    Article Title: Adopting STING agonist cyclic dinucleotides as a potential adjuvant for SARS-CoV-2 vaccine

    Journal: bioRxiv

    doi: 10.1101/2020.07.24.217570

    Immune analysis of CDG SF as an adjuvant for SARS-CoV-2 vaccine. (a) Immunization scheme of the vaccine group: blank, S, S+Alum and S+CDG SF . (b) IFN-γ ELISPOT test of splenocytes and (c) spleen weight data from immunized mice. ns: no significant difference, *p
    Figure Legend Snippet: Immune analysis of CDG SF as an adjuvant for SARS-CoV-2 vaccine. (a) Immunization scheme of the vaccine group: blank, S, S+Alum and S+CDG SF . (b) IFN-γ ELISPOT test of splenocytes and (c) spleen weight data from immunized mice. ns: no significant difference, *p

    Techniques Used: Enzyme-linked Immunospot, Mouse Assay

    (a) The SARS-CoV-2 vaccine adjuvant based on a modified STING agonist. (b) Chemical structure of CDG SF and dithio CDG. B1/B2: base.
    Figure Legend Snippet: (a) The SARS-CoV-2 vaccine adjuvant based on a modified STING agonist. (b) Chemical structure of CDG SF and dithio CDG. B1/B2: base.

    Techniques Used: Modification

    SARS-CoV-2 specific antibody titers (a) and isotypes (b) evaluation in the antisera of each vaccine group. ns: no significant difference, **p
    Figure Legend Snippet: SARS-CoV-2 specific antibody titers (a) and isotypes (b) evaluation in the antisera of each vaccine group. ns: no significant difference, **p

    Techniques Used:

    10) Product Images from "SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus"

    Article Title: SARS-CoV-2 specific antibody and neutralization assays reveal the wide range of the humoral immune response to virus

    Journal: Communications Biology

    doi: 10.1038/s42003-021-01649-6

    Neutralizing titers for SARS-CoV-2 and SARS-CoV in COVID-19 subject plasma. a Neutralization assay with S-RBD-specific NAb, healthy control plasma, and a COVID-19 patient plasma. Threefold serial dilutions of NAb from 10 μg/ml to 1 ng/ml or the plasma from 1:10 to 1:10,000 were pre-incubated with spike protein pseudovirus and added to 293-ACE2 cells. GFP expression was analyzed by flow cytometry 3 days post infection. b SARS-CoV-2 neutralization titers (NT50) of COVID-19 plasma grouped as an outpatient, hospitalized, ICU or deceased and convalescent plasma donor groups ( n = 113). c NT50 of COVID-19 patient and plasma donor groups subdivided into males and females ( n = 113). d Comparison of NT50 of COVID-19 plasma for SARS-CoV-2 and SARS-CoV neutralization. SARS-CoV-2 or SARS-CoV pseudoviruses were pre-incubated with COVID-19 plasma from all severity groups ( n = 104), 293-ACE2 cells were infected and RFP expression was determined at day 3 using flow cytometry. e Graph of SARS-CoV-2 NT50 values from hospitalized subjects plotted against SARS-CoV ( n = 46). Two-tailed Mann–Whitney U test was used to determine the statistical significances in figures ( b ), ( c ) and ( d ) and two-tailed Spearman’s was used for figure ( e ). Horizontal bars in ( b ), ( c ) and ( d ) indicate mean values.
    Figure Legend Snippet: Neutralizing titers for SARS-CoV-2 and SARS-CoV in COVID-19 subject plasma. a Neutralization assay with S-RBD-specific NAb, healthy control plasma, and a COVID-19 patient plasma. Threefold serial dilutions of NAb from 10 μg/ml to 1 ng/ml or the plasma from 1:10 to 1:10,000 were pre-incubated with spike protein pseudovirus and added to 293-ACE2 cells. GFP expression was analyzed by flow cytometry 3 days post infection. b SARS-CoV-2 neutralization titers (NT50) of COVID-19 plasma grouped as an outpatient, hospitalized, ICU or deceased and convalescent plasma donor groups ( n = 113). c NT50 of COVID-19 patient and plasma donor groups subdivided into males and females ( n = 113). d Comparison of NT50 of COVID-19 plasma for SARS-CoV-2 and SARS-CoV neutralization. SARS-CoV-2 or SARS-CoV pseudoviruses were pre-incubated with COVID-19 plasma from all severity groups ( n = 104), 293-ACE2 cells were infected and RFP expression was determined at day 3 using flow cytometry. e Graph of SARS-CoV-2 NT50 values from hospitalized subjects plotted against SARS-CoV ( n = 46). Two-tailed Mann–Whitney U test was used to determine the statistical significances in figures ( b ), ( c ) and ( d ) and two-tailed Spearman’s was used for figure ( e ). Horizontal bars in ( b ), ( c ) and ( d ) indicate mean values.

    Techniques Used: Neutralization, Incubation, Expressing, Flow Cytometry, Infection, Two Tailed Test, MANN-WHITNEY

    SARS-CoV-2 specific antibody detection assay. a Illustration of antibody detection assay. Biotinylated S-RBD or Nucleocapsid proteins are captured by streptavidin-coated beads, then incubated with plasma samples and stained with PE-conjugated anti-IgG, IgA, IgM, IgG1, IgG2, IgG3, IgG4 antibodies. Fluorescence intensity analyzed by flow cytometry. b Histogram overlays demonstrating the detection of anti-S-RBD human IgG antibody (left) and soluble ACE2-Fc (right) as positive controls for plasma antibody assay. c Representative patient plasma titration. Healthy control plasma at 1:100 dilution was used as a negative control. Serial dilutions were used in the flow cytometry overlay. d Comparison of IgG antibody levels captured by S-RBD, S1 subunit of spike, S1 N terminal domain (NTD), S2 extracellular domain (ECD) and nucleocapsid protein coated beads ( n = 46 biologically independent samples). e Correlation and comparison of bead-based assay S-RBD IgG antibody levels with ELISA-based assay ( n = 44). Two-tailed Mann–Whitney U test was used to determine the statistical significance in ( d ) and two-tailed Spearman’s was used for correlation significance in ( e ). Horizontal bars in ( d ) and ( e ) indicate mean values.
    Figure Legend Snippet: SARS-CoV-2 specific antibody detection assay. a Illustration of antibody detection assay. Biotinylated S-RBD or Nucleocapsid proteins are captured by streptavidin-coated beads, then incubated with plasma samples and stained with PE-conjugated anti-IgG, IgA, IgM, IgG1, IgG2, IgG3, IgG4 antibodies. Fluorescence intensity analyzed by flow cytometry. b Histogram overlays demonstrating the detection of anti-S-RBD human IgG antibody (left) and soluble ACE2-Fc (right) as positive controls for plasma antibody assay. c Representative patient plasma titration. Healthy control plasma at 1:100 dilution was used as a negative control. Serial dilutions were used in the flow cytometry overlay. d Comparison of IgG antibody levels captured by S-RBD, S1 subunit of spike, S1 N terminal domain (NTD), S2 extracellular domain (ECD) and nucleocapsid protein coated beads ( n = 46 biologically independent samples). e Correlation and comparison of bead-based assay S-RBD IgG antibody levels with ELISA-based assay ( n = 44). Two-tailed Mann–Whitney U test was used to determine the statistical significance in ( d ) and two-tailed Spearman’s was used for correlation significance in ( e ). Horizontal bars in ( d ) and ( e ) indicate mean values.

    Techniques Used: Detection Assay, Incubation, Staining, Fluorescence, Flow Cytometry, Titration, Negative Control, Bead-based Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

    SARS-CoV-2 specific antibody detection in COVID-19 and convalescent plasma samples. a Measurement of spike protein and nucleocapsid protein-specific IgG and spike protein-specific IgM and IgA antibodies as described in Fig. 1 . Area under the curve (AUC) values of plasma antibodies were calculated from reciprocal dilution curves in antibody detection assay ( n = 256 for S-RBD IgG and Nucleocapsid IgG, n = 50 for S-RBD IgM, n = 144 for S-RBD IgA). Dotted lines indicate the negative threshold calculated by adding 1 standard deviation to the mean AUC values of healthy controls’ plasma. Horizontal bars show the mean value. Green, blue, salmon, red and yellow dots indicate negative controls, outpatient, hospitalized, ICU/deceased and plasma donor subjects, respectively. b S-RBD-specific IgG subclass AUC levels ( n = 144 for S-RBD IgG1, n = 74 for S-RBD IgG2, S-RBD IgG3 and S-RBD IgG4) c S-RBD IgG AUC values of subject plasma grouped by outpatient, hospitalized, ICU or deceased and plasma donors ( n = 115) d Nucleocapsid protein IgG AUC values of subject plasma grouped by outpatient, hospitalized, ICU or deceased and convalescent plasma donors ( n = 115). e S-RBD IgA AUC values of subject plasma grouped by outpatient, hospitalized, ICU or deceased and plasma donors ( n = 115). f S-RBD IgG AUC values of severity groups and plasma donors subdivided into males and females ( n = 115). Green dots show female subjects while purple squares indicate male subjects. Statistical significances were determined using two-tailed Mann–Whitney U test.
    Figure Legend Snippet: SARS-CoV-2 specific antibody detection in COVID-19 and convalescent plasma samples. a Measurement of spike protein and nucleocapsid protein-specific IgG and spike protein-specific IgM and IgA antibodies as described in Fig. 1 . Area under the curve (AUC) values of plasma antibodies were calculated from reciprocal dilution curves in antibody detection assay ( n = 256 for S-RBD IgG and Nucleocapsid IgG, n = 50 for S-RBD IgM, n = 144 for S-RBD IgA). Dotted lines indicate the negative threshold calculated by adding 1 standard deviation to the mean AUC values of healthy controls’ plasma. Horizontal bars show the mean value. Green, blue, salmon, red and yellow dots indicate negative controls, outpatient, hospitalized, ICU/deceased and plasma donor subjects, respectively. b S-RBD-specific IgG subclass AUC levels ( n = 144 for S-RBD IgG1, n = 74 for S-RBD IgG2, S-RBD IgG3 and S-RBD IgG4) c S-RBD IgG AUC values of subject plasma grouped by outpatient, hospitalized, ICU or deceased and plasma donors ( n = 115) d Nucleocapsid protein IgG AUC values of subject plasma grouped by outpatient, hospitalized, ICU or deceased and convalescent plasma donors ( n = 115). e S-RBD IgA AUC values of subject plasma grouped by outpatient, hospitalized, ICU or deceased and plasma donors ( n = 115). f S-RBD IgG AUC values of severity groups and plasma donors subdivided into males and females ( n = 115). Green dots show female subjects while purple squares indicate male subjects. Statistical significances were determined using two-tailed Mann–Whitney U test.

    Techniques Used: Detection Assay, Standard Deviation, Two Tailed Test, MANN-WHITNEY

    Neutralization of SARS-CoV-2 and SARS-CoV pseudoviruses with soluble ACE2 and Nabs. a Illustration of spike-protein pseudovirus blocked by soluble ACE2 or neutralizing antibodies. b SARS-CoV-2 and SARS-CoV pseudovirus neutralization with soluble ACE2. SARS-CoV-2 RFP and SARS-CoV GFP pseudoviruses were pre-incubated with soluble ACE2 for 1 h and added to 293 cells expressing ACE2-IRES-GFP or ACE2-mKO2 fusion, respectively. c Neutralization of SARS-CoV-2 and SARS-CoV with S-RBD-specific antibodies and soluble ACE2 (sACE2). Viruses were pre-incubated with antibodies (NAb#1 and SARS-CoV-2 S-RBD non-NAb) or soluble ACE2 (sACE2) proteins for 1 h at the concentrations shown and subsequently added to target cells. Expression of RFP was determined at day 3 post-infection. Infection percentages were normalized to negative controls which are the infection conditions with no blocking agent. Triangles and circles represent SARS-CoV and SARS-CoV-2 data, respectively. Red, green, blue and turquoise colored lines show SARS-CoV-2 S-RBD NAb#1, SARS-CoV-2 S-RBD non-NAb, soluble ACE2 #1 and #2, respectively. d Neutralization of SARS-CoV-2 pseudoviruses using 4 different S-RBD NAbs and two different soluble ACE2 proteins. NAb #1 and #4 were human antibodies whereas NAb #2 and #3 were mouse. Red lines represent antibodies while blue lines show soluble ACE2 molecules. Dot, triangle, square, asterisk, circle and star symbols indicate SARS-CoV-2 S-RBD NAb #1, #2, #3, #4, soluble ACE2 #1 and #2, respectively. Graphs in ( c ) and ( d ) represent three replicates of the experiments. Error bars indicate one standard deviation of mean values.
    Figure Legend Snippet: Neutralization of SARS-CoV-2 and SARS-CoV pseudoviruses with soluble ACE2 and Nabs. a Illustration of spike-protein pseudovirus blocked by soluble ACE2 or neutralizing antibodies. b SARS-CoV-2 and SARS-CoV pseudovirus neutralization with soluble ACE2. SARS-CoV-2 RFP and SARS-CoV GFP pseudoviruses were pre-incubated with soluble ACE2 for 1 h and added to 293 cells expressing ACE2-IRES-GFP or ACE2-mKO2 fusion, respectively. c Neutralization of SARS-CoV-2 and SARS-CoV with S-RBD-specific antibodies and soluble ACE2 (sACE2). Viruses were pre-incubated with antibodies (NAb#1 and SARS-CoV-2 S-RBD non-NAb) or soluble ACE2 (sACE2) proteins for 1 h at the concentrations shown and subsequently added to target cells. Expression of RFP was determined at day 3 post-infection. Infection percentages were normalized to negative controls which are the infection conditions with no blocking agent. Triangles and circles represent SARS-CoV and SARS-CoV-2 data, respectively. Red, green, blue and turquoise colored lines show SARS-CoV-2 S-RBD NAb#1, SARS-CoV-2 S-RBD non-NAb, soluble ACE2 #1 and #2, respectively. d Neutralization of SARS-CoV-2 pseudoviruses using 4 different S-RBD NAbs and two different soluble ACE2 proteins. NAb #1 and #4 were human antibodies whereas NAb #2 and #3 were mouse. Red lines represent antibodies while blue lines show soluble ACE2 molecules. Dot, triangle, square, asterisk, circle and star symbols indicate SARS-CoV-2 S-RBD NAb #1, #2, #3, #4, soluble ACE2 #1 and #2, respectively. Graphs in ( c ) and ( d ) represent three replicates of the experiments. Error bars indicate one standard deviation of mean values.

    Techniques Used: Neutralization, Incubation, Expressing, Infection, Blocking Assay, Standard Deviation

    Development of SARS-CoV-2 and SARS-CoV spike-protein pseudotyped lentiviruses. a Schematic illustration of spike protein expression on the cell surface and soluble ACE2-Fc staining followed by an anti-Fc antibody staining. b 293 cells transfected with spike protein with or without endoplasmic reticulum retention signal (ERRS) or with VSV-G as a negative control. The cells were stained with ACE2-Fc and anti-Fc-APC secondary antibody, flow cytometry data overlays are shown. c Schematic representation of spike protein pseudovirus generation and subsequent infection of ACE2-expressing host cells. A lentivector plasmid and a spike protein over-expressing envelope plasmid are used to co-transfect 293 cells to generate spike pseudovirus that in turn infect engineered cells over-expressing wild-type ACE2 or ACE2-mKO2 fusion. d Infection of wild-type 293 cells with either bald lentiviruses generated without envelope plasmid or spike protein pseudovirus. e Infection of 293-ACE2 cells with bald and spike lentiviruses. GFP and mKO2 markers are used to determine ACE2 over-expressing cells in ACE2-IRES-GFP and ACE2-mKO2, respectively. f The titrations of SARS-CoV-2 and SARS-CoV spike protein pseudoviruses encoding RFP. Triangles and circles represent SARS-CoV and SARS-CoV-2 data, respectively. Brown, red, salmon and orange-colored lines show direct infection, first, second and third freeze/thaw cycles, respectively. ACE2-IRES-GFP expressing 293 cells were incubated with threefold serial dilutions of virus supernatant, stored for several hours at 4 °C or serially frozen and thawed for 1, 2 and 3 cycles, and analyzed for RFP expression by flow cytometry on day 3 post-infection. Percent infection is % RFP+ cells after gating on GFP+ cells (i.e., ACE2+). Titration experiments were replicated twice except for the ‘1 freeze/thaw cycle’ for which titrations were replicated four times. Error bars represent 1 standard deviation of mean values.
    Figure Legend Snippet: Development of SARS-CoV-2 and SARS-CoV spike-protein pseudotyped lentiviruses. a Schematic illustration of spike protein expression on the cell surface and soluble ACE2-Fc staining followed by an anti-Fc antibody staining. b 293 cells transfected with spike protein with or without endoplasmic reticulum retention signal (ERRS) or with VSV-G as a negative control. The cells were stained with ACE2-Fc and anti-Fc-APC secondary antibody, flow cytometry data overlays are shown. c Schematic representation of spike protein pseudovirus generation and subsequent infection of ACE2-expressing host cells. A lentivector plasmid and a spike protein over-expressing envelope plasmid are used to co-transfect 293 cells to generate spike pseudovirus that in turn infect engineered cells over-expressing wild-type ACE2 or ACE2-mKO2 fusion. d Infection of wild-type 293 cells with either bald lentiviruses generated without envelope plasmid or spike protein pseudovirus. e Infection of 293-ACE2 cells with bald and spike lentiviruses. GFP and mKO2 markers are used to determine ACE2 over-expressing cells in ACE2-IRES-GFP and ACE2-mKO2, respectively. f The titrations of SARS-CoV-2 and SARS-CoV spike protein pseudoviruses encoding RFP. Triangles and circles represent SARS-CoV and SARS-CoV-2 data, respectively. Brown, red, salmon and orange-colored lines show direct infection, first, second and third freeze/thaw cycles, respectively. ACE2-IRES-GFP expressing 293 cells were incubated with threefold serial dilutions of virus supernatant, stored for several hours at 4 °C or serially frozen and thawed for 1, 2 and 3 cycles, and analyzed for RFP expression by flow cytometry on day 3 post-infection. Percent infection is % RFP+ cells after gating on GFP+ cells (i.e., ACE2+). Titration experiments were replicated twice except for the ‘1 freeze/thaw cycle’ for which titrations were replicated four times. Error bars represent 1 standard deviation of mean values.

    Techniques Used: Expressing, Staining, Transfection, Negative Control, Flow Cytometry, Infection, Plasmid Preparation, Generated, Incubation, Titration, Standard Deviation

    11) Product Images from "Degradation of SARS-CoV-2 receptor ACE2 by tobacco carcinogen-induced Skp2 in lung epithelial cells"

    Article Title: Degradation of SARS-CoV-2 receptor ACE2 by tobacco carcinogen-induced Skp2 in lung epithelial cells

    Journal: bioRxiv

    doi: 10.1101/2020.10.13.337774

    Inhibition of SARS-CoV-2 S protein pseudoviron entry by CSE, BaP, and NNK. (A) Entry of SARS-CoV-2 S protein pseudovirions into 16HBE cells treated with indicated agents. (B) Entry of SARS-CoV-2 S protein pseudovirions into 293T- ACE2 cells treated with indicated agents. (C) Schematic representation of tobacco smoke-induced ACE2 degradation.
    Figure Legend Snippet: Inhibition of SARS-CoV-2 S protein pseudoviron entry by CSE, BaP, and NNK. (A) Entry of SARS-CoV-2 S protein pseudovirions into 16HBE cells treated with indicated agents. (B) Entry of SARS-CoV-2 S protein pseudovirions into 293T- ACE2 cells treated with indicated agents. (C) Schematic representation of tobacco smoke-induced ACE2 degradation.

    Techniques Used: Inhibition

    12) Product Images from "In vitro study of BromAc on SARS-CoV-2 spike and envelope protein shows synergy and disintegration at modest concentrations"

    Article Title: In vitro study of BromAc on SARS-CoV-2 spike and envelope protein shows synergy and disintegration at modest concentrations

    Journal: bioRxiv

    doi: 10.1101/2020.09.07.286906

    A) The recombinant SARS-CoV-2 spike protein (red arrow) S1+S2 subunits treated with Bromelain or Acetylcysteine alone or BromAc ® combinations. B) The recombinant SARS-CoV-2 envelope protein (red arrow) treated with Bromelain or Acetylcysteine alone or BromAc ® combinations.
    Figure Legend Snippet: A) The recombinant SARS-CoV-2 spike protein (red arrow) S1+S2 subunits treated with Bromelain or Acetylcysteine alone or BromAc ® combinations. B) The recombinant SARS-CoV-2 envelope protein (red arrow) treated with Bromelain or Acetylcysteine alone or BromAc ® combinations.

    Techniques Used: Recombinant

    13) Product Images from "A Novel DNA Vaccine Against SARS-CoV-2 Encoding a Chimeric Protein of Its Receptor-Binding Domain (RBD) Fused to the Amino-Terminal Region of Hepatitis B Virus preS1 With a W4P Mutation"

    Article Title: A Novel DNA Vaccine Against SARS-CoV-2 Encoding a Chimeric Protein of Its Receptor-Binding Domain (RBD) Fused to the Amino-Terminal Region of Hepatitis B Virus preS1 With a W4P Mutation

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.637654

    W4P-RBD potentiates functional T cells specific to SARS-CoV-2 S1 proteins. C57BL/6 mice were intramuscularly injected with W-RBD, W4P-RBD (50 μ g /mouse), or mock, and the spleens were collected 5 weeks post-vaccination for analysis by flow cytometry. (A,B) Splenocytes were incubated with SARS-CoV-2 S1 protein (5 μ g / ml ) for 24 h and stained to detect IFNγ-producing CD8 + T cells and CD4 + T cells. (C) Correlation between RBD-specific IgG in serum and the S1-specific T-cell population in splenocytes. Significance differences (* P
    Figure Legend Snippet: W4P-RBD potentiates functional T cells specific to SARS-CoV-2 S1 proteins. C57BL/6 mice were intramuscularly injected with W-RBD, W4P-RBD (50 μ g /mouse), or mock, and the spleens were collected 5 weeks post-vaccination for analysis by flow cytometry. (A,B) Splenocytes were incubated with SARS-CoV-2 S1 protein (5 μ g / ml ) for 24 h and stained to detect IFNγ-producing CD8 + T cells and CD4 + T cells. (C) Correlation between RBD-specific IgG in serum and the S1-specific T-cell population in splenocytes. Significance differences (* P

    Techniques Used: Functional Assay, Mouse Assay, Injection, Flow Cytometry, Incubation, Staining

    Schematic representation of W4P-RBD as a vaccine candidate against SARS-CoV-2. A novel platform of N-terminal addition of HBV W4P preS1 33-bp sequences for a DNA vaccine against SARS-CoV-2 were developed. The W4P-RBD led to enhanced both humoral and cell-mediated immune response against SARS-CoV-2 in vaccinated mice, demonstrating its feasibility as a DNA vaccine to protect against SARS-CoV-2.
    Figure Legend Snippet: Schematic representation of W4P-RBD as a vaccine candidate against SARS-CoV-2. A novel platform of N-terminal addition of HBV W4P preS1 33-bp sequences for a DNA vaccine against SARS-CoV-2 were developed. The W4P-RBD led to enhanced both humoral and cell-mediated immune response against SARS-CoV-2 in vaccinated mice, demonstrating its feasibility as a DNA vaccine to protect against SARS-CoV-2.

    Techniques Used: Mouse Assay

    W4P-RBD elicits RBD-specific antibody responses in serum and induces potent neutralizing activity against pseudotyped SARS-CoV-2. C57BL/6 mice were immunized with W-RBD, W4P-RBD (50 μ g /mouse), or empty pcDNA3.3 (Mock) three times at 1-week intervals. (A,C) Antibody responses in serum specific to SARS-CoV-2 RBD proteins were detected by ELISA. (B) Serum at 5 weeks after the last immunization was assessed using different dilution factors for IgG against the SARS-CoV-2 RBD protein using ELISA. (D) The 50% neutralizing antibody titer (NT 50 ) was calculated using the SARS-CoV-2 pseudovirus neutralization assay in Calu-3 cells. (E) Correlation between SARS-CoV-2 RBD-specific IgG and pseudotyped SARS-CoV-2 neutralization titers for immunized mice. Significance differences (* P
    Figure Legend Snippet: W4P-RBD elicits RBD-specific antibody responses in serum and induces potent neutralizing activity against pseudotyped SARS-CoV-2. C57BL/6 mice were immunized with W-RBD, W4P-RBD (50 μ g /mouse), or empty pcDNA3.3 (Mock) three times at 1-week intervals. (A,C) Antibody responses in serum specific to SARS-CoV-2 RBD proteins were detected by ELISA. (B) Serum at 5 weeks after the last immunization was assessed using different dilution factors for IgG against the SARS-CoV-2 RBD protein using ELISA. (D) The 50% neutralizing antibody titer (NT 50 ) was calculated using the SARS-CoV-2 pseudovirus neutralization assay in Calu-3 cells. (E) Correlation between SARS-CoV-2 RBD-specific IgG and pseudotyped SARS-CoV-2 neutralization titers for immunized mice. Significance differences (* P

    Techniques Used: Activity Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Neutralization

    W4P-RBD exerts potent neutralizing activity against live SARS-CoV-2. C57BL/6 mice were immunized with W-RBD, W4P-RBD (50 μ g /mouse), or empty pcDNA3.3 (Mock) three times at 1-week intervals. Serum from the immunized mice was diluted and incubated with live SARS-CoV-2 for neutralization assays. (A) A 50% plaque reduction neutralizing antibody (PRNT 50 ) titer against live SARS-CoV-2 was calculated against SARS-CoV-2 infection in Vero E6 cells. (B) Reduction in plaque formation in Vero E6 cells infected with SARS-CoV-2. (C) Correlation between SARS-CoV-2 RBD-specific IgG and SARS-CoV-2 neutralization titers in immunized mice. Significance differences (** P
    Figure Legend Snippet: W4P-RBD exerts potent neutralizing activity against live SARS-CoV-2. C57BL/6 mice were immunized with W-RBD, W4P-RBD (50 μ g /mouse), or empty pcDNA3.3 (Mock) three times at 1-week intervals. Serum from the immunized mice was diluted and incubated with live SARS-CoV-2 for neutralization assays. (A) A 50% plaque reduction neutralizing antibody (PRNT 50 ) titer against live SARS-CoV-2 was calculated against SARS-CoV-2 infection in Vero E6 cells. (B) Reduction in plaque formation in Vero E6 cells infected with SARS-CoV-2. (C) Correlation between SARS-CoV-2 RBD-specific IgG and SARS-CoV-2 neutralization titers in immunized mice. Significance differences (** P

    Techniques Used: Activity Assay, Mouse Assay, Incubation, Neutralization, Plaque Reduction Neutralization Test, Infection

    The W4P-RBD vaccine exerts potent neutralizing activity against live SARS-CoV-2 and inhibits viral infection and replication of SARS-CoV-2. C57BL/6 mice were immunized with W-RBD, W4P-RBD (50 μ g /mouse), or empty pcDNA3.3 (Mock) three times at 1-week intervals. Serum from the immunized mice was diluted and incubated with live SARS-CoV-2 for neutralization assays. (A) The neutralization efficacy of serum from immunized mice against live SARS-CoV-2 RNA copy number in Vero E6 cells was determined by qRT-PCR. (B) The neutralization efficacy of the diluted serum against live SARS-CoV-2 in Vero E6 cells was determined by Western blotting. (C) Pooled serum (diluted 1:500) from each mouse group was tested in the neutralization assay against live SARS-CoV-2. Representative images (40-fold magnification) show live SARS-CoV-2-infected cells after neutralization in each group. Significance differences (* P
    Figure Legend Snippet: The W4P-RBD vaccine exerts potent neutralizing activity against live SARS-CoV-2 and inhibits viral infection and replication of SARS-CoV-2. C57BL/6 mice were immunized with W-RBD, W4P-RBD (50 μ g /mouse), or empty pcDNA3.3 (Mock) three times at 1-week intervals. Serum from the immunized mice was diluted and incubated with live SARS-CoV-2 for neutralization assays. (A) The neutralization efficacy of serum from immunized mice against live SARS-CoV-2 RNA copy number in Vero E6 cells was determined by qRT-PCR. (B) The neutralization efficacy of the diluted serum against live SARS-CoV-2 in Vero E6 cells was determined by Western blotting. (C) Pooled serum (diluted 1:500) from each mouse group was tested in the neutralization assay against live SARS-CoV-2. Representative images (40-fold magnification) show live SARS-CoV-2-infected cells after neutralization in each group. Significance differences (* P

    Techniques Used: Activity Assay, Infection, Mouse Assay, Incubation, Neutralization, Quantitative RT-PCR, Western Blot

    W4P-RBD induces proinflammatory cytokine production in S1-stimulated splenocytes. Splenocytes from immunized mice were stimulated with SARS-CoV-2 S1 protein (5 μ g / ml ) for 5 days. The cytokine production of (A) TNFα, (B) IFNγ, (C) IL-12p40, (D) IFNβ, (E) IL-6, and (F) IL-2 from splenocytes was detected by ELISA. Significance differences (* P
    Figure Legend Snippet: W4P-RBD induces proinflammatory cytokine production in S1-stimulated splenocytes. Splenocytes from immunized mice were stimulated with SARS-CoV-2 S1 protein (5 μ g / ml ) for 5 days. The cytokine production of (A) TNFα, (B) IFNγ, (C) IL-12p40, (D) IFNβ, (E) IL-6, and (F) IL-2 from splenocytes was detected by ELISA. Significance differences (* P

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Construction of the HBV W4P preS1-fused pcDNA3.3-RBD plasmid (W4P-RBD) as a candidate for SARS-CoV-2. (A) Design of pcDNA3.3-RBD and pcDNA3.3-W4P-RBD. The W4P region comprises 33 bp from the first site of the preS1 region of the HBV genome and encodes 11 amino acids. (B) The protein expression of SARS-CoV-2 RBD and W4P-conjugated RBD was detected by the Western blot assay. pcDNA3.3-RBD, pcDNA3.3-W4P-RBD, and empty pcDNA3.3 were transfected into Vero E6, Huh7, and 293T cells, and cell lysates were collected 48 h post transfection to detect protein expression. (C) The mRNA expression levels of IL-6 and in pcDNA3.3-transfected cells were detected by qRT-PCR. Significance differences (* P
    Figure Legend Snippet: Construction of the HBV W4P preS1-fused pcDNA3.3-RBD plasmid (W4P-RBD) as a candidate for SARS-CoV-2. (A) Design of pcDNA3.3-RBD and pcDNA3.3-W4P-RBD. The W4P region comprises 33 bp from the first site of the preS1 region of the HBV genome and encodes 11 amino acids. (B) The protein expression of SARS-CoV-2 RBD and W4P-conjugated RBD was detected by the Western blot assay. pcDNA3.3-RBD, pcDNA3.3-W4P-RBD, and empty pcDNA3.3 were transfected into Vero E6, Huh7, and 293T cells, and cell lysates were collected 48 h post transfection to detect protein expression. (C) The mRNA expression levels of IL-6 and in pcDNA3.3-transfected cells were detected by qRT-PCR. Significance differences (* P

    Techniques Used: Plasmid Preparation, Expressing, Western Blot, Transfection, Quantitative RT-PCR

    14) Product Images from "FN3-based monobodies selective for the receptor binding domain of the SARS-CoV-2 spike protein"

    Article Title: FN3-based monobodies selective for the receptor binding domain of the SARS-CoV-2 spike protein

    Journal: New Biotechnology

    doi: 10.1016/j.nbt.2021.01.010

    Isolation of four monobodies that bind the RBD of the SARS-CoV-2 virus by phage-display. (a) The 3D visualization of the fibronectin type III (FN3) domain (PDB: 1TTG) as shown in PyMOL, with the BC, DE, and FG loops labelled in different colors [ 66 ]. (b) Virions displaying the four monobody sequences (A, B, C, and D) were confirmed by ELISA to bind the RBD-Fc fusion protein and not to the Fc (negative control). Error bars represent standard error (SE) of triplicate measurements. (c) The amino acid sequences of the BC, DE, and FG loops within the four monobodies. Frequency represents the number of times a given monobody was identified among 9 confirmed binders. The complete primary structures of the four monobodies are shown in Suppl. Figure S3.
    Figure Legend Snippet: Isolation of four monobodies that bind the RBD of the SARS-CoV-2 virus by phage-display. (a) The 3D visualization of the fibronectin type III (FN3) domain (PDB: 1TTG) as shown in PyMOL, with the BC, DE, and FG loops labelled in different colors [ 66 ]. (b) Virions displaying the four monobody sequences (A, B, C, and D) were confirmed by ELISA to bind the RBD-Fc fusion protein and not to the Fc (negative control). Error bars represent standard error (SE) of triplicate measurements. (c) The amino acid sequences of the BC, DE, and FG loops within the four monobodies. Frequency represents the number of times a given monobody was identified among 9 confirmed binders. The complete primary structures of the four monobodies are shown in Suppl. Figure S3.

    Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay, Negative Control

    Specificity of anti-RBD monobodies. The four MBP-FN3 fusions were adsorbed on microtiter plate wells and incubated with chemically biotinylated SARS-CoV-1 and SARS-CoV-2 RBD proteins mixed with a bacterial cell lysate. Wells coated with MBP alone served as a negative control and wells coated with an anti-spike monoclonal antibody, clone CR3022 [ 35 ], which binds equally well to the RBDs of both SARS-CoV-1 and SARS-CoV-2, served as a positive control. Binding of SARS-CoV-1 and SARS-CoV-2 RBD-Fc fusion proteins was revealed with streptavidin-HRP. Error bars represent standard error (SE) of triplicate measurements.
    Figure Legend Snippet: Specificity of anti-RBD monobodies. The four MBP-FN3 fusions were adsorbed on microtiter plate wells and incubated with chemically biotinylated SARS-CoV-1 and SARS-CoV-2 RBD proteins mixed with a bacterial cell lysate. Wells coated with MBP alone served as a negative control and wells coated with an anti-spike monoclonal antibody, clone CR3022 [ 35 ], which binds equally well to the RBDs of both SARS-CoV-1 and SARS-CoV-2, served as a positive control. Binding of SARS-CoV-1 and SARS-CoV-2 RBD-Fc fusion proteins was revealed with streptavidin-HRP. Error bars represent standard error (SE) of triplicate measurements.

    Techniques Used: Incubation, Negative Control, Positive Control, Binding Assay

    Purification of RBD-Fc, ACE2-Fc, and spike protein. (a) Recombinant. SARS-CoV-2 spike RBD-Fc fusion protein. The predicted molecular weight (MW) is ∼ 65 kDa, when resolved by SDS-PAGE under reducing conditions with sized standards (MW shown in kDa); > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. (b) Recombinant ACE2-Fc fusion protein. The predicted MW is ∼110 kDa, when resolved by SDS-PAGE under reducing conditions, and judged to be > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. (c) Spike protein. The near full-length protein resolved as a doublet with a MW of ∼170 kDa under reducing conditions and was judged to be > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. The doublet bands are thought to differ in post-translational modifications. Composite image of two lanes from the same gel.
    Figure Legend Snippet: Purification of RBD-Fc, ACE2-Fc, and spike protein. (a) Recombinant. SARS-CoV-2 spike RBD-Fc fusion protein. The predicted molecular weight (MW) is ∼ 65 kDa, when resolved by SDS-PAGE under reducing conditions with sized standards (MW shown in kDa); > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. (b) Recombinant ACE2-Fc fusion protein. The predicted MW is ∼110 kDa, when resolved by SDS-PAGE under reducing conditions, and judged to be > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. (c) Spike protein. The near full-length protein resolved as a doublet with a MW of ∼170 kDa under reducing conditions and was judged to be > 90 % pure by quantitative densitometry of the Coomassie Blue stained gel. The doublet bands are thought to differ in post-translational modifications. Composite image of two lanes from the same gel.

    Techniques Used: Purification, Recombinant, Molecular Weight, SDS Page, Staining

    Detection of the SARS-CoV-2 RBD in a complex biological mixture. An E. coli cell lysate was mixed with various concentrations of SARS-CoV-2 RBD and added to microtiter wells coated with the FN3A-MBP fusion protein. After incubation and washing of the wells, the ectodomain of ACE2, conjugated to HRP, was added. Negative controls consisted of MBP in lieu of FN3A and Fc alone in lieu of RBD. Error bars represent SE of triplicate measurements.
    Figure Legend Snippet: Detection of the SARS-CoV-2 RBD in a complex biological mixture. An E. coli cell lysate was mixed with various concentrations of SARS-CoV-2 RBD and added to microtiter wells coated with the FN3A-MBP fusion protein. After incubation and washing of the wells, the ectodomain of ACE2, conjugated to HRP, was added. Negative controls consisted of MBP in lieu of FN3A and Fc alone in lieu of RBD. Error bars represent SE of triplicate measurements.

    Techniques Used: Incubation

    15) Product Images from "Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses"

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses

    Journal: bioRxiv

    doi: 10.1101/2020.06.08.140871

    A replication-competent VSV/SARS-CoV-2 chimera. A . Schematic representation of the rVSV/SARS-CoV-2/GFP genome in which G-encoding sequences were replaced by SARS-CoV-2 SΔ18 coding sequences. GFP-encoding sequences were introduced between the SARS-CoV-2 SΔ18 and L open reading frames. B . Representative images of 293T/ACE2(B) cells infected with the indicated volumes of plaque purified, adapted derivatives (2E1 and 1D7) of VSV/SARS-CoV-2/GFP following passage in the same cell line. Left and center images show contents of an entire well of a 96-well plate, the right image shows expanded view of the boxed areas containing individual plaques. C . Infectivity measurements of rVSV/SARS-CoV-2/GFP virus stocks on 293T/ACE2(B) or control 293T cells, quantified by measuring % GFP-positive cells at 16h after infection. Average and standard deviation from two technical replicates is shown. D . Schematic representation of the adaptive changes acquired in rVSV/SARS-CoV-2/GFP during passage. Changes in 1D7 and 2E1 are shown in blue and red, respectively.
    Figure Legend Snippet: A replication-competent VSV/SARS-CoV-2 chimera. A . Schematic representation of the rVSV/SARS-CoV-2/GFP genome in which G-encoding sequences were replaced by SARS-CoV-2 SΔ18 coding sequences. GFP-encoding sequences were introduced between the SARS-CoV-2 SΔ18 and L open reading frames. B . Representative images of 293T/ACE2(B) cells infected with the indicated volumes of plaque purified, adapted derivatives (2E1 and 1D7) of VSV/SARS-CoV-2/GFP following passage in the same cell line. Left and center images show contents of an entire well of a 96-well plate, the right image shows expanded view of the boxed areas containing individual plaques. C . Infectivity measurements of rVSV/SARS-CoV-2/GFP virus stocks on 293T/ACE2(B) or control 293T cells, quantified by measuring % GFP-positive cells at 16h after infection. Average and standard deviation from two technical replicates is shown. D . Schematic representation of the adaptive changes acquired in rVSV/SARS-CoV-2/GFP during passage. Changes in 1D7 and 2E1 are shown in blue and red, respectively.

    Techniques Used: Infection, Purification, Standard Deviation

    Examples of neutralization of HIV-1 and VSV pseudotyped virus particles by monoclonal antibodies targeting SARS-CoV-2 S. A . Images of Huh7.5 cells following infection with rVSVΔG/NG-NanoLuc pseudotyped virus (∼10 3 IU/well) in the presence of the indicated concentrations of a human monoclonal antibody (C144) targeting SARS-CoV-2 S RBD. B . Quantification of rVSVΔG/NG-NanoLuc pseudotyped virus infection (measured by flow cytometry (% mNeonGreen positive cells, green) or by NanoLuc luciferase activity (RLU, blue) in the presence of the indicated concentrations of a human monoclonal antibody (C102) targeting SARS-CoV-2 S RBD, or a control monoclonal antibody against the Zika virus envelope glycoprotein. C . Quantification of HIV-1 NL ΔEnv-NanoLuc or CCNanoLuc/GFP pseudotyped virus infection on the indicated cell lines in the presence of the indicated concentrations of a human monoclonal antibody (C121) targeting SARS-CoV-2 S RBD Infectivity was quantified by measuring NanoLuc luciferase levels (RLU).
    Figure Legend Snippet: Examples of neutralization of HIV-1 and VSV pseudotyped virus particles by monoclonal antibodies targeting SARS-CoV-2 S. A . Images of Huh7.5 cells following infection with rVSVΔG/NG-NanoLuc pseudotyped virus (∼10 3 IU/well) in the presence of the indicated concentrations of a human monoclonal antibody (C144) targeting SARS-CoV-2 S RBD. B . Quantification of rVSVΔG/NG-NanoLuc pseudotyped virus infection (measured by flow cytometry (% mNeonGreen positive cells, green) or by NanoLuc luciferase activity (RLU, blue) in the presence of the indicated concentrations of a human monoclonal antibody (C102) targeting SARS-CoV-2 S RBD, or a control monoclonal antibody against the Zika virus envelope glycoprotein. C . Quantification of HIV-1 NL ΔEnv-NanoLuc or CCNanoLuc/GFP pseudotyped virus infection on the indicated cell lines in the presence of the indicated concentrations of a human monoclonal antibody (C121) targeting SARS-CoV-2 S RBD Infectivity was quantified by measuring NanoLuc luciferase levels (RLU).

    Techniques Used: Neutralization, Infection, Flow Cytometry, Luciferase, Activity Assay

    Measurement of neutralization activity in COVID19 convalescent donor plasma. A . Plasma neutralization of SARS-CoV-2: serial 5-fold dilutions of plasma samples from convalescent donors were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using VeroE6 target cells, expressed as % infected cells by immunostaining. B . Plasma neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP-positive cells, as indicated. C . Correlation between NT 50 values for each of the 20 plasmas for each of the surrogate viruses (x-axis) and NT 50 values for the same plasmas for SARS-CoV-2 (y-axis).
    Figure Legend Snippet: Measurement of neutralization activity in COVID19 convalescent donor plasma. A . Plasma neutralization of SARS-CoV-2: serial 5-fold dilutions of plasma samples from convalescent donors were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using VeroE6 target cells, expressed as % infected cells by immunostaining. B . Plasma neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP-positive cells, as indicated. C . Correlation between NT 50 values for each of the 20 plasmas for each of the surrogate viruses (x-axis) and NT 50 values for the same plasmas for SARS-CoV-2 (y-axis).

    Techniques Used: Neutralization, Activity Assay, Incubation, Infection, Immunostaining, Luciferase

    Generation of and HIV-1 pseudotype infection of ACE2-expressing cell lines. A . 293T cells were stably transduced with a lentivirus vector CSIB, expressing either wild type ACE2 or catalytically active mutant ACE2*. Following selection, cells were used as uncloned bulk populations (B) or single cell clones were isolated. Flow cytometry histograms show staining with an antibody against huACE2 (purple) or an isotype control (grey). B . HT1080 cells were stably transduced as in A and a single cell clone used throughout this study is shown, stained as in A. C . Infectivity of CCNanoLuc/GFP viruses, pseudotyped with either full length or C-terminally truncated SARS-CoV and SARS-CoV-2 S proteins on 293T/ACE2*(B) cells. Virus particles generated in the absence of an S protein (No S) were used as background controls. Infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. D . Infectivity of HIV-1 NL ΔEnv-NanoLuc in the various cell lines. Virus generated in the absence of S is used as a background control and infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. E . Same as D except that CCNanoLuc/GFP virus was used F . Effect of virus ultracentrifugation on the infectivity of HIV-1-based pseudotyped virus particles. 293T/ACE2*(B) cells were infected with equivalent doses of unconcentrated HIV-1 NL ΔEnv-NanoLuc, or the same virus that had be pelleted through 20% sucrose and then diluted to the original volume.
    Figure Legend Snippet: Generation of and HIV-1 pseudotype infection of ACE2-expressing cell lines. A . 293T cells were stably transduced with a lentivirus vector CSIB, expressing either wild type ACE2 or catalytically active mutant ACE2*. Following selection, cells were used as uncloned bulk populations (B) or single cell clones were isolated. Flow cytometry histograms show staining with an antibody against huACE2 (purple) or an isotype control (grey). B . HT1080 cells were stably transduced as in A and a single cell clone used throughout this study is shown, stained as in A. C . Infectivity of CCNanoLuc/GFP viruses, pseudotyped with either full length or C-terminally truncated SARS-CoV and SARS-CoV-2 S proteins on 293T/ACE2*(B) cells. Virus particles generated in the absence of an S protein (No S) were used as background controls. Infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. D . Infectivity of HIV-1 NL ΔEnv-NanoLuc in the various cell lines. Virus generated in the absence of S is used as a background control and infectivity was quantified by measuring NanoLuc luciferase activity (RLU). Average and standard deviation from two technical replicates is shown. E . Same as D except that CCNanoLuc/GFP virus was used F . Effect of virus ultracentrifugation on the infectivity of HIV-1-based pseudotyped virus particles. 293T/ACE2*(B) cells were infected with equivalent doses of unconcentrated HIV-1 NL ΔEnv-NanoLuc, or the same virus that had be pelleted through 20% sucrose and then diluted to the original volume.

    Techniques Used: Infection, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Mutagenesis, Selection, Clone Assay, Isolation, Flow Cytometry, Staining, Generated, Luciferase, Activity Assay, Standard Deviation

    Variables determining HIV-1 pseudotype infection signal. A . Images (GFP) of confluent monolayers of the indicated cell lines after infection with equivalent amounts of CCNanoLuc/GFP pseudotyped with SARS-CoV-2 SΔ19 or no S protein, as indicated. B . Relationship between NanoLuc luciferase activity (RLU) and ACE2 cell surface expression levels (quantified by flow cytometry, Fig S1A ) following infection the cell lines depicted in Fig. S1A with HIV-1 NL ΔEnv-NanoLuc pseudotyped virus. C . Infectivity of CCNanoLuc/GFP pseudotyped virus generated by cotransfection with the indicated amounts of SARS-CoV-2 SΔ19 expression plasmid. D . Quantification of infectivity of HIV-1 NL4-3 ΔEnv-NanoLuc pseudotype infection by immunostaining of 293T/ACE2(B) target cells with antibodies against the HIV-1 capsid (CA) protein. Nuclei were visualized by Hoechst staining.
    Figure Legend Snippet: Variables determining HIV-1 pseudotype infection signal. A . Images (GFP) of confluent monolayers of the indicated cell lines after infection with equivalent amounts of CCNanoLuc/GFP pseudotyped with SARS-CoV-2 SΔ19 or no S protein, as indicated. B . Relationship between NanoLuc luciferase activity (RLU) and ACE2 cell surface expression levels (quantified by flow cytometry, Fig S1A ) following infection the cell lines depicted in Fig. S1A with HIV-1 NL ΔEnv-NanoLuc pseudotyped virus. C . Infectivity of CCNanoLuc/GFP pseudotyped virus generated by cotransfection with the indicated amounts of SARS-CoV-2 SΔ19 expression plasmid. D . Quantification of infectivity of HIV-1 NL4-3 ΔEnv-NanoLuc pseudotype infection by immunostaining of 293T/ACE2(B) target cells with antibodies against the HIV-1 capsid (CA) protein. Nuclei were visualized by Hoechst staining.

    Techniques Used: Infection, Luciferase, Activity Assay, Expressing, Flow Cytometry, Generated, Cotransfection, Plasmid Preparation, Immunostaining, Staining

    Measurement of neutralization potency of human monoclonal antibodies. A . Neutralization of SARS-CoV-2: the indicated concentrations of monoclonal antibodies were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using Vero E6 target cells, expressed as % infected cells, by immunostaining B . Monoclonal antibody neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP positive cells, as indicated. C . Correlation between IC 50 values for each of the 15 monoclonal antibodies for each of the surrogate viruses (x-axis) and IC 50 values for the same antibodies for SARS-CoV-2 (y-axis).
    Figure Legend Snippet: Measurement of neutralization potency of human monoclonal antibodies. A . Neutralization of SARS-CoV-2: the indicated concentrations of monoclonal antibodies were incubated with SARS-CoV-2 n=3 replicates and residual infectivity determined using Vero E6 target cells, expressed as % infected cells, by immunostaining B . Monoclonal antibody neutralization of HIV-1 NL ΔEnv-NanoLuc pseudotyped virus using 293T/ACE2*(B) target cells, rVSVΔG/NG-NanoLuc pseudotyped virus using Huh7.5 target cells or replication competent rVSV/SARS-CoV-2/GFP using 293T/ACE2(B) target cells. Residual infectivity was quantified by measuring either NanoLuc luciferase (RLU) or the % GFP positive cells, as indicated. C . Correlation between IC 50 values for each of the 15 monoclonal antibodies for each of the surrogate viruses (x-axis) and IC 50 values for the same antibodies for SARS-CoV-2 (y-axis).

    Techniques Used: Neutralization, Incubation, Infection, Immunostaining, Luciferase

    VSV-based SARS-CoV-2 pseudotyped viruses. A . Schematic representation of the rVSVΔG/NG-NanoLuc genome in which G-coding sequences were replaced by an mNeonGreen-2A-NanoLuc luciferase reporter cassette. Infectious virus particles were generated by passaging G-complemented rVSVΔG/NG-NanoLuc virus stocks through 293T cells transfected with a plasmid encoding SARS-CoV-2 SΔ19. B . Infectivity of pseudotyped rVSVΔG/NG-NanoLuc particles on Huh7.5 cells was quantified by measuring luciferase activity (RLU) or the % GFP-positive cells. Mean and standard deviation from two technical replicates is plotted. Virus particles generated by passage through cells that were not transfected with SARS-CoV-2 S were used as a control. C . NanoLuc luciferase activity (RLU) in Huh7.5 cells measured at various times after infection with pseudotyped rVSVΔG/NG-NanoLuc particles. Average and standard deviation from two technical replicates is shown. D . Infectivity of pseudotyped rVSVΔG/NG-NanoLuc particles on the indicated cell lines. Infectivity was quantified by measuring NanoLuc luciferase activity (RLU) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. Average and standard deviation from two technical replicates is shown.
    Figure Legend Snippet: VSV-based SARS-CoV-2 pseudotyped viruses. A . Schematic representation of the rVSVΔG/NG-NanoLuc genome in which G-coding sequences were replaced by an mNeonGreen-2A-NanoLuc luciferase reporter cassette. Infectious virus particles were generated by passaging G-complemented rVSVΔG/NG-NanoLuc virus stocks through 293T cells transfected with a plasmid encoding SARS-CoV-2 SΔ19. B . Infectivity of pseudotyped rVSVΔG/NG-NanoLuc particles on Huh7.5 cells was quantified by measuring luciferase activity (RLU) or the % GFP-positive cells. Mean and standard deviation from two technical replicates is plotted. Virus particles generated by passage through cells that were not transfected with SARS-CoV-2 S were used as a control. C . NanoLuc luciferase activity (RLU) in Huh7.5 cells measured at various times after infection with pseudotyped rVSVΔG/NG-NanoLuc particles. Average and standard deviation from two technical replicates is shown. D . Infectivity of pseudotyped rVSVΔG/NG-NanoLuc particles on the indicated cell lines. Infectivity was quantified by measuring NanoLuc luciferase activity (RLU) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. Average and standard deviation from two technical replicates is shown.

    Techniques Used: Luciferase, Generated, Passaging, Transfection, Plasmid Preparation, Infection, Activity Assay, Standard Deviation

    Two-plasmid and three-plasmid HIV-1-based pseudotyped viruses. A . Schematic representation of the modified HIV-1 NL ΔEnv-NanoLuc genome in which a deletion in env was introduced and Nef-coding sequences were replaced by those encoding a NanoLuc luciferase reporter. Infectious virus particles were generated by cotransfection of pHIV-1 NL4 ΔEnv-NanoLuc and a plasmid encoding the SARS-CoV-2 S lacking the 19 amino acids at the C-terminus of the cytoplasmic tail (SΔ19). B . Schematic representation of constructs used to generate SARS-CoV-2 S pseudotyped HIV-1-based particles in which HIV-1 NL GagPol, an HIV-1 reporter vector (pCCNanoLuc/GFP) encoding both NanoLuc luciferase and EGFP reporter and the SARS-CoV-2 SΔ19 are each expressed on separate plasmids. C . Infectivity measurements of HIV-1 NL ΔEnv-NanoLuc particles (generated using the plasmids depicted in A) on the indicated cell lines. Infectivity was quantified by measuring NanoLuc luciferase activity (Relative Light Units, RLU) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. The mean and standard deviation of two technical replicates is shown. Target cells 293T/ACE2cl.22 and HT1080/ACE2cl.14 are single-cell clones engineered to express human ACE2 (see Fig S1A ). Virus particles generated in the absence of viral envelope glycoproteins were used as background controls. D . Same as, C but viruses were generated using the 3 plasmids depicted in B. E . Infectivity meaurements of CCNanoLuc/GFP containing SARS-CoV-2 pseudotyped particles generated using plasmids depicted in B on 293ACE2*(B) cells, quantified by measuring NanoLuc luciferase activity (RLU) or GFP levels (% of GFP positive cells). Mean and standard deviation from two technical replicates is shown.
    Figure Legend Snippet: Two-plasmid and three-plasmid HIV-1-based pseudotyped viruses. A . Schematic representation of the modified HIV-1 NL ΔEnv-NanoLuc genome in which a deletion in env was introduced and Nef-coding sequences were replaced by those encoding a NanoLuc luciferase reporter. Infectious virus particles were generated by cotransfection of pHIV-1 NL4 ΔEnv-NanoLuc and a plasmid encoding the SARS-CoV-2 S lacking the 19 amino acids at the C-terminus of the cytoplasmic tail (SΔ19). B . Schematic representation of constructs used to generate SARS-CoV-2 S pseudotyped HIV-1-based particles in which HIV-1 NL GagPol, an HIV-1 reporter vector (pCCNanoLuc/GFP) encoding both NanoLuc luciferase and EGFP reporter and the SARS-CoV-2 SΔ19 are each expressed on separate plasmids. C . Infectivity measurements of HIV-1 NL ΔEnv-NanoLuc particles (generated using the plasmids depicted in A) on the indicated cell lines. Infectivity was quantified by measuring NanoLuc luciferase activity (Relative Light Units, RLU) following infection of cells in 96-well plates with the indicated volumes of pseudotyped viruses. The mean and standard deviation of two technical replicates is shown. Target cells 293T/ACE2cl.22 and HT1080/ACE2cl.14 are single-cell clones engineered to express human ACE2 (see Fig S1A ). Virus particles generated in the absence of viral envelope glycoproteins were used as background controls. D . Same as, C but viruses were generated using the 3 plasmids depicted in B. E . Infectivity meaurements of CCNanoLuc/GFP containing SARS-CoV-2 pseudotyped particles generated using plasmids depicted in B on 293ACE2*(B) cells, quantified by measuring NanoLuc luciferase activity (RLU) or GFP levels (% of GFP positive cells). Mean and standard deviation from two technical replicates is shown.

    Techniques Used: Plasmid Preparation, Modification, Luciferase, Generated, Cotransfection, Construct, Infection, Activity Assay, Standard Deviation, Clone Assay

    rVSVΔG/NG-NanoLuc pseudotyped virus infection. A . Infection of Huh7.5 cells with the indicated volumes of rVSVΔG/NG-NanoLuc pseudotyped virus. Images of the entire well of 96-well plates are shown. B . Infectivity of rVSVΔG/NG-NanoLuc pseudotyped with SARS-CoV-2 SΔ19 or no S (background control) on the indicated cell lines. Infectivity was quantified at the indicated times post-inoculation by measuring NanoLuc luciferase levels (RLU). C and D HT1080-derived cell lines (C) or 293T-derived cell lines (D) were infected with varying amounts of rVSVΔG/NG-NanoLuc pseudotyped with SARS-CoV-2 SΔ19 or no S (background control) and NanoLuc luciferase levels were measured at 16h after infection. E . Relationship between NanoLuc luciferase activity (RLU) and ACE2 cell surface expression levels (quantified by flow cytometry, Fig S1A ) following infection the cell lines depicted in Fig. S1A with rVSVΔG/NG-NanoLuc. F . Effect of virus concentration on the infectivity of rVSVΔG/NG-NanoLuc pseudotyped virus. Huh7.5 cells were infected with equivalent doses of either unmanipulated virus-containing supernatant or virions that had been pelleted by ultracentifugation or using Lenti-X and diluted to the original volume.
    Figure Legend Snippet: rVSVΔG/NG-NanoLuc pseudotyped virus infection. A . Infection of Huh7.5 cells with the indicated volumes of rVSVΔG/NG-NanoLuc pseudotyped virus. Images of the entire well of 96-well plates are shown. B . Infectivity of rVSVΔG/NG-NanoLuc pseudotyped with SARS-CoV-2 SΔ19 or no S (background control) on the indicated cell lines. Infectivity was quantified at the indicated times post-inoculation by measuring NanoLuc luciferase levels (RLU). C and D HT1080-derived cell lines (C) or 293T-derived cell lines (D) were infected with varying amounts of rVSVΔG/NG-NanoLuc pseudotyped with SARS-CoV-2 SΔ19 or no S (background control) and NanoLuc luciferase levels were measured at 16h after infection. E . Relationship between NanoLuc luciferase activity (RLU) and ACE2 cell surface expression levels (quantified by flow cytometry, Fig S1A ) following infection the cell lines depicted in Fig. S1A with rVSVΔG/NG-NanoLuc. F . Effect of virus concentration on the infectivity of rVSVΔG/NG-NanoLuc pseudotyped virus. Huh7.5 cells were infected with equivalent doses of either unmanipulated virus-containing supernatant or virions that had been pelleted by ultracentifugation or using Lenti-X and diluted to the original volume.

    Techniques Used: Infection, Luciferase, Derivative Assay, Activity Assay, Expressing, Flow Cytometry, Concentration Assay

    16) Product Images from "Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity nanobody"

    Article Title: Potent neutralization of clinical isolates of SARS-CoV-2 D614 and G614 variants by a monomeric, sub-nanomolar affinity nanobody

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-82833-w

    Binding characterization and neutralization of SARS-CoV-2 by the nanobody W25. ( a) Pulldown of the W25 nanobody. A recombinant Spike RBD domain of the SARS-CoV-2 spike protein or control BSA protein were covalently bound to NHS-sepharose beads. Further, the W25 nanobody was incubated with control and spike RBD beads, washed, and further eluted in LSD lysis buffer (Invitrogen). Original SDS-Page as supplemental Fig. 3 b. ( b) Unfolding profiles of 2 µM SARS-CoV-2 S1, spike RBD in the absence (black) and presence (red) of 2 µM W25, measured with Tycho NT.6. Binding of W25 to spike RBD leads to strong stabilization and shifts the inflection unfolding temperature (T i ) from 52.1 to 66.3 °C. ( c) MST binding curve for the titration of 1 nM fluorescently labeled W25 into a 16-point serial dilution of SARS-CoV-2 S1, Spike RBD (250 nM to 7.6 pM). W25 binds Spike RBD with sub-nanomolar affinity (K d = 295 ± 84 pM). Error bars show the SD calculated from experiments performed in triplicate. ( d) MST competitive curve for 2 nM of fluorescently labeled W25 incubated with 4 nM SARS-CoV-2 RBD, titrated with a 16-point dilution series of hACE2 (1 µM to 30.5 pM). W25 is displaced by hACE2 with nanomolar concentration (EC50 = 33 ± 9 nM). Error bar show the SD calculated from triplicate experiments. ( e) Diagram of W25 and ACE2 competition for RBD of spike of SARS-CoV-2. ( f) Neutralization assay of SARS-CoV-2 life virus D624 variant with nanobody W25, W25 fused to monomeric Fc (W25FcM) and W25 fused to dimeric Fc (W25Fc) and the previously reported nanobodies VHH-72-Fc (monomeric) and VHH-55-Fc (monomeric). The independent experiments were normalized by percentage of neutralization. ( g) Neutralization assay of SARS-CoV-2 life virus D624 under similar condition as in ( g ). ( h) Comparative neutralization values of W25, W25FcM, W25Fc and VHH-72 FcM against SARS-CoV-2 D614 and G614 virus variants. Illustration (e) by Felipe G. Serrano BSc., MSc Scientific illustrator.
    Figure Legend Snippet: Binding characterization and neutralization of SARS-CoV-2 by the nanobody W25. ( a) Pulldown of the W25 nanobody. A recombinant Spike RBD domain of the SARS-CoV-2 spike protein or control BSA protein were covalently bound to NHS-sepharose beads. Further, the W25 nanobody was incubated with control and spike RBD beads, washed, and further eluted in LSD lysis buffer (Invitrogen). Original SDS-Page as supplemental Fig. 3 b. ( b) Unfolding profiles of 2 µM SARS-CoV-2 S1, spike RBD in the absence (black) and presence (red) of 2 µM W25, measured with Tycho NT.6. Binding of W25 to spike RBD leads to strong stabilization and shifts the inflection unfolding temperature (T i ) from 52.1 to 66.3 °C. ( c) MST binding curve for the titration of 1 nM fluorescently labeled W25 into a 16-point serial dilution of SARS-CoV-2 S1, Spike RBD (250 nM to 7.6 pM). W25 binds Spike RBD with sub-nanomolar affinity (K d = 295 ± 84 pM). Error bars show the SD calculated from experiments performed in triplicate. ( d) MST competitive curve for 2 nM of fluorescently labeled W25 incubated with 4 nM SARS-CoV-2 RBD, titrated with a 16-point dilution series of hACE2 (1 µM to 30.5 pM). W25 is displaced by hACE2 with nanomolar concentration (EC50 = 33 ± 9 nM). Error bar show the SD calculated from triplicate experiments. ( e) Diagram of W25 and ACE2 competition for RBD of spike of SARS-CoV-2. ( f) Neutralization assay of SARS-CoV-2 life virus D624 variant with nanobody W25, W25 fused to monomeric Fc (W25FcM) and W25 fused to dimeric Fc (W25Fc) and the previously reported nanobodies VHH-72-Fc (monomeric) and VHH-55-Fc (monomeric). The independent experiments were normalized by percentage of neutralization. ( g) Neutralization assay of SARS-CoV-2 life virus D624 under similar condition as in ( g ). ( h) Comparative neutralization values of W25, W25FcM, W25Fc and VHH-72 FcM against SARS-CoV-2 D614 and G614 virus variants. Illustration (e) by Felipe G. Serrano BSc., MSc Scientific illustrator.

    Techniques Used: Binding Assay, Neutralization, Recombinant, Incubation, Lysis, SDS Page, Titration, Labeling, Serial Dilution, Concentration Assay, Variant Assay

    Diagram of W25 neutralization of SARS-CoV-2. Illustration by Felipe G. Serrano BSc., MSc Scientific illustrator.
    Figure Legend Snippet: Diagram of W25 neutralization of SARS-CoV-2. Illustration by Felipe G. Serrano BSc., MSc Scientific illustrator.

    Techniques Used: Neutralization

    Immunization of the spike of SARS-CoV-2 and a simple density gradient method for the selection of nanobodies. (a) SDS-Page to ensure protein integrity of full-length spike of SARS-CoV-2 before immunization. ( b) Adult alpaca immunized with spike . (c) Evaluation of the alpaca´s immune response by dot blot. Image shows the reaction to decreasing amounts of Spike and bovine serum albumin (negative control) using a pre-immunization control, and after one immunization (1 week), or two immunizations (3 weeks) with full-length SARS-CoV-2 spike, using alpaca serums as a primary antibody source followed by an anti-camelid IgG-HRP secondary antibody. (d) ELISA before and after the second immunization (3 weeks) n = 4 error bars indicate standard deviation statistic t-test, ** P ≤ 0.005. (e) Schematic representation of novel protocol for isolation of nanobodies using density gradient separation. The bacterial display library expressing the nanobodies on the surface of bacteria is briefly incubated with conventional sepharose beads coated with the antigen of interest. Directly after the mixture is deposited on a Ficoll gradient conic tube and centrifuged at 200 g for 1 min, the beads drive through the gradient to the bottom of the tube with the bacteria expressing specific nanobodies, while unbound bacteria remain on the surface of the gradient. The beads are then resuspended, and bacterial clones are isolated for biochemical binding confirmation. Illustration (e) by Felipe G. Serrano BSc., MSc Scientific illustrator.
    Figure Legend Snippet: Immunization of the spike of SARS-CoV-2 and a simple density gradient method for the selection of nanobodies. (a) SDS-Page to ensure protein integrity of full-length spike of SARS-CoV-2 before immunization. ( b) Adult alpaca immunized with spike . (c) Evaluation of the alpaca´s immune response by dot blot. Image shows the reaction to decreasing amounts of Spike and bovine serum albumin (negative control) using a pre-immunization control, and after one immunization (1 week), or two immunizations (3 weeks) with full-length SARS-CoV-2 spike, using alpaca serums as a primary antibody source followed by an anti-camelid IgG-HRP secondary antibody. (d) ELISA before and after the second immunization (3 weeks) n = 4 error bars indicate standard deviation statistic t-test, ** P ≤ 0.005. (e) Schematic representation of novel protocol for isolation of nanobodies using density gradient separation. The bacterial display library expressing the nanobodies on the surface of bacteria is briefly incubated with conventional sepharose beads coated with the antigen of interest. Directly after the mixture is deposited on a Ficoll gradient conic tube and centrifuged at 200 g for 1 min, the beads drive through the gradient to the bottom of the tube with the bacteria expressing specific nanobodies, while unbound bacteria remain on the surface of the gradient. The beads are then resuspended, and bacterial clones are isolated for biochemical binding confirmation. Illustration (e) by Felipe G. Serrano BSc., MSc Scientific illustrator.

    Techniques Used: Selection, SDS Page, Dot Blot, Negative Control, Enzyme-linked Immunosorbent Assay, Standard Deviation, Isolation, Expressing, Incubation, Clone Assay, Binding Assay

    Dual biochemical and microscopy-based selection of nanobodies. ( a) Dot blot immunodetection of full-length SARS-CoV-2 Spike using direct total protein extracts of clones W25 and W23 as the primary antibody. Mouse anti-Myc (1:3000) followed by anti-mouse-HRP were used for detection. Protein extract from E. coli (BL21 strain) was used as a negative control. Lineal contrast and grey scale were applied to the image, original dot blot scan is shown in the supplemental Fig. 3 a. ( b) Immunodetection of Spike-GFP transiently transfected in HeLa cells using total protein extract selected clones as the primary antibody, followed by mouse anti-Myc (1:3000) and anti-mouse-Alexa 647. The image shows two positive clones (W25 and W23), and an example of a negative Nanobody the screening assay was performed once, scale bar indicates 20 µm. ( c) Sequence alignment of aminoacidic sequence of W25 and W23. CDR sequences are marked with a black line. ( d) Purification from periplasm of bacteria, elution fraction of a single liter of bacterial culture n = 5. ( e) Immunodetection as in (b) , using purified protein n = 3, scale bar indicates 20 µm. ( f) ELISA assay of full-length Spike of SARS-CoV-2 using conjugated W25-HRP nanobody n = 3. ( g) ELISA assay of RBD of Spike using W25-HRP conjugate Nanobody n = 3. Statistic t-test, *** P ≤ 0.001; ** P ≤ 0.005; * P ≤ 0.01 to -W25 control. Illustrations (f,g) by Felipe G. Serrano BSc., MSc Scientific illustrator.
    Figure Legend Snippet: Dual biochemical and microscopy-based selection of nanobodies. ( a) Dot blot immunodetection of full-length SARS-CoV-2 Spike using direct total protein extracts of clones W25 and W23 as the primary antibody. Mouse anti-Myc (1:3000) followed by anti-mouse-HRP were used for detection. Protein extract from E. coli (BL21 strain) was used as a negative control. Lineal contrast and grey scale were applied to the image, original dot blot scan is shown in the supplemental Fig. 3 a. ( b) Immunodetection of Spike-GFP transiently transfected in HeLa cells using total protein extract selected clones as the primary antibody, followed by mouse anti-Myc (1:3000) and anti-mouse-Alexa 647. The image shows two positive clones (W25 and W23), and an example of a negative Nanobody the screening assay was performed once, scale bar indicates 20 µm. ( c) Sequence alignment of aminoacidic sequence of W25 and W23. CDR sequences are marked with a black line. ( d) Purification from periplasm of bacteria, elution fraction of a single liter of bacterial culture n = 5. ( e) Immunodetection as in (b) , using purified protein n = 3, scale bar indicates 20 µm. ( f) ELISA assay of full-length Spike of SARS-CoV-2 using conjugated W25-HRP nanobody n = 3. ( g) ELISA assay of RBD of Spike using W25-HRP conjugate Nanobody n = 3. Statistic t-test, *** P ≤ 0.001; ** P ≤ 0.005; * P ≤ 0.01 to -W25 control. Illustrations (f,g) by Felipe G. Serrano BSc., MSc Scientific illustrator.

    Techniques Used: Microscopy, Selection, Dot Blot, Immunodetection, Clone Assay, Negative Control, Transfection, Screening Assay, Sequencing, Purification, Enzyme-linked Immunosorbent Assay

    17) Product Images from "Exploring beyond clinical routine SARS-CoV-2 serology using MultiCoV-Ab to evaluate endemic coronavirus cross-reactivity"

    Article Title: Exploring beyond clinical routine SARS-CoV-2 serology using MultiCoV-Ab to evaluate endemic coronavirus cross-reactivity

    Journal: Nature Communications

    doi: 10.1038/s41467-021-20973-3

    Combination of 2 spike protein variants and isotype profiling by multiplex assay increases accuracy to identify SARS-CoV-2 antibody-positive individuals. a , b Scatterplot detailing MultiCoV-Ab cut-offs. Signal to cut-off (S/CO) values are displayed for Spike Trimer against RBD on a logarithmic scale. For IgG ( a ), cut-offs are visualized by straight lines and SARS-CoV-2-infected and uninfected samples are separated by color (black circles – SARS-CoV-2-uninfected; red circles – SARS-CoV-2-infected). For IgA ( b ) cut-offs are visualized as dashed lines and S/CO of 2 used for the combined cut-off is shown as straight lines. SARS-CoV-2-infected samples are split into IgG-positives and -negatives by color as indicated in the plot. c , d Scatterplots display IgG response to additional SARS-CoV-2 antigens contained in the MultiCoV-Ab panel: MFI for spike subdomains S1 vs S2 ( c ) or nucleocapsid antigens N vs N-NTD ( d ) are displayed on a logarithmic scale. SARS-CoV-2-uninfected samples are distinguished from SARS-CoV-2-infected and MultiCoV-Ab classification into positives or negatives as indicated by color. Source data are provided as a Source Data file.
    Figure Legend Snippet: Combination of 2 spike protein variants and isotype profiling by multiplex assay increases accuracy to identify SARS-CoV-2 antibody-positive individuals. a , b Scatterplot detailing MultiCoV-Ab cut-offs. Signal to cut-off (S/CO) values are displayed for Spike Trimer against RBD on a logarithmic scale. For IgG ( a ), cut-offs are visualized by straight lines and SARS-CoV-2-infected and uninfected samples are separated by color (black circles – SARS-CoV-2-uninfected; red circles – SARS-CoV-2-infected). For IgA ( b ) cut-offs are visualized as dashed lines and S/CO of 2 used for the combined cut-off is shown as straight lines. SARS-CoV-2-infected samples are split into IgG-positives and -negatives by color as indicated in the plot. c , d Scatterplots display IgG response to additional SARS-CoV-2 antigens contained in the MultiCoV-Ab panel: MFI for spike subdomains S1 vs S2 ( c ) or nucleocapsid antigens N vs N-NTD ( d ) are displayed on a logarithmic scale. SARS-CoV-2-uninfected samples are distinguished from SARS-CoV-2-infected and MultiCoV-Ab classification into positives or negatives as indicated by color. Source data are provided as a Source Data file.

    Techniques Used: Multiplex Assay, Infection

    Correlation of seasonal hCoV and SARS CoV-2 antibody responses. a Correlation of IgG response for the entire sample set ( n = 1176) is visualized as heatmap based on Spearman’s ρ coefficient; dendrogram on the right side displays antigens after hierarchical clustering was performed. b-c, Immune responses (IgG and IgA) towards hCoV S1 ( b ) and N ( c ) proteins are presented as Box-Whisker plots of sample MFI on a logarithmic scale for SARS-CoV-2-infected (red, n = 310) and uninfected (blue, n = 866) individuals. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR of log-transformed data are depicted as circles. d-e, Relative levels of IgG-specific immune response towards hCoV S1 ( d ) and N ( e ) proteins are presented as Box-Whisker plots/strip chart overlays of log-transformed and per-antigen scaled and centered MFI for the sample subsets of Spike Trimer false positives (blue, n = 17) and combined IgG + IgA false negatives (red, n = 31). Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values, excluding outliers as determined by 1.5 times IQR. Source data are provided as a Source Data file.
    Figure Legend Snippet: Correlation of seasonal hCoV and SARS CoV-2 antibody responses. a Correlation of IgG response for the entire sample set ( n = 1176) is visualized as heatmap based on Spearman’s ρ coefficient; dendrogram on the right side displays antigens after hierarchical clustering was performed. b-c, Immune responses (IgG and IgA) towards hCoV S1 ( b ) and N ( c ) proteins are presented as Box-Whisker plots of sample MFI on a logarithmic scale for SARS-CoV-2-infected (red, n = 310) and uninfected (blue, n = 866) individuals. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR of log-transformed data are depicted as circles. d-e, Relative levels of IgG-specific immune response towards hCoV S1 ( d ) and N ( e ) proteins are presented as Box-Whisker plots/strip chart overlays of log-transformed and per-antigen scaled and centered MFI for the sample subsets of Spike Trimer false positives (blue, n = 17) and combined IgG + IgA false negatives (red, n = 31). Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values, excluding outliers as determined by 1.5 times IQR. Source data are provided as a Source Data file.

    Techniques Used: Whisker Assay, Infection, Transformation Assay, Stripping Membranes

    Multiplex-based seroprofiling allows in-depth characterization of SARS-CoV-2 antibody responses. a Kinetic of SARS-CoV-2 antigen-specific IgA and IgG responses is shown for indicated days after symptom onset for six SARS-CoV-2-specific antigens for five different patients. Patients are indicated by color. b , c Samples of SARS-CoV-2-infected individuals were analyzed to identify antigen- and isotype-specific antibody responses based on hospitalization indicating disease severity ( b ) or age ( c ). Data is presented as Box-Whisker plots of sample MFI on a logarithmic scale. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR of log-transformed data are depicted as circles. p -value (Mann–Whitney U test, two-sided) is displayed at the top of the boxes, indicating differences between signal distribution for respective groups. Cut-off values for MultiCoV-Ab classification are displayed as horizontal lines (Spike Trimer IgG: 3,000 MFI, IgA: 400 MFI; RBD IgG: 450 MFI, IgA: 250 MFI). Source data are provided as a Source Data file.
    Figure Legend Snippet: Multiplex-based seroprofiling allows in-depth characterization of SARS-CoV-2 antibody responses. a Kinetic of SARS-CoV-2 antigen-specific IgA and IgG responses is shown for indicated days after symptom onset for six SARS-CoV-2-specific antigens for five different patients. Patients are indicated by color. b , c Samples of SARS-CoV-2-infected individuals were analyzed to identify antigen- and isotype-specific antibody responses based on hospitalization indicating disease severity ( b ) or age ( c ). Data is presented as Box-Whisker plots of sample MFI on a logarithmic scale. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR of log-transformed data are depicted as circles. p -value (Mann–Whitney U test, two-sided) is displayed at the top of the boxes, indicating differences between signal distribution for respective groups. Cut-off values for MultiCoV-Ab classification are displayed as horizontal lines (Spike Trimer IgG: 3,000 MFI, IgA: 400 MFI; RBD IgG: 450 MFI, IgA: 250 MFI). Source data are provided as a Source Data file.

    Techniques Used: Multiplex Assay, Infection, Whisker Assay, Transformation Assay, MANN-WHITNEY

    MultiCoV-Ab, a sensitive and specific tool to monitor SARS-CoV-2 antibody responses. a Control sera (blue, n = 72) and sera from individuals with PCR-confirmed SARS-CoV-2 infection (red, n = 205) were screened in a multiplex bead-based assay using Luminex technology (MultiCoV-Ab) to quantify IgG or IgA responses to various antigens. Reactivity towards trimeric SARS-CoV-2 spike protein (Spike Trimer) or SARS-CoV-2 receptor binding domain of spike (RBD) was found to be the best predictor of SARS-CoV-2 infection. Data are presented as Box-Whisker plots of a sample’s median fluorescence intensity (MFI) on a logarithmic scale. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR of log-transformed data are depicted as circles. Cut-off values for classification for single antigens are displayed as horizontal lines (Spike Trimer IgG: 3,000 MFI, IgA: 400 MFI; RBD IgG: 450 MFI, IgA: 250 MFI). b Sample set from a , was used to compare assay performance of the MultiCoV-Ab using Spike Trimer and RBD antigens with commercially available single analyte SARS-CoV-2 IVD assays which detect total Ig (Elecsys Anti-SARS-CoV-2 (Roche); ADVIA Centaur SARS-CoV-2 Total (COV2T) (Siemens Healthineers)) or IgG (Anti-SARS-CoV-2-ELISA - IgG (Euroimmun)) or IgA (Anti-SARS-CoV-2-ELISA - IgA (Euroimmun)). SARS-CoV-2 infection status of samples based on PCR diagnostic is indicated as SARS-CoV-2 positive or negative. Antibody test results were classified as negative (blue), positive (red), or borderline (gray) as per the manufacturer’s definition. Only samples with divergent antibody test results are shown. c Performance and specifications as stated in the manufacturer’s IVD assay manual. For the manufacturer sensitivity specification, information for samples > 14 days post-infection are presented. Respective sensitivity and specificity values calculated in this study are given with 95% Clopper-Pearson confidence intervals 52 . Positive and negative predictive values (PPV/NPV) were calculated based on a seropositivity of 3%. Source data are provided as a Source Data file.
    Figure Legend Snippet: MultiCoV-Ab, a sensitive and specific tool to monitor SARS-CoV-2 antibody responses. a Control sera (blue, n = 72) and sera from individuals with PCR-confirmed SARS-CoV-2 infection (red, n = 205) were screened in a multiplex bead-based assay using Luminex technology (MultiCoV-Ab) to quantify IgG or IgA responses to various antigens. Reactivity towards trimeric SARS-CoV-2 spike protein (Spike Trimer) or SARS-CoV-2 receptor binding domain of spike (RBD) was found to be the best predictor of SARS-CoV-2 infection. Data are presented as Box-Whisker plots of a sample’s median fluorescence intensity (MFI) on a logarithmic scale. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR of log-transformed data are depicted as circles. Cut-off values for classification for single antigens are displayed as horizontal lines (Spike Trimer IgG: 3,000 MFI, IgA: 400 MFI; RBD IgG: 450 MFI, IgA: 250 MFI). b Sample set from a , was used to compare assay performance of the MultiCoV-Ab using Spike Trimer and RBD antigens with commercially available single analyte SARS-CoV-2 IVD assays which detect total Ig (Elecsys Anti-SARS-CoV-2 (Roche); ADVIA Centaur SARS-CoV-2 Total (COV2T) (Siemens Healthineers)) or IgG (Anti-SARS-CoV-2-ELISA - IgG (Euroimmun)) or IgA (Anti-SARS-CoV-2-ELISA - IgA (Euroimmun)). SARS-CoV-2 infection status of samples based on PCR diagnostic is indicated as SARS-CoV-2 positive or negative. Antibody test results were classified as negative (blue), positive (red), or borderline (gray) as per the manufacturer’s definition. Only samples with divergent antibody test results are shown. c Performance and specifications as stated in the manufacturer’s IVD assay manual. For the manufacturer sensitivity specification, information for samples > 14 days post-infection are presented. Respective sensitivity and specificity values calculated in this study are given with 95% Clopper-Pearson confidence intervals 52 . Positive and negative predictive values (PPV/NPV) were calculated based on a seropositivity of 3%. Source data are provided as a Source Data file.

    Techniques Used: Polymerase Chain Reaction, Infection, Multiplex Assay, Bead-based Assay, Luminex, Binding Assay, Whisker Assay, Fluorescence, Transformation Assay, Enzyme-linked Immunosorbent Assay, Diagnostic Assay

    Analysis of seasonal hCoV high and low responders. a From the entire study population, groups of α- or β-hCoV high and low responders were built as indicated. High responder were defined as samples with above average MFI values for S1 and N-specific IgGs of the respective hCoV clade. Low responders were defined with below MFI values, correspondingly. Responder groups (i) α-hCoV ↑, red, n = 233, (ii) β-hCoV ↑, green, n = 254, (iii) α-hCoV ↓, blue, n = 172 (iv) β-hCoV ↓, purple, n = 210 are shown as Box-Whisker plots of log-transformed and per-antigen scaled and centered MFI values across hCoV N and S1 antigens. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR are depicted as circles. b The over- or under-representation of SARS-CoV-2 responders (SARS-CoV-2 + , n = 279, as determined by positive MultiCoV-Ab classification) within the four sample groups is visualized in Venn diagrams, stochastic significance was calculated using Fisher’s exact test (two-sided). Source data are provided as a Source Data file.
    Figure Legend Snippet: Analysis of seasonal hCoV high and low responders. a From the entire study population, groups of α- or β-hCoV high and low responders were built as indicated. High responder were defined as samples with above average MFI values for S1 and N-specific IgGs of the respective hCoV clade. Low responders were defined with below MFI values, correspondingly. Responder groups (i) α-hCoV ↑, red, n = 233, (ii) β-hCoV ↑, green, n = 254, (iii) α-hCoV ↓, blue, n = 172 (iv) β-hCoV ↓, purple, n = 210 are shown as Box-Whisker plots of log-transformed and per-antigen scaled and centered MFI values across hCoV N and S1 antigens. Box represents the median and the 25th and 75th percentiles, whiskers show the largest and smallest values. Outliers determined by 1.5 times IQR are depicted as circles. b The over- or under-representation of SARS-CoV-2 responders (SARS-CoV-2 + , n = 279, as determined by positive MultiCoV-Ab classification) within the four sample groups is visualized in Venn diagrams, stochastic significance was calculated using Fisher’s exact test (two-sided). Source data are provided as a Source Data file.

    Techniques Used: Whisker Assay, Transformation Assay

    18) Product Images from "SARS-CoV-2 spike protein promotes IL-6 trans-signaling by activation of angiotensin II receptor signaling in epithelial cells"

    Article Title: SARS-CoV-2 spike protein promotes IL-6 trans-signaling by activation of angiotensin II receptor signaling in epithelial cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1009128

    SARS-CoV-2 spike protein induces alarmin secretion from epithelial cells. (A, B) The extra-cellular level of IL-1α and HMGB1 were measured by ELISA from culture supernatant of Huh7.5 and A549 cells after transfection of SARS-CoV-2 spike gene construct or empty vector as a control for comparison. The results are presented as mean ± standard deviation. ‘*’ ( p
    Figure Legend Snippet: SARS-CoV-2 spike protein induces alarmin secretion from epithelial cells. (A, B) The extra-cellular level of IL-1α and HMGB1 were measured by ELISA from culture supernatant of Huh7.5 and A549 cells after transfection of SARS-CoV-2 spike gene construct or empty vector as a control for comparison. The results are presented as mean ± standard deviation. ‘*’ ( p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Transfection, Construct, Plasmid Preparation, Standard Deviation

    SARS-CoV-2 spike protein stimulates IL-6 and soluble IL-6R production. (A) The extra-cellular level of IL-6 was measured by ELISA from culture supernatant of Huh7.5 and A549 cells after transfection of SARS-CoV-2 spike gene construct with or without Candesartan cilexetil treatment. (B) The extra-cellular level of soluble IL-6R was similarly measured by ELISA from culture supernatant of Huh7.5 and A549 cells after transfection of SARS-CoV-2 spike gene construct with or without Candesartan cilexetil treatment as shown in panel A. The results are presented as means ± standard deviations. ‘*’ and ‘**’ represent statistical significance p
    Figure Legend Snippet: SARS-CoV-2 spike protein stimulates IL-6 and soluble IL-6R production. (A) The extra-cellular level of IL-6 was measured by ELISA from culture supernatant of Huh7.5 and A549 cells after transfection of SARS-CoV-2 spike gene construct with or without Candesartan cilexetil treatment. (B) The extra-cellular level of soluble IL-6R was similarly measured by ELISA from culture supernatant of Huh7.5 and A549 cells after transfection of SARS-CoV-2 spike gene construct with or without Candesartan cilexetil treatment as shown in panel A. The results are presented as means ± standard deviations. ‘*’ and ‘**’ represent statistical significance p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Transfection, Construct

    SARS-CoV-2 spike protein regulates ADAM-17 activity. (A) ADAM-17 enzymatic activity was measured from crude extracts of Huh7.5 and A549 cells after transfection of SARS-CoV-2 spike gene constructs. (B) ADAM-17 enzymatic activity was measured from crude extracts of Huh7.5 and A549 cells after transfection of SARS-CoV-2 spike S1 or S2 gene construct or empty vector. The results are presented as mean ± standard deviation. ‘*’ represent statistical significance ( p
    Figure Legend Snippet: SARS-CoV-2 spike protein regulates ADAM-17 activity. (A) ADAM-17 enzymatic activity was measured from crude extracts of Huh7.5 and A549 cells after transfection of SARS-CoV-2 spike gene constructs. (B) ADAM-17 enzymatic activity was measured from crude extracts of Huh7.5 and A549 cells after transfection of SARS-CoV-2 spike S1 or S2 gene construct or empty vector. The results are presented as mean ± standard deviation. ‘*’ represent statistical significance ( p

    Techniques Used: Activity Assay, Transfection, Construct, Plasmid Preparation, Standard Deviation

    Candesartan cilexetil as an AT1 receptor antagonist prevent MAPK activation. (A) Western blot analysis of phospho-p38 MAPK (Thr180/Tyr182) and phospho-p42/44 MAPK (Thr202/Tyr204) in Huh7.5 cell lysates prepared after 48 h of transfection of SARS-CoV-2 spike gene construct with or without Candesartan cilexetil treatment. Expression level of actin in each lane is shown as a total protein loading control for comparison. (B) Western blot analysis of phospho-p38 MAPK (Thr180/Tyr182) and phospho-p42/44 MAPK (Thr202/Tyr204) in A549 cell lysates prepared after 48 h of transfection of SARS-CoV-2 spike gene construct with or without Candesartan cilexetil treatment. Expression level of actin or tubulin in each lane from the same gel is shown as a total protein load for comparison.
    Figure Legend Snippet: Candesartan cilexetil as an AT1 receptor antagonist prevent MAPK activation. (A) Western blot analysis of phospho-p38 MAPK (Thr180/Tyr182) and phospho-p42/44 MAPK (Thr202/Tyr204) in Huh7.5 cell lysates prepared after 48 h of transfection of SARS-CoV-2 spike gene construct with or without Candesartan cilexetil treatment. Expression level of actin in each lane is shown as a total protein loading control for comparison. (B) Western blot analysis of phospho-p38 MAPK (Thr180/Tyr182) and phospho-p42/44 MAPK (Thr202/Tyr204) in A549 cell lysates prepared after 48 h of transfection of SARS-CoV-2 spike gene construct with or without Candesartan cilexetil treatment. Expression level of actin or tubulin in each lane from the same gel is shown as a total protein load for comparison.

    Techniques Used: Activation Assay, Western Blot, Transfection, Construct, Expressing

    SARS-CoV-2 spike protein inhibits ACE2 expression. (A, B) Western blot analysis of SARS-CoV-2 spike protein expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock- or SARS-CoV-2 virus infection, or transiently transfected with an empty vector or SARS-CoV-2 spike gene construct. (C, D) Western blot analysis of ACE2 and AT1 receptor expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock- or SARS-CoV-2 virus infection, or transiently transfected with empty vector or SARS-CoV-2 spike gene construct. (E, F) Western blot analysis of ACE2 expression in Huh7.5 and A549 cell lysates prepared after 48 h of transfection of empty vector or SARS-CoV-2 spike S1 or S2 gene construct. Expression level of actin or tubulin in each lane from the same gel is shown as a total protein load for comparison.
    Figure Legend Snippet: SARS-CoV-2 spike protein inhibits ACE2 expression. (A, B) Western blot analysis of SARS-CoV-2 spike protein expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock- or SARS-CoV-2 virus infection, or transiently transfected with an empty vector or SARS-CoV-2 spike gene construct. (C, D) Western blot analysis of ACE2 and AT1 receptor expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock- or SARS-CoV-2 virus infection, or transiently transfected with empty vector or SARS-CoV-2 spike gene construct. (E, F) Western blot analysis of ACE2 expression in Huh7.5 and A549 cell lysates prepared after 48 h of transfection of empty vector or SARS-CoV-2 spike S1 or S2 gene construct. Expression level of actin or tubulin in each lane from the same gel is shown as a total protein load for comparison.

    Techniques Used: Expressing, Western Blot, Infection, Transfection, Plasmid Preparation, Construct

    SARS-CoV-2 spike protein activates the transcription factors for IL-6 synthesis. (A, B) Western blot analysis of phospho-p38 MAPK (Thr180/Tyr182) and phospho-p42/44 MAPK (Thr202/Tyr204) in Huh7.5 and A649 cell lysates prepared after 48 h of mock- or SARS-CoV-2 virus infection, or transiently transfected with empty vector or SARS-CoV-2 spike gene construct. (C, D) Western blot analysis of phospho-NF-κB (Ser276), IκBα and c-Fos expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock- or SARS-CoV-2 virus infection, or transiently transfected with empty vector or SARS-CoV-2 spike gene construct. Expression level of actin or tubulin in each lane from the same gel is shown as a total protein load for comparison.
    Figure Legend Snippet: SARS-CoV-2 spike protein activates the transcription factors for IL-6 synthesis. (A, B) Western blot analysis of phospho-p38 MAPK (Thr180/Tyr182) and phospho-p42/44 MAPK (Thr202/Tyr204) in Huh7.5 and A649 cell lysates prepared after 48 h of mock- or SARS-CoV-2 virus infection, or transiently transfected with empty vector or SARS-CoV-2 spike gene construct. (C, D) Western blot analysis of phospho-NF-κB (Ser276), IκBα and c-Fos expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock- or SARS-CoV-2 virus infection, or transiently transfected with empty vector or SARS-CoV-2 spike gene construct. Expression level of actin or tubulin in each lane from the same gel is shown as a total protein load for comparison.

    Techniques Used: Western Blot, Infection, Transfection, Plasmid Preparation, Construct, Expressing

    SARS-CoV-2 spike protein causes inhibition of tyrosine phosphorylation of STAT3. (A, B) Western blot analysis of phospho-STAT3 (Tyr705) and total STAT3 expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock-treated or transient transfection of SARS-CoV-2 spike gene constructs or infection with SARS-CoV-2 virus. (C, D) Western blot analysis of phospho-STAT3 (Ser727) and total STAT3 expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock-treated or transiently transfection of SARS-CoV-2 spike gene construct or infection with SARS-CoV-2 virus. (E, F) Western blot analysis of SOCS3 expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock-treated or transiently transfected with SARS-CoV-2 spike constructs or infected with SARS-CoV-2 virus. Expression level of actin in each lane from the same gel is shown as a total protein loading control for comparison.
    Figure Legend Snippet: SARS-CoV-2 spike protein causes inhibition of tyrosine phosphorylation of STAT3. (A, B) Western blot analysis of phospho-STAT3 (Tyr705) and total STAT3 expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock-treated or transient transfection of SARS-CoV-2 spike gene constructs or infection with SARS-CoV-2 virus. (C, D) Western blot analysis of phospho-STAT3 (Ser727) and total STAT3 expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock-treated or transiently transfection of SARS-CoV-2 spike gene construct or infection with SARS-CoV-2 virus. (E, F) Western blot analysis of SOCS3 expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock-treated or transiently transfected with SARS-CoV-2 spike constructs or infected with SARS-CoV-2 virus. Expression level of actin in each lane from the same gel is shown as a total protein loading control for comparison.

    Techniques Used: Inhibition, Western Blot, Expressing, Transfection, Construct, Infection

    (A, B) Overview of IL-6 mediated classical and trans-signaling mechanisms. (C) Schematic presentation of molecular changes occurring upon SARS-CoV-2 infection or spike protein expression in epithelial cells.
    Figure Legend Snippet: (A, B) Overview of IL-6 mediated classical and trans-signaling mechanisms. (C) Schematic presentation of molecular changes occurring upon SARS-CoV-2 infection or spike protein expression in epithelial cells.

    Techniques Used: Infection, Expressing

    IL-6 trans-signaling induces MCP-1 expression. (A, B) Western blot analysis of MCP-1 expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock-treated or infected with SARS-CoV-2 virus. (C) Western blot analysis for IL-6Rα expression in Huh7.5, A549, or TMNK-1 cell lysates. (D) Western blot analysis of phospho-STAT3 (Tyr705) and MCP-1 expression in TMNK-1 liver endothelial cell lysates prepared after treated with or without culture supernatant from SARS-CoV-2 spike gene expressed A549 cells in presence or absence of Candesartan cilexetil and LPS. (E) Western blot analysis for phospho-STAT3 (Tyr705) and MCP-1 expression status in TMNK-1 liver endothelial cell lysates prepared after treatment with LPS and culture supernatant from SARS-CoV-2 spike gene expressed A549 cells in the presence or absence of Tocilizumab. Expression level of actin in each lane from the same gel is shown as a total protein load for comparison. (F) The extra-cellular level of MCP-1 was measured by ELISA in culture supernatant of TMNK-1 cells after treatment with LPS alone or together with culture supernatant from SARS-CoV-2 spike gene expressing A549 cells in the presence or absence of Tocilizumab. The MCP-1 expression level in the culture supernatant from SARS-CoV-2 spike gene expressing A549 cells was also detected. (G) Comparative analysis of cellular migration of human monocytes (THP-1) in the presence of culture supernatant from TMNK-1 cells treated with LPS, and from culture supernatant of SARS-CoV-2 spike gene expressed A549 cells in the presence or absence of Tocilizumab. The results are presented as mean ± standard deviation. ‘*’ ( p
    Figure Legend Snippet: IL-6 trans-signaling induces MCP-1 expression. (A, B) Western blot analysis of MCP-1 expression in Huh7.5 and A549 cell lysates prepared after 48 h of mock-treated or infected with SARS-CoV-2 virus. (C) Western blot analysis for IL-6Rα expression in Huh7.5, A549, or TMNK-1 cell lysates. (D) Western blot analysis of phospho-STAT3 (Tyr705) and MCP-1 expression in TMNK-1 liver endothelial cell lysates prepared after treated with or without culture supernatant from SARS-CoV-2 spike gene expressed A549 cells in presence or absence of Candesartan cilexetil and LPS. (E) Western blot analysis for phospho-STAT3 (Tyr705) and MCP-1 expression status in TMNK-1 liver endothelial cell lysates prepared after treatment with LPS and culture supernatant from SARS-CoV-2 spike gene expressed A549 cells in the presence or absence of Tocilizumab. Expression level of actin in each lane from the same gel is shown as a total protein load for comparison. (F) The extra-cellular level of MCP-1 was measured by ELISA in culture supernatant of TMNK-1 cells after treatment with LPS alone or together with culture supernatant from SARS-CoV-2 spike gene expressing A549 cells in the presence or absence of Tocilizumab. The MCP-1 expression level in the culture supernatant from SARS-CoV-2 spike gene expressing A549 cells was also detected. (G) Comparative analysis of cellular migration of human monocytes (THP-1) in the presence of culture supernatant from TMNK-1 cells treated with LPS, and from culture supernatant of SARS-CoV-2 spike gene expressed A549 cells in the presence or absence of Tocilizumab. The results are presented as mean ± standard deviation. ‘*’ ( p

    Techniques Used: Expressing, Western Blot, Infection, Enzyme-linked Immunosorbent Assay, Migration, Standard Deviation

    19) Product Images from "SARS-CoV-2 causes severe alveolar inflammation and barrier dysfunction"

    Article Title: SARS-CoV-2 causes severe alveolar inflammation and barrier dysfunction

    Journal: bioRxiv

    doi: 10.1101/2020.08.31.276725

    SARS-CoV-2 infection results in induction of antiviral and proinflammatory mRNA synthesis. Calu-3 cells were left uninfected (mock) or were infected with a SARS-CoV-2 patient isolate (5159, 5587, 5588) (MOI=1). RNA-lysates were performed 24h p.i. Levels of IFNα, IFNβ, IFNλ1, IFNλ2,3, IL6, IL8, IP10, TNFα, cIAP2, TRAIL, and RIPK1 Mrna were measured of three patient isolate (5159, 5587, 5588) and two technical samples in 3 independent experiments. Means ± SD of three independent experiments are shown. Levels of mock-treated samples were arbitrarily set as 1. After normalization, two-tailed unpaired t-tests were performed for comparison of mock-treated and SARS-CoV-2-infected and samples. (*p
    Figure Legend Snippet: SARS-CoV-2 infection results in induction of antiviral and proinflammatory mRNA synthesis. Calu-3 cells were left uninfected (mock) or were infected with a SARS-CoV-2 patient isolate (5159, 5587, 5588) (MOI=1). RNA-lysates were performed 24h p.i. Levels of IFNα, IFNβ, IFNλ1, IFNλ2,3, IL6, IL8, IP10, TNFα, cIAP2, TRAIL, and RIPK1 Mrna were measured of three patient isolate (5159, 5587, 5588) and two technical samples in 3 independent experiments. Means ± SD of three independent experiments are shown. Levels of mock-treated samples were arbitrarily set as 1. After normalization, two-tailed unpaired t-tests were performed for comparison of mock-treated and SARS-CoV-2-infected and samples. (*p

    Techniques Used: Infection, Two Tailed Test

    Infection with SARS-CoV-2 results in the disruption of the epithelial- and endothelial barrier. The epithelial side of the alveolus-on-a-chip model was left uninfected (mock) or infected with three different SARS-CoV-2 patient isolates (5159, 5587, 5588) (MOI=1). Immunofluorescence staining was performed 40h p.i., (A) The E-cadherin of the epithelial layer and the (B) VE-cadherin of the endothelial layer were visualized by an anti-E-Cadherin-specific antibody or an anti-VE-Cadherin antiserum, respectively, and a Cy5 goat anti-rabbit IgG (red). (A, B) The SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Scale bars represent 100 μm.
    Figure Legend Snippet: Infection with SARS-CoV-2 results in the disruption of the epithelial- and endothelial barrier. The epithelial side of the alveolus-on-a-chip model was left uninfected (mock) or infected with three different SARS-CoV-2 patient isolates (5159, 5587, 5588) (MOI=1). Immunofluorescence staining was performed 40h p.i., (A) The E-cadherin of the epithelial layer and the (B) VE-cadherin of the endothelial layer were visualized by an anti-E-Cadherin-specific antibody or an anti-VE-Cadherin antiserum, respectively, and a Cy5 goat anti-rabbit IgG (red). (A, B) The SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Scale bars represent 100 μm.

    Techniques Used: Infection, Chromatin Immunoprecipitation, Immunofluorescence, Staining

    Phylogenetic tree of SARS-CoV-2. (A) Phylogenetic analysis revealed a close relationship of SARS-CoV-2 to the SARS-related coronaviruses RaTG13, bat-SL-CoVZXC21 and bat-SL-CoVZC45. Sequences of strains 5587 and 5588 exhibit two base substitutions T8,782C ( nsp1ab: synonymous) and C28,144T ( nsp8: S84L). (B) Accordingly, 5587 and 5588 clustered with lineage L/lineage B strains in the phylogenetic analysis. Both strains exhibit deletion of nsp1ab D448 and two synonymous substitutions (T514C, C5512T). Beside the nsp8 S84L substitution, strain 5159 has accumulated three additional amino acid substitutions ( S: D614G, nsp1ab: P4715L and N: R203K/G204R) which place this virus in lineage B.1.1.
    Figure Legend Snippet: Phylogenetic tree of SARS-CoV-2. (A) Phylogenetic analysis revealed a close relationship of SARS-CoV-2 to the SARS-related coronaviruses RaTG13, bat-SL-CoVZXC21 and bat-SL-CoVZC45. Sequences of strains 5587 and 5588 exhibit two base substitutions T8,782C ( nsp1ab: synonymous) and C28,144T ( nsp8: S84L). (B) Accordingly, 5587 and 5588 clustered with lineage L/lineage B strains in the phylogenetic analysis. Both strains exhibit deletion of nsp1ab D448 and two synonymous substitutions (T514C, C5512T). Beside the nsp8 S84L substitution, strain 5159 has accumulated three additional amino acid substitutions ( S: D614G, nsp1ab: P4715L and N: R203K/G204R) which place this virus in lineage B.1.1.

    Techniques Used:

    SARS-CoV-2 infects epithelial cells productively. (A) Vero-76, Calu-3, and HUVECs were infected with a SARS-CoV-2 patient isolate (5159, 5587, 5588) (MOI=1). RNA-lysates were performed 24h p.i. and copies of viral RNA (E-gene) were determined by r-biopharm qRT-PCR. Means ± SD of three independent experiments are shown. (B) HUVECs were infected with a SARS-CoV-2 patient isolate (5159) (MOI=1) for 4h, 8h, and 24h. SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Immunofluorescence (IF) microscopy was acquired by use of the Axio Observer.Z1 (Zeiss) with a 200×magnification.
    Figure Legend Snippet: SARS-CoV-2 infects epithelial cells productively. (A) Vero-76, Calu-3, and HUVECs were infected with a SARS-CoV-2 patient isolate (5159, 5587, 5588) (MOI=1). RNA-lysates were performed 24h p.i. and copies of viral RNA (E-gene) were determined by r-biopharm qRT-PCR. Means ± SD of three independent experiments are shown. (B) HUVECs were infected with a SARS-CoV-2 patient isolate (5159) (MOI=1) for 4h, 8h, and 24h. SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Immunofluorescence (IF) microscopy was acquired by use of the Axio Observer.Z1 (Zeiss) with a 200×magnification.

    Techniques Used: Infection, Quantitative RT-PCR, Staining, Immunofluorescence, Microscopy

    SARS-CoV-2 efficiently infects epithelial cells of the human-alveolus-on-a chip model and provokes type I and III interferon production. (A-C) The epithelial chamber of the alveolus-on-a-chip model was left uninfected (mock) or infected with three different SARS-CoV-2 patient isolates (5159, 5587, 5588) (MOI=1). (A, B) Immunofluorescence staining was performed 28h p.i. and analyzed by immunofluorescence microscopy (Axio Observer.Z1 (Zeiss)). (A) The E-cadherin of the epithelial layer and the (B) VE-cadherin of the endothelial layer were visualized by an anti-E-Cadherin-specific antibody or an anti-VE-Cadherin antiserum, respectively, and a Cy5 goat anti-rabbit IgG (red). (A, B) The SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Scale bars represent 100 μm. (C) Production of antiviral cytokines derived from the epithelial side was determined by use of Legendplex Panel (Biolegend, CA, USA). SARS-CoV-2 induced IFNβ, IFNλ1 and IFNλ2,3 release (pg/ml) was measured. Means ± SD of three independent experiments each infected with another patient isolate (5159, 5587, 5588) are shown. Levels of mock-treated samples were arbitrarily set as 1. After normalization, two-tailed unpaired t-tests were performed for comparison of mock-treated and SARS-CoV-2-infected and samples. (**p
    Figure Legend Snippet: SARS-CoV-2 efficiently infects epithelial cells of the human-alveolus-on-a chip model and provokes type I and III interferon production. (A-C) The epithelial chamber of the alveolus-on-a-chip model was left uninfected (mock) or infected with three different SARS-CoV-2 patient isolates (5159, 5587, 5588) (MOI=1). (A, B) Immunofluorescence staining was performed 28h p.i. and analyzed by immunofluorescence microscopy (Axio Observer.Z1 (Zeiss)). (A) The E-cadherin of the epithelial layer and the (B) VE-cadherin of the endothelial layer were visualized by an anti-E-Cadherin-specific antibody or an anti-VE-Cadherin antiserum, respectively, and a Cy5 goat anti-rabbit IgG (red). (A, B) The SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Scale bars represent 100 μm. (C) Production of antiviral cytokines derived from the epithelial side was determined by use of Legendplex Panel (Biolegend, CA, USA). SARS-CoV-2 induced IFNβ, IFNλ1 and IFNλ2,3 release (pg/ml) was measured. Means ± SD of three independent experiments each infected with another patient isolate (5159, 5587, 5588) are shown. Levels of mock-treated samples were arbitrarily set as 1. After normalization, two-tailed unpaired t-tests were performed for comparison of mock-treated and SARS-CoV-2-infected and samples. (**p

    Techniques Used: Chromatin Immunoprecipitation, Infection, Immunofluorescence, Staining, Microscopy, Derivative Assay, Two Tailed Test

    SARS-CoV-2 infection results in the disruption of barrier integrity in the human-alveolus-on-a chip model. The epithelial side of the alveolus-on-a-chip model was left uninfected (mock) or infected with the SARS-CoV-2 patient isolate (5159) (MOI=1) for 28h. An overview (upper panel) of the (A) epithelial layer and (B) endothelial layer are depicted. Dead cells (middle panel) are focused. The surface of dead cells (lower panel) shows particles (arrows) attached to the plasma membranes of the epithelial cells only. Scale bars represent 50 μm (200×magnification), 5 μm (2.000×magnification) and 200 nm (60.000×magnification). (C) Barrier function of the human alveolus-on-a-chip model was analyzed by a permeability assay of mock-infected and SARS-CoV-2-infected human alveolus-on-a-chip model using FITC-dextran at 28h p.i., FITC-dextran was measured via the fluorescence intensity (exc. 488nm; em. 518 nm) and depicted as the permeability coefficient ( P app ), calculated according to P app (cm s -1 ) = (dQ/d t ) (1/AC o ). Results show significant higher barrier permeability after SARS-CoV-2 infection. (D) Supernatants of the epithelial- and endothelial side of SARS-CoV-2 infected human alveolus-on-a-chip models were used to perform LDH-assays indicating cell membrane rupture at 28h and 40h p.i.. (E) Progeny virus titers were analyzed in the supernatants of the epithelial- and endothelial layer by standard plaque assay. Shown are means (±SD) of (C) three independent experiments each infected with another patient isolate (5159, 5587, 5588), (D) LDH release, and (E) plaque forming units (PFU/ml). Statistical significance was analyzed by unpaired, two-tailed t-test (*p
    Figure Legend Snippet: SARS-CoV-2 infection results in the disruption of barrier integrity in the human-alveolus-on-a chip model. The epithelial side of the alveolus-on-a-chip model was left uninfected (mock) or infected with the SARS-CoV-2 patient isolate (5159) (MOI=1) for 28h. An overview (upper panel) of the (A) epithelial layer and (B) endothelial layer are depicted. Dead cells (middle panel) are focused. The surface of dead cells (lower panel) shows particles (arrows) attached to the plasma membranes of the epithelial cells only. Scale bars represent 50 μm (200×magnification), 5 μm (2.000×magnification) and 200 nm (60.000×magnification). (C) Barrier function of the human alveolus-on-a-chip model was analyzed by a permeability assay of mock-infected and SARS-CoV-2-infected human alveolus-on-a-chip model using FITC-dextran at 28h p.i., FITC-dextran was measured via the fluorescence intensity (exc. 488nm; em. 518 nm) and depicted as the permeability coefficient ( P app ), calculated according to P app (cm s -1 ) = (dQ/d t ) (1/AC o ). Results show significant higher barrier permeability after SARS-CoV-2 infection. (D) Supernatants of the epithelial- and endothelial side of SARS-CoV-2 infected human alveolus-on-a-chip models were used to perform LDH-assays indicating cell membrane rupture at 28h and 40h p.i.. (E) Progeny virus titers were analyzed in the supernatants of the epithelial- and endothelial layer by standard plaque assay. Shown are means (±SD) of (C) three independent experiments each infected with another patient isolate (5159, 5587, 5588), (D) LDH release, and (E) plaque forming units (PFU/ml). Statistical significance was analyzed by unpaired, two-tailed t-test (*p

    Techniques Used: Infection, Chromatin Immunoprecipitation, Permeability, Fluorescence, Plaque Assay, Two Tailed Test

    SARS-CoV-2 replicates in Vero-76 and Calu-3 cells. Vero-76 (A-D) and Calu-3 (B-D) cells were left uninfected (mock) (B-D) or were infected (A-D) with a SARS-CoV-2 patient isolate (5159) (MOI=1). (A) Transmission electron microscopy was performed 24h post infection (p.i.): (upper panel, scale bar: 5 μm) overview of 3 SARS-CoV-2-infected Vero-76 cells; (lower left panel, scale bar: 200 nm) generation of double membrane vesicles; (lower middle panel, scale bar: 200 nm) virion assembly in the ER–Golgi-intermediate compartment (ERGIC); (lower right panel, scale bar: 200 nm) viral release. (B) SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Immunofluorescence (IF) microscopy was acquired by use of the Axio Observer.Z1 (Zeiss) with a 200×magnification. (C) Total cell lysates were harvested at the times indicated and expression of the spike protein was analyzed by western-blot assay. ERK2 served as loading control. (D) Progeny virus particles were measured in the supernatant by standard plaque assay at the indicated times post infection. Shown are means (±SD) of plaque forming units (PFU) ml -1 of three independent experiments including two biological samples. Statistical significance was analyzed by unpaired, two-tailed t-test (***p
    Figure Legend Snippet: SARS-CoV-2 replicates in Vero-76 and Calu-3 cells. Vero-76 (A-D) and Calu-3 (B-D) cells were left uninfected (mock) (B-D) or were infected (A-D) with a SARS-CoV-2 patient isolate (5159) (MOI=1). (A) Transmission electron microscopy was performed 24h post infection (p.i.): (upper panel, scale bar: 5 μm) overview of 3 SARS-CoV-2-infected Vero-76 cells; (lower left panel, scale bar: 200 nm) generation of double membrane vesicles; (lower middle panel, scale bar: 200 nm) virion assembly in the ER–Golgi-intermediate compartment (ERGIC); (lower right panel, scale bar: 200 nm) viral release. (B) SARS-CoV-2 was visualized by detection of the spike protein via a spike-specific antibody and an Alexa Fluor™ 488-conjugated goat anti-mouse IgG (green). The nuclei were stained with Hoechst 33342 (blue). Immunofluorescence (IF) microscopy was acquired by use of the Axio Observer.Z1 (Zeiss) with a 200×magnification. (C) Total cell lysates were harvested at the times indicated and expression of the spike protein was analyzed by western-blot assay. ERK2 served as loading control. (D) Progeny virus particles were measured in the supernatant by standard plaque assay at the indicated times post infection. Shown are means (±SD) of plaque forming units (PFU) ml -1 of three independent experiments including two biological samples. Statistical significance was analyzed by unpaired, two-tailed t-test (***p

    Techniques Used: Infection, Transmission Assay, Electron Microscopy, Staining, Immunofluorescence, Microscopy, Expressing, Western Blot, Plaque Assay, Two Tailed Test

    20) Product Images from "Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies"

    Article Title: Identification of four linear B-cell epitopes on the SARS-CoV-2 spike protein able to elicit neutralizing antibodies

    Journal: bioRxiv

    doi: 10.1101/2020.12.13.422550

    The predicted linear B-cell epitopes in the Spike protein of SARS-CoV-2. a , The number of linear B-cell epitopes shared among the distinct methods and literature mining. The pink, green and light blue represent epitopes with antigenicity scores > 0.9, 0.4 and 0.9, and
    Figure Legend Snippet: The predicted linear B-cell epitopes in the Spike protein of SARS-CoV-2. a , The number of linear B-cell epitopes shared among the distinct methods and literature mining. The pink, green and light blue represent epitopes with antigenicity scores > 0.9, 0.4 and 0.9, and

    Techniques Used:

    Measurements of the selected Linear B cell epitope binding to antibody and neutralization efficiency of selected epitopes against SARS-CoV-2. a-d, The binding affinity assessed by ELISA between linear B-cell epitopes and serum antibodies from immunized horse with S1-based vaccines (a), immunized mouse with RBD-based vaccines (b), immunized monkey with RBD-based vaccines (c), and a patient recovering from COVID-19 (d). e, The binding affinity assessed by ELISA between the linear B-cell epitopes and serum antibodies from immunized mice with corresponding epitopes of ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. f, Neutralization assay against SARS-CoV-2 pseudovirus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of EC 50 . g, Neutralization assay against SARS-CoV-2 live virus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of NT 50 .
    Figure Legend Snippet: Measurements of the selected Linear B cell epitope binding to antibody and neutralization efficiency of selected epitopes against SARS-CoV-2. a-d, The binding affinity assessed by ELISA between linear B-cell epitopes and serum antibodies from immunized horse with S1-based vaccines (a), immunized mouse with RBD-based vaccines (b), immunized monkey with RBD-based vaccines (c), and a patient recovering from COVID-19 (d). e, The binding affinity assessed by ELISA between the linear B-cell epitopes and serum antibodies from immunized mice with corresponding epitopes of ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. f, Neutralization assay against SARS-CoV-2 pseudovirus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of EC 50 . g, Neutralization assay against SARS-CoV-2 live virus in ‘YNSASFSTFKCYGVSPTKLNDLCFT’, ‘GDEVRQIAPGQTGKIADYNYKLP’, ‘YQPYRVVVLSFELLH’, and ‘CVNFNFNGL’. y-axis is the value of NT 50 .

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

    The characteristics of the 18 selected linear B cell epitopes. a , The sequences of 18 selected linear B-cell epitopes. The bold is the mutated site in less than ten of 118,694 virus strains; The red is the predicted discontinuous residues. The bars on the right side are the Wilcoxon test p value for the comparisons of IgG or IgA antibody enrichment scores associated with each linear B-cell epitope between COVID-19 patients and negative controls. b , The digesting enzymes profile of the epitope sequence. Red indicated not digest, blue indicated digest. c-d , The localization of the 18 selected epitopes mapped on SARS-CoV-2 S (PDB: 6VSB) protein (c) and ACE-RBD complex (d). e-f , The localizations of B cell discontinuous epitopes on SARS-CoV-2 S (PDB: 6VSB) protein (e) and ACE-RBD complex (f). The spike protein is grey, the RBD region is wheat color, the selected epitopes are green, the mutation sites are red, the human ACE domain is blue, and the discontinuous B-cell epitopes are purple.
    Figure Legend Snippet: The characteristics of the 18 selected linear B cell epitopes. a , The sequences of 18 selected linear B-cell epitopes. The bold is the mutated site in less than ten of 118,694 virus strains; The red is the predicted discontinuous residues. The bars on the right side are the Wilcoxon test p value for the comparisons of IgG or IgA antibody enrichment scores associated with each linear B-cell epitope between COVID-19 patients and negative controls. b , The digesting enzymes profile of the epitope sequence. Red indicated not digest, blue indicated digest. c-d , The localization of the 18 selected epitopes mapped on SARS-CoV-2 S (PDB: 6VSB) protein (c) and ACE-RBD complex (d). e-f , The localizations of B cell discontinuous epitopes on SARS-CoV-2 S (PDB: 6VSB) protein (e) and ACE-RBD complex (f). The spike protein is grey, the RBD region is wheat color, the selected epitopes are green, the mutation sites are red, the human ACE domain is blue, and the discontinuous B-cell epitopes are purple.

    Techniques Used: Sequencing, Mutagenesis

    21) Product Images from "Neutralizing Human Antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 Isolated from a Human Synthetic Fab Phage Display Library"

    Article Title: Neutralizing Human Antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 Isolated from a Human Synthetic Fab Phage Display Library

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22041913

    Characterization of human anti-SARS-2 RBD Fabs. ( a ) Soluble ELISA of ten serially diluted human anti-SARS-2 RBD Fabs on immobilized SARS-2 RBD surfaces to measure their apparent affinities ( EC 50 , nM). ( b ) Schematic drawings of a competitive ELISA of human anti-SARS-2 RBD Fabs between the SARS-2 RBD and ACE2 protein (left) or ACE2-overexpressed cells (right). ( c ) Competitive ELISA of human anti-SARS-2 RBD Fabs antagonizing the interaction between ACE2 and the SARS-CoV-2 RBD. ( d ) Competitive flow cytometry analysis of human anti-SARS-2 RBD Fabs antagonizing the interaction between ACE2 on cells and the SARS-CoV-2 RBD (tagged with mouse Fc (mFc)). Arrows indicate potentially neutralizing clones. mFc-PE: anti-mouse PE (phycoerythrin) conjugate; MFI: mean fluorescence intensity; n.s: not significant ( p > 0.05); NC: negative control. * and **: p
    Figure Legend Snippet: Characterization of human anti-SARS-2 RBD Fabs. ( a ) Soluble ELISA of ten serially diluted human anti-SARS-2 RBD Fabs on immobilized SARS-2 RBD surfaces to measure their apparent affinities ( EC 50 , nM). ( b ) Schematic drawings of a competitive ELISA of human anti-SARS-2 RBD Fabs between the SARS-2 RBD and ACE2 protein (left) or ACE2-overexpressed cells (right). ( c ) Competitive ELISA of human anti-SARS-2 RBD Fabs antagonizing the interaction between ACE2 and the SARS-CoV-2 RBD. ( d ) Competitive flow cytometry analysis of human anti-SARS-2 RBD Fabs antagonizing the interaction between ACE2 on cells and the SARS-CoV-2 RBD (tagged with mouse Fc (mFc)). Arrows indicate potentially neutralizing clones. mFc-PE: anti-mouse PE (phycoerythrin) conjugate; MFI: mean fluorescence intensity; n.s: not significant ( p > 0.05); NC: negative control. * and **: p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Competitive ELISA, Flow Cytometry, Clone Assay, Fluorescence, Negative Control

    Characterization of anti-SARS-2 RBD immunoglobulin Gs (IgGs). ( a ) Binding analysis of five human anti-SARS-2 RBD IgGs—C12 (IgG), H1 (IgG), C2 (IgG), D12 (IgG), and F7 (IgG)—to the SARS-2 RBD and its variants (top) and the SARS-CoV-2 S1 (D614G) and other coronavirus S1 proteins (bottom), respectively. ( b ) Soluble ELISA of five serially diluted human anti-SARS-2 RBD IgGs on immobilized SARS-2 RBD surfaces to measure their apparent affinities ( EC 50 , nM). ( c ) ELISA detection for five human anti-SARS-2 RBD IgGs blocking the binding of the ACE2 protein with the SARS-CoV-2 RBD (top) and analysis of the flow cytometry for the blocking effect between the SARS-CoV-2 RBD and an ACE2-overexpressed cell (bottom). ( d ) Size-exclusion chromatography analysis of five human anti-SARS-2 RBD IgGs. The positions of the molecular mass markers, shown as kDa, on the retention time x -axis are indicated above the peaks. The data are presented as the mean ± standard error (SEM). MFI: mean fluorescence intensity; NC: negative control; *, **, and ***: p
    Figure Legend Snippet: Characterization of anti-SARS-2 RBD immunoglobulin Gs (IgGs). ( a ) Binding analysis of five human anti-SARS-2 RBD IgGs—C12 (IgG), H1 (IgG), C2 (IgG), D12 (IgG), and F7 (IgG)—to the SARS-2 RBD and its variants (top) and the SARS-CoV-2 S1 (D614G) and other coronavirus S1 proteins (bottom), respectively. ( b ) Soluble ELISA of five serially diluted human anti-SARS-2 RBD IgGs on immobilized SARS-2 RBD surfaces to measure their apparent affinities ( EC 50 , nM). ( c ) ELISA detection for five human anti-SARS-2 RBD IgGs blocking the binding of the ACE2 protein with the SARS-CoV-2 RBD (top) and analysis of the flow cytometry for the blocking effect between the SARS-CoV-2 RBD and an ACE2-overexpressed cell (bottom). ( d ) Size-exclusion chromatography analysis of five human anti-SARS-2 RBD IgGs. The positions of the molecular mass markers, shown as kDa, on the retention time x -axis are indicated above the peaks. The data are presented as the mean ± standard error (SEM). MFI: mean fluorescence intensity; NC: negative control; *, **, and ***: p

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Flow Cytometry, Size-exclusion Chromatography, Fluorescence, Negative Control

    In vitro neutralization assay of human anti-SARS-2 RBD IgGs. Pseudo-typed virus-based neutralization ( a ) and a neutralization assay using authentic SARS-CoV-2 ( b ). ( c ) Correlation in neutralization potencies between pseudo-typed virus- and authentic virus-based assays. ( d ) Correlation between affinities of anti-SARS-2 RBD IgGs and their neutralization potencies for the authentic virus. The data are showed as the mean ± standard error (SEM).
    Figure Legend Snippet: In vitro neutralization assay of human anti-SARS-2 RBD IgGs. Pseudo-typed virus-based neutralization ( a ) and a neutralization assay using authentic SARS-CoV-2 ( b ). ( c ) Correlation in neutralization potencies between pseudo-typed virus- and authentic virus-based assays. ( d ) Correlation between affinities of anti-SARS-2 RBD IgGs and their neutralization potencies for the authentic virus. The data are showed as the mean ± standard error (SEM).

    Techniques Used: In Vitro, Neutralization

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    Recombinant:

    Article Title: Patients with immune-mediated inflammatory diseases receiving cytokine inhibitors have low prevalence of SARS-CoV-2 seroconversion
    Article Snippet: In detail, 100 µl of the coating solution, consisting of 1.5 µg/ml of one of the above indicated SARS-CoV-2 antigens in 1xPBS, were applied to wells of a 96 well plates (Costar), incubated overnight at 4 °C. .. The following antigens were used: recombinant SARS-CoV-2 Spike Protein, S1 Subunit (1-Us-Tag); SARS-CoV-2 Spike S1 receptor binding domain (His-Tag) (both Sino Biological, Beijing, China); SARS-CoV-2 (2019-nCoV) Meridian Biosciences (Memphis, TX); Spike Protein (S2 ECD, His tag); SARS-CoV-2 (COVID-19) nucleocapsid protein(Sino Biological, Beijing, China). .. After removing the coating solution Liquid plate sealer animal free (#163 050, Candor, Germany) was added to the plates at room temperature for 1 h as blocking solution.

    Article Title: Next generation vaccine platform: polymersomes as stable nanocarriers for a highly immunogenic and durable SARS-CoV-2 spike protein subunit vaccine
    Article Snippet: .. Mouse serum raised against a recombinant SARS-CoV-2 spike protein (purchased from Sino Biological) was diluted 1:6,000 and incubated with the membrane for 1 h at room temperature. .. The membrane was washed thrice with TBST for a total of 30 min before incubating 1 h at room temperature with HRP-conjugated goat anti-mouse secondary antibody at a 1:10,000 dilution.

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses
    Article Snippet: A second construct was derived from a codon-optimized plasmid (SinoBiological) behaved identically in our assays and were used interchangeably. .. To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ). .. An ACE2 lentivirus expression CS(ACE2)IB vector was constructed by inserting a cDNA encoding an unaltered ACE2 (Addgene:1786) or an catalytically inactive ACE2 mutant (ACE2-H374N & H378N) into the lentivirus expression vector CSIB ( ).

    Article Title: Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies
    Article Snippet: The binding signals of the mutants to the mAbs were compared to those of the wild-type virus proteins. .. Receptor blocking assay To investigate the ability of the mAbs and sera from immunized mice to block SARS-CoV-2 spike protein binding to ACE2, serially diluted mAbs (3-fold dilution of initial 30 µg/ml) and sera (3-fold dilution of initial 1:10) were added to plates pre-coated with 100 ng/well of the recombinant SARS-CoV RBD-his, SARS-CoV-2 RBD-his (Sino Biological) and incubated for 1 h at 37 °C. .. Then, 150 ng/ml of the biotin-labelled recombinant hACE2-his (Novoprotein) expressed by 293 T cells was added to the plates.

    Article Title: Next generation vaccine platform: polymersomes as stable nanocarriers for a highly immunogenic and durable SARS-CoV-2 spike protein subunit vaccine
    Article Snippet: S2 was ideal as a negative control since it lacked strongly neutralizing epitopes whereas trimeric spike was used as a positive control given that it best represented the natural configuration of this viral protein. .. The three spike variants were analysed by SDS-PAGE followed by SYPRO Ruby staining ( ) and western blot using mouse immune serum raised against a recombinant SARS-CoV-2 spike protein purchased from Sino Biological ( ). .. Total protein staining using SYPRO dye showed S1S2 protein to consist of several bands, including two closely migrating major bands at the 150 kDa position, as well as two smaller bands at 75 kDa and 50 kDa ( ).

    Binding Assay:

    Article Title: Patients with immune-mediated inflammatory diseases receiving cytokine inhibitors have low prevalence of SARS-CoV-2 seroconversion
    Article Snippet: In detail, 100 µl of the coating solution, consisting of 1.5 µg/ml of one of the above indicated SARS-CoV-2 antigens in 1xPBS, were applied to wells of a 96 well plates (Costar), incubated overnight at 4 °C. .. The following antigens were used: recombinant SARS-CoV-2 Spike Protein, S1 Subunit (1-Us-Tag); SARS-CoV-2 Spike S1 receptor binding domain (His-Tag) (both Sino Biological, Beijing, China); SARS-CoV-2 (2019-nCoV) Meridian Biosciences (Memphis, TX); Spike Protein (S2 ECD, His tag); SARS-CoV-2 (COVID-19) nucleocapsid protein(Sino Biological, Beijing, China). .. After removing the coating solution Liquid plate sealer animal free (#163 050, Candor, Germany) was added to the plates at room temperature for 1 h as blocking solution.

    Incubation:

    Article Title: Next generation vaccine platform: polymersomes as stable nanocarriers for a highly immunogenic and durable SARS-CoV-2 spike protein subunit vaccine
    Article Snippet: .. Mouse serum raised against a recombinant SARS-CoV-2 spike protein (purchased from Sino Biological) was diluted 1:6,000 and incubated with the membrane for 1 h at room temperature. .. The membrane was washed thrice with TBST for a total of 30 min before incubating 1 h at room temperature with HRP-conjugated goat anti-mouse secondary antibody at a 1:10,000 dilution.

    Article Title: Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies
    Article Snippet: The binding signals of the mutants to the mAbs were compared to those of the wild-type virus proteins. .. Receptor blocking assay To investigate the ability of the mAbs and sera from immunized mice to block SARS-CoV-2 spike protein binding to ACE2, serially diluted mAbs (3-fold dilution of initial 30 µg/ml) and sera (3-fold dilution of initial 1:10) were added to plates pre-coated with 100 ng/well of the recombinant SARS-CoV RBD-his, SARS-CoV-2 RBD-his (Sino Biological) and incubated for 1 h at 37 °C. .. Then, 150 ng/ml of the biotin-labelled recombinant hACE2-his (Novoprotein) expressed by 293 T cells was added to the plates.

    Concentration Assay:

    Article Title: SARS-CoV-2 Entry Receptor ACE2 Is Expressed on Very Small CD45− Precursors of Hematopoietic and Endothelial Cells and in Response to Virus Spike Protein Activates the Nlrp3 Inflammasome
    Article Snippet: Amplification of the synthesized cDNA fragments was carried out using TaKaRa Taq™ DNA Polymerase (Takara Bio USA, Inc.) and sequence-specific ACE2 primers with 1 cycle of 8 min at 95 °C; 2 cycles of 2 min at 95 °C, 1 min at 60 °C, and 1 min at 72 °C; 45 cycles of 30 s at 95 °C, 1 min at 60 °C, 1 min at 72 °C; and 1 cycle of 10 min at 72 °C. .. Exposure of Cells to SARS-CoV-2 Spike Protein and Angiotensin Angiotensin Fragment 1–7 UCB-derived HSCs and VSELs were plated in 96-well plates and stimulated with NCP-CoV (2019-nCoV) spike protein (S1 + S2 ECD, with His-tag; Sino Biological) at a concentration of 10 nM. .. After 16 h of incubation, the cells were lysed, and total RNA was isolated for qRT-PCR analysis of Nlrp3, ASC, AIM2, IL1β, IL18, and Nlrp1 expression.

    Construct:

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses
    Article Snippet: A second construct was derived from a codon-optimized plasmid (SinoBiological) behaved identically in our assays and were used interchangeably. .. To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ). .. An ACE2 lentivirus expression CS(ACE2)IB vector was constructed by inserting a cDNA encoding an unaltered ACE2 (Addgene:1786) or an catalytically inactive ACE2 mutant (ACE2-H374N & H378N) into the lentivirus expression vector CSIB ( ).

    Sequencing:

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses
    Article Snippet: A second construct was derived from a codon-optimized plasmid (SinoBiological) behaved identically in our assays and were used interchangeably. .. To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ). .. An ACE2 lentivirus expression CS(ACE2)IB vector was constructed by inserting a cDNA encoding an unaltered ACE2 (Addgene:1786) or an catalytically inactive ACE2 mutant (ACE2-H374N & H378N) into the lentivirus expression vector CSIB ( ).

    Clone Assay:

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses
    Article Snippet: A second construct was derived from a codon-optimized plasmid (SinoBiological) behaved identically in our assays and were used interchangeably. .. To construct a replication competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 spike protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ). .. An ACE2 lentivirus expression CS(ACE2)IB vector was constructed by inserting a cDNA encoding an unaltered ACE2 (Addgene:1786) or an catalytically inactive ACE2 mutant (ACE2-H374N & H378N) into the lentivirus expression vector CSIB ( ).

    Blocking Assay:

    Article Title: Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies
    Article Snippet: The binding signals of the mutants to the mAbs were compared to those of the wild-type virus proteins. .. Receptor blocking assay To investigate the ability of the mAbs and sera from immunized mice to block SARS-CoV-2 spike protein binding to ACE2, serially diluted mAbs (3-fold dilution of initial 30 µg/ml) and sera (3-fold dilution of initial 1:10) were added to plates pre-coated with 100 ng/well of the recombinant SARS-CoV RBD-his, SARS-CoV-2 RBD-his (Sino Biological) and incubated for 1 h at 37 °C. .. Then, 150 ng/ml of the biotin-labelled recombinant hACE2-his (Novoprotein) expressed by 293 T cells was added to the plates.

    Mouse Assay:

    Article Title: Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies
    Article Snippet: The binding signals of the mutants to the mAbs were compared to those of the wild-type virus proteins. .. Receptor blocking assay To investigate the ability of the mAbs and sera from immunized mice to block SARS-CoV-2 spike protein binding to ACE2, serially diluted mAbs (3-fold dilution of initial 30 µg/ml) and sera (3-fold dilution of initial 1:10) were added to plates pre-coated with 100 ng/well of the recombinant SARS-CoV RBD-his, SARS-CoV-2 RBD-his (Sino Biological) and incubated for 1 h at 37 °C. .. Then, 150 ng/ml of the biotin-labelled recombinant hACE2-his (Novoprotein) expressed by 293 T cells was added to the plates.

    Protein Binding:

    Article Title: Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies
    Article Snippet: The binding signals of the mutants to the mAbs were compared to those of the wild-type virus proteins. .. Receptor blocking assay To investigate the ability of the mAbs and sera from immunized mice to block SARS-CoV-2 spike protein binding to ACE2, serially diluted mAbs (3-fold dilution of initial 30 µg/ml) and sera (3-fold dilution of initial 1:10) were added to plates pre-coated with 100 ng/well of the recombinant SARS-CoV RBD-his, SARS-CoV-2 RBD-his (Sino Biological) and incubated for 1 h at 37 °C. .. Then, 150 ng/ml of the biotin-labelled recombinant hACE2-his (Novoprotein) expressed by 293 T cells was added to the plates.

    SDS Page:

    Article Title: Next generation vaccine platform: polymersomes as stable nanocarriers for a highly immunogenic and durable SARS-CoV-2 spike protein subunit vaccine
    Article Snippet: S2 was ideal as a negative control since it lacked strongly neutralizing epitopes whereas trimeric spike was used as a positive control given that it best represented the natural configuration of this viral protein. .. The three spike variants were analysed by SDS-PAGE followed by SYPRO Ruby staining ( ) and western blot using mouse immune serum raised against a recombinant SARS-CoV-2 spike protein purchased from Sino Biological ( ). .. Total protein staining using SYPRO dye showed S1S2 protein to consist of several bands, including two closely migrating major bands at the 150 kDa position, as well as two smaller bands at 75 kDa and 50 kDa ( ).

    Staining:

    Article Title: Next generation vaccine platform: polymersomes as stable nanocarriers for a highly immunogenic and durable SARS-CoV-2 spike protein subunit vaccine
    Article Snippet: S2 was ideal as a negative control since it lacked strongly neutralizing epitopes whereas trimeric spike was used as a positive control given that it best represented the natural configuration of this viral protein. .. The three spike variants were analysed by SDS-PAGE followed by SYPRO Ruby staining ( ) and western blot using mouse immune serum raised against a recombinant SARS-CoV-2 spike protein purchased from Sino Biological ( ). .. Total protein staining using SYPRO dye showed S1S2 protein to consist of several bands, including two closely migrating major bands at the 150 kDa position, as well as two smaller bands at 75 kDa and 50 kDa ( ).

    Western Blot:

    Article Title: Next generation vaccine platform: polymersomes as stable nanocarriers for a highly immunogenic and durable SARS-CoV-2 spike protein subunit vaccine
    Article Snippet: S2 was ideal as a negative control since it lacked strongly neutralizing epitopes whereas trimeric spike was used as a positive control given that it best represented the natural configuration of this viral protein. .. The three spike variants were analysed by SDS-PAGE followed by SYPRO Ruby staining ( ) and western blot using mouse immune serum raised against a recombinant SARS-CoV-2 spike protein purchased from Sino Biological ( ). .. Total protein staining using SYPRO dye showed S1S2 protein to consist of several bands, including two closely migrating major bands at the 150 kDa position, as well as two smaller bands at 75 kDa and 50 kDa ( ).

    Article Title: SARS-CoV-2 spike protein promotes IL-6 trans-signaling by activation of angiotensin II receptor signaling in epithelial cells
    Article Snippet: The blot from the same run was reprobed with β-actin (Sigma) or α-tubulin (Santa Cruz Biotechnology) HRP conjugated antibody to compare protein load in each lane. .. Commercially available antibodies for ACE2, AT1 receptor, c-Fos, IκBα, ADAM-17, MCP-1 and IL-6Rα were procured from Santa Cruz Biotechnology; phospho-p38 MAPK (Thr180/Tyr182), phospho-p42/44 MAPK (Thr202/Tyr204), phospho-NF-κB (Ser276), SOCS3, phospho-STAT3 (Tyr705), phospho-STAT3 (Ser727), total STAT3 were procured from Cell Signaling Technologies; and SARS-CoV-2 spike protein (Sino Biological) were also procured for western blot analyses. .. ELISAPatient serum samples and cell culture supernatants from SARS-CoV-2 spike protein transfected cells were analyzed for the presence of secreted IL-6 (Sigma), soluble IL-6R (Life Technologies) IL-1α (Sigma) and HMGB-1 (Novus Biologicals) using ELISA kits following the manufacturer’s instructions.

    Produced:

    Article Title: Degradation of SARS-CoV-2 receptor ACE2 by tobacco carcinogen-induced Skp2 in lung epithelial cells
    Article Snippet: The proteins were incubated with 50 μl reaction mixture (Boston Biochem), 2 mM Mg-ATP (abcam), 0.5 μg E1 (abcam), 2.5 μg UbcH5a/UBE2D1 (Boston Biochem), and 2.5 μg ubiquitin (abcam) at 37°C for 2 h. After incubation, the reaction was terminated with SDS loading buffer, and the ubiquitination was detected using Western blot assays. .. SARS-CoV-2 Spike protein pseudovirions Pseudovirions were purchased from Sino Biological Inc. (Beijing, China), and were produced by transfection of 293T cells with psPAX2, pLenti-GFP, and plasmids encoding SARS-CoV-2 Spike protein using polyetherimide. .. To infect 16HBE and 293T-ACE2 cells with pseudovirions, the cells were seeded onto 96-well plates, pre-treated with CSE (10%), BaP (5 μM), or NNK (5 μM) for 48 h, followed by co-incubation with 100 μl media containing pseudovirions for 48 h. The cells were lysed with 60 μl medium containing 50% Steady-glo (promega) and measured by quantification of the luciferase activity using a Multi-Mode Reader (BioTek, Sunnyvale, CA, USA).

    Transfection:

    Article Title: Degradation of SARS-CoV-2 receptor ACE2 by tobacco carcinogen-induced Skp2 in lung epithelial cells
    Article Snippet: The proteins were incubated with 50 μl reaction mixture (Boston Biochem), 2 mM Mg-ATP (abcam), 0.5 μg E1 (abcam), 2.5 μg UbcH5a/UBE2D1 (Boston Biochem), and 2.5 μg ubiquitin (abcam) at 37°C for 2 h. After incubation, the reaction was terminated with SDS loading buffer, and the ubiquitination was detected using Western blot assays. .. SARS-CoV-2 Spike protein pseudovirions Pseudovirions were purchased from Sino Biological Inc. (Beijing, China), and were produced by transfection of 293T cells with psPAX2, pLenti-GFP, and plasmids encoding SARS-CoV-2 Spike protein using polyetherimide. .. To infect 16HBE and 293T-ACE2 cells with pseudovirions, the cells were seeded onto 96-well plates, pre-treated with CSE (10%), BaP (5 μM), or NNK (5 μM) for 48 h, followed by co-incubation with 100 μl media containing pseudovirions for 48 h. The cells were lysed with 60 μl medium containing 50% Steady-glo (promega) and measured by quantification of the luciferase activity using a Multi-Mode Reader (BioTek, Sunnyvale, CA, USA).

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    Sino Biological sars cov 2 rbd protein
    Immunogenicity evaluation of a single mRNA-RBD vaccination. a – c Groups of BALB/c mice ( n = 6) were immunized with a single injection of mRNA-RBD at different doses or with a placebo via the i.m. route. Sera at 4 weeks post immunization were collected. <t>SARS-CoV-2</t> RBD-specific IgG ( a ) and neutralizing antibody titers in sera against pseudovirus ( b ) and live virus ( c ) infection were determined. d – h C57BL/6 mice ( n = 6) were inoculated with a single mRNA-RBD vaccination or a placebo. Serum samples were collected from mice at 4 weeks following vaccination. RBD-specific IgG titers and pseudovirus-neutralizing antibodies were measured as shown in d and e , respectively. f An ELISPOT assay was performed to evaluate the capacity of splenocytes to secrete IFNγ following re-stimulation with SARS-CoV-2 RBD peptide pools. g , h An ICS assay was conducted to quantify the proportions of IFNγ-secreting CD8 + ( g ) and CD4 + ( h ) T cells. mRNA-RBD-L indicates the low dose (2 μg). mRNA-RBD-H indicates the high dose (15 μg). HCS represents human convalescent sera. Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; mRNA-RBD-L vaccinated animals = blue triangles; mRNA-RBD-H vaccinated animals = red squares; HCS = brown circles; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.
    Sars Cov 2 Rbd Protein, 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
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    95
    Sino Biological anti s2
    N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. A . Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] − and [N] − 293T cells. All 9 viruses were applied at equal titer to stable 293T/ACE2. B - C . O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. D . Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. E . Western blot of Spike protein using <t>anti-S2</t> Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] − 293T products due to truncation of glycan biosynthesis. F . Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] − 293Ts is almost fully proteolyzed during viral production (red arrowhead). * P
    Anti S2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Sino Biological sars cov2 s1
    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with <t>SARS‐CoV2‐RBG</t> and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and <t>SARS‐CoV2‐S1</t> proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.
    Sars Cov2 S1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Immunogenicity evaluation of a single mRNA-RBD vaccination. a – c Groups of BALB/c mice ( n = 6) were immunized with a single injection of mRNA-RBD at different doses or with a placebo via the i.m. route. Sera at 4 weeks post immunization were collected. SARS-CoV-2 RBD-specific IgG ( a ) and neutralizing antibody titers in sera against pseudovirus ( b ) and live virus ( c ) infection were determined. d – h C57BL/6 mice ( n = 6) were inoculated with a single mRNA-RBD vaccination or a placebo. Serum samples were collected from mice at 4 weeks following vaccination. RBD-specific IgG titers and pseudovirus-neutralizing antibodies were measured as shown in d and e , respectively. f An ELISPOT assay was performed to evaluate the capacity of splenocytes to secrete IFNγ following re-stimulation with SARS-CoV-2 RBD peptide pools. g , h An ICS assay was conducted to quantify the proportions of IFNγ-secreting CD8 + ( g ) and CD4 + ( h ) T cells. mRNA-RBD-L indicates the low dose (2 μg). mRNA-RBD-H indicates the high dose (15 μg). HCS represents human convalescent sera. Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; mRNA-RBD-L vaccinated animals = blue triangles; mRNA-RBD-H vaccinated animals = red squares; HCS = brown circles; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2

    doi: 10.1038/s41467-021-21037-2

    Figure Lengend Snippet: Immunogenicity evaluation of a single mRNA-RBD vaccination. a – c Groups of BALB/c mice ( n = 6) were immunized with a single injection of mRNA-RBD at different doses or with a placebo via the i.m. route. Sera at 4 weeks post immunization were collected. SARS-CoV-2 RBD-specific IgG ( a ) and neutralizing antibody titers in sera against pseudovirus ( b ) and live virus ( c ) infection were determined. d – h C57BL/6 mice ( n = 6) were inoculated with a single mRNA-RBD vaccination or a placebo. Serum samples were collected from mice at 4 weeks following vaccination. RBD-specific IgG titers and pseudovirus-neutralizing antibodies were measured as shown in d and e , respectively. f An ELISPOT assay was performed to evaluate the capacity of splenocytes to secrete IFNγ following re-stimulation with SARS-CoV-2 RBD peptide pools. g , h An ICS assay was conducted to quantify the proportions of IFNγ-secreting CD8 + ( g ) and CD4 + ( h ) T cells. mRNA-RBD-L indicates the low dose (2 μg). mRNA-RBD-H indicates the high dose (15 μg). HCS represents human convalescent sera. Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; mRNA-RBD-L vaccinated animals = blue triangles; mRNA-RBD-H vaccinated animals = red squares; HCS = brown circles; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Article Snippet: Briefly, a monoclonal antibody specific for SARS-CoV-2 RBD protein was pre-coated onto plate wells.

    Techniques: Mouse Assay, Injection, Infection, Enzyme-linked Immunospot, Two Tailed Test

    Duration and long-term protection of humoral response induced by mRNA-RBD. a Passive immunization and challenge schedule. The blue and red arrow indicates the time of vaccination and sera transfer, respectively. b , c Groups of BALB/c mice ( n = 10) received 15 μg of mRNA-RBD or a placebo. Half of the mice per group were euthanized at 8 weeks (short term) post vaccination, and massive sera were collected for further passive immunization. The other mice of the group were bled as desired and eventually euthanized at 26 weeks (long term) post vaccination to collect massive sera for further passive immunization. All serum samples were detected for IgG ( b ) and neutralizing antibodies ( c ) titers. d–e hACE2 transgenic mice ( n = 5) were administered 350 μl per mouse of pooled short- and long-term immune sera and one day later were challenged with 1 × 10 5 FFU of SARS-CoV-2 via the i.n. route. d The hACE2 mice weight change was recorded after challenge. e Virus titers in lung. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; animals for long-term study = blue triangles; animals for short-term study = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2

    doi: 10.1038/s41467-021-21037-2

    Figure Lengend Snippet: Duration and long-term protection of humoral response induced by mRNA-RBD. a Passive immunization and challenge schedule. The blue and red arrow indicates the time of vaccination and sera transfer, respectively. b , c Groups of BALB/c mice ( n = 10) received 15 μg of mRNA-RBD or a placebo. Half of the mice per group were euthanized at 8 weeks (short term) post vaccination, and massive sera were collected for further passive immunization. The other mice of the group were bled as desired and eventually euthanized at 26 weeks (long term) post vaccination to collect massive sera for further passive immunization. All serum samples were detected for IgG ( b ) and neutralizing antibodies ( c ) titers. d–e hACE2 transgenic mice ( n = 5) were administered 350 μl per mouse of pooled short- and long-term immune sera and one day later were challenged with 1 × 10 5 FFU of SARS-CoV-2 via the i.n. route. d The hACE2 mice weight change was recorded after challenge. e Virus titers in lung. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; animals for long-term study = blue triangles; animals for short-term study = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Article Snippet: Briefly, a monoclonal antibody specific for SARS-CoV-2 RBD protein was pre-coated onto plate wells.

    Techniques: Mouse Assay, Transgenic Assay, Two Tailed Test

    Protection efficacy of mRNA-RBD in hACE2 transgenic mice against SARS-CoV-2. a-d Groups of hACE2 transgenic mice ( n = 6) received one (prime group) or two (boost group) doses of mRNA-RBD-H or placebo via the i.m. route. Four weeks post initial vaccination, mice were challenged with 1 × 10 5 FFU of SARS-CoV-2 virus. a Mice immunization and challenge schedule. The blue arrows indicate the time of vaccination. b , c Sera collected at 4 weeks post initial vaccination were examined for IgG ( b ) and neutralizing antibody ( c ) titers. d Mice weight change after challenge. e Virus titers in lungs of challenged mice ( n = 4). f Representative histopathology (H E) of lungs in SARS-CoV-2-infected hACE2 mice (5 dpi). Infiltration of lymphocytes within alveolar spaces is indicated by yellow arrows. Scale bar, 100 μm. g Representative immunohistochemistry (IHC) of lung tissues with SARS-CoV-2 N-specific monoclonal antibodies. Virus is indicated by yellow arrows. Scale bar, 100 μm. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; one injection-animals = blue triangles; two injections-vaccinated animals = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2

    doi: 10.1038/s41467-021-21037-2

    Figure Lengend Snippet: Protection efficacy of mRNA-RBD in hACE2 transgenic mice against SARS-CoV-2. a-d Groups of hACE2 transgenic mice ( n = 6) received one (prime group) or two (boost group) doses of mRNA-RBD-H or placebo via the i.m. route. Four weeks post initial vaccination, mice were challenged with 1 × 10 5 FFU of SARS-CoV-2 virus. a Mice immunization and challenge schedule. The blue arrows indicate the time of vaccination. b , c Sera collected at 4 weeks post initial vaccination were examined for IgG ( b ) and neutralizing antibody ( c ) titers. d Mice weight change after challenge. e Virus titers in lungs of challenged mice ( n = 4). f Representative histopathology (H E) of lungs in SARS-CoV-2-infected hACE2 mice (5 dpi). Infiltration of lymphocytes within alveolar spaces is indicated by yellow arrows. Scale bar, 100 μm. g Representative immunohistochemistry (IHC) of lung tissues with SARS-CoV-2 N-specific monoclonal antibodies. Virus is indicated by yellow arrows. Scale bar, 100 μm. mRNA-RBD-H indicates the high-dose vaccine (15 μg). Data are means ± SEM (standard error of the mean). Comparisons were performed by Student’s t -test (unpaired, two tailed). Placebo animals = black circles; one injection-animals = blue triangles; two injections-vaccinated animals = red squares; dotted line = the limit of detection. Data are one representative result of two independent experiments. Source data are provided as a Source Data file.

    Article Snippet: Briefly, a monoclonal antibody specific for SARS-CoV-2 RBD protein was pre-coated onto plate wells.

    Techniques: Transgenic Assay, Mouse Assay, Histopathology, Infection, Immunohistochemistry, Two Tailed Test, Injection

    Construction and characterization of mRNA-RBD vaccine. a Schematic of the mRNA-RBD vaccine design. The SARS-CoV-2 mRNA encodes the signal peptide (SP), receptor-binding domain (RBD) from SARS-CoV-2 strain Wuhan/IVDC-HB-01/2019. b mRNA-RBD was transfected into HEK293T cells. RBD expression in the cell lysate and supernatant was analyzed by western blotting. c Particle size of LNPs by dynamic light scattering. d A representative cryo-electron microscopy image of a LNPs solution following mRNA encapsulation. Scale bar, 100 nm. e Zeta potential for LNPs at pH 4.0 and 7.4. For b and d , two independent experiments were carried out with similar results. For c and e , one representative result from three independent experiments is shown. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: A single-dose mRNA vaccine provides a long-term protection for hACE2 transgenic mice from SARS-CoV-2

    doi: 10.1038/s41467-021-21037-2

    Figure Lengend Snippet: Construction and characterization of mRNA-RBD vaccine. a Schematic of the mRNA-RBD vaccine design. The SARS-CoV-2 mRNA encodes the signal peptide (SP), receptor-binding domain (RBD) from SARS-CoV-2 strain Wuhan/IVDC-HB-01/2019. b mRNA-RBD was transfected into HEK293T cells. RBD expression in the cell lysate and supernatant was analyzed by western blotting. c Particle size of LNPs by dynamic light scattering. d A representative cryo-electron microscopy image of a LNPs solution following mRNA encapsulation. Scale bar, 100 nm. e Zeta potential for LNPs at pH 4.0 and 7.4. For b and d , two independent experiments were carried out with similar results. For c and e , one representative result from three independent experiments is shown. Source data are provided as a Source Data file.

    Article Snippet: Briefly, a monoclonal antibody specific for SARS-CoV-2 RBD protein was pre-coated onto plate wells.

    Techniques: Binding Assay, Transfection, Expressing, Western Blot, Electron Microscopy

    N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. A . Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] − and [N] − 293T cells. All 9 viruses were applied at equal titer to stable 293T/ACE2. B - C . O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. D . Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. E . Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] − 293T products due to truncation of glycan biosynthesis. F . Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] − 293Ts is almost fully proteolyzed during viral production (red arrowhead). * P

    Journal: bioRxiv

    Article Title: Inhibition of SARS-CoV-2 viral entry in vitro upon blocking N- and O-glycan elaboration

    doi: 10.1101/2020.10.15.339838

    Figure Lengend Snippet: N-glycan modification of SARS-CoV-2 pseudovirus abolishes entry into 293T/ACE2 cells. A . Pseudovirus expressing VSVG envelope protein, Spike-WT and Spike-mutant were produced in wild-type, [O] − and [N] − 293T cells. All 9 viruses were applied at equal titer to stable 293T/ACE2. B - C . O-glycan truncation of Spike partially reduced viral entry. N-glycan truncation abolished viral entry. In order to combine data from multiple viral preparations and independent runs in a single plot, all data were normalized by setting DsRed signal produced by virus generated in wild-type 293T to 10,000 normalized MFI or 100% normalized DsRed positive value. D . Viral titration study performed with Spike-mutant virus shows complete loss of viral infection over a wide range. E . Western blot of Spike protein using anti-S2 Ab shows reduced proteolysis of Spike-mut compared to Spike-WT. The full Spike protein and free S2-subunit resulting from S1-S2 cleavage is indicated. Molecular mass is reduced in [N] − 293T products due to truncation of glycan biosynthesis. F . Anti-FLAG Ab binds the C-terminus of Spike-mutant. Spike produced in [N] − 293Ts is almost fully proteolyzed during viral production (red arrowhead). * P

    Article Snippet: Identity of expressed protein and also viral Spike was determined using western blotting with anti-Fc (Jackson), anti-RBD (Sino Biologicals), anti-S2 (Sino Biologicals) and anti-ACE2 (R & D Systems) pAbs.

    Techniques: Modification, Expressing, Mutagenesis, Produced, Generated, Titration, Infection, Western Blot

    Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.

    Journal: Small Methods

    Article Title: Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors, Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

    doi: 10.1002/smtd.202001031

    Figure Lengend Snippet: Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.

    Article Snippet: Generation and Production of Antibodies against SARS‐CoV‐2 S Balb/c mice were intraperitoneal immunized with 5 µg of SARS‐CoV2‐RBD (expression in this study, n = 5), SARS‐CoV2‐S1 (Sino Biological, 40591‐V08H, n = 3), and SARS‐CoV2‐S2 (Sino Biological, 40590‐V08B, n = 3), respectively.

    Techniques: Construct, Modification, Derivative Assay, Plasmid Preparation, Western Blot, Stable Transfection, Transfection, Fluorescence, Incubation, Imaging, Inhibition, Recombinant, Binding Assay, Blocking Assay