sars cov 2 2019 ncov spike s1 s2 ecd his recombinant protein covid 19 spike research  (Sino Biological)


Bioz Verified Symbol Sino Biological is a verified supplier
Bioz Manufacturer Symbol Sino Biological manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 98
    Name:
    SARS CoV 2 2019 nCoV Spike S1 S2 ECD His Recombinant Protein COVID 19 Spike Research
    Description:
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein S1 S2 ECD YP 009724390 1 Val 16 Pro1213 was expressed with a polyhistidine tag at the C terminus
    Catalog Number:
    40589-V08B1
    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:
    Baculovirus-Insect Cells
    Buy from Supplier


    Structured Review

    Sino Biological sars cov 2 2019 ncov spike s1 s2 ecd his recombinant protein covid 19 spike research
    Adjuvanted S and S1 immune sera exhibit high titers of <t>SARS-CoV-2</t> pseudovirus neutralization and receptor binding inhibition activities. ( A – C ) Reduction percentage (%) in relative luminometer units (RLU) as a measure of luciferase activity for SARS-CoV-2 spike pseudotyped lentivirus infection in HEK293 cells expressing human ACE2 receptor. Data were obtained from pooled sera ( n = 6 to 8) with triplicate wells. S-0.8 (y): S 0.8 µg boost sera of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant boost sera of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant boost sera of old aged mice, S1-4 (y): S1 4 µg boost immune sera of young adult mice, S1-4 + adj (y): S1 4 µg + adjuvant boost immune sera of young adult mice, S2-4 (y): S2 4 µg boost immune sera of young adult mice, S2-4 + adj (y): S2 4 µg + adjuvant boost immune sera of young adult mice, S-0.8 + adj (y, x3): S 0.8 µg + adjuvant 2nd boost immune sera of young adult mice, S-0.8 + adj (y, x3, 19W): S 0.8 µg + adjuvant immune sera collected at week 19 post 2nd boost of young adult mice, S-4 + adj (a, x3): S 4 µg + adjuvant 2nd boost sera of old aged mice. IV-0.8-10 + adj (y, x3): inactivated adjuvanted SARS-CoV-2 vaccination in young age mice (prime 0.8 µg of heat-inactivated and gamma-irradiated virus, 2 times boost with 10 µg inactivated adjuvanted SARS-CoV-2 of heat-inactivated and gamma-irradiated virus). Adj: adjuvants (MPL + QS-21, 1 µg + 10 µg). Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. ( D – F ) ACE2 receptor binding inhibition titers in pooled immune sera ( n = 6–8) with triplicate wells. Inhibition percentage (%) of hACE2 binding to RBD was measured after incubation with serially diluted immune sera in the plate precoated with hACE2 protein. Immune sera of groups are the same as in ( A – C ). Statistical significance was calculated using two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. **; p
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike Protein S1 S2 ECD YP 009724390 1 Val 16 Pro1213 was expressed with a polyhistidine tag at the C terminus
    https://www.bioz.com/result/sars cov 2 2019 ncov spike s1 s2 ecd his recombinant protein covid 19 spike research/product/Sino Biological
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 2019 ncov spike s1 s2 ecd his recombinant protein covid 19 spike research - by Bioz Stars, 2021-07
    98/100 stars

    Images

    1) Product Images from "Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice"

    Article Title: Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice

    Journal: Vaccines

    doi: 10.3390/vaccines9040316

    Adjuvanted S and S1 immune sera exhibit high titers of SARS-CoV-2 pseudovirus neutralization and receptor binding inhibition activities. ( A – C ) Reduction percentage (%) in relative luminometer units (RLU) as a measure of luciferase activity for SARS-CoV-2 spike pseudotyped lentivirus infection in HEK293 cells expressing human ACE2 receptor. Data were obtained from pooled sera ( n = 6 to 8) with triplicate wells. S-0.8 (y): S 0.8 µg boost sera of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant boost sera of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant boost sera of old aged mice, S1-4 (y): S1 4 µg boost immune sera of young adult mice, S1-4 + adj (y): S1 4 µg + adjuvant boost immune sera of young adult mice, S2-4 (y): S2 4 µg boost immune sera of young adult mice, S2-4 + adj (y): S2 4 µg + adjuvant boost immune sera of young adult mice, S-0.8 + adj (y, x3): S 0.8 µg + adjuvant 2nd boost immune sera of young adult mice, S-0.8 + adj (y, x3, 19W): S 0.8 µg + adjuvant immune sera collected at week 19 post 2nd boost of young adult mice, S-4 + adj (a, x3): S 4 µg + adjuvant 2nd boost sera of old aged mice. IV-0.8-10 + adj (y, x3): inactivated adjuvanted SARS-CoV-2 vaccination in young age mice (prime 0.8 µg of heat-inactivated and gamma-irradiated virus, 2 times boost with 10 µg inactivated adjuvanted SARS-CoV-2 of heat-inactivated and gamma-irradiated virus). Adj: adjuvants (MPL + QS-21, 1 µg + 10 µg). Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. ( D – F ) ACE2 receptor binding inhibition titers in pooled immune sera ( n = 6–8) with triplicate wells. Inhibition percentage (%) of hACE2 binding to RBD was measured after incubation with serially diluted immune sera in the plate precoated with hACE2 protein. Immune sera of groups are the same as in ( A – C ). Statistical significance was calculated using two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. **; p
    Figure Legend Snippet: Adjuvanted S and S1 immune sera exhibit high titers of SARS-CoV-2 pseudovirus neutralization and receptor binding inhibition activities. ( A – C ) Reduction percentage (%) in relative luminometer units (RLU) as a measure of luciferase activity for SARS-CoV-2 spike pseudotyped lentivirus infection in HEK293 cells expressing human ACE2 receptor. Data were obtained from pooled sera ( n = 6 to 8) with triplicate wells. S-0.8 (y): S 0.8 µg boost sera of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant boost sera of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant boost sera of old aged mice, S1-4 (y): S1 4 µg boost immune sera of young adult mice, S1-4 + adj (y): S1 4 µg + adjuvant boost immune sera of young adult mice, S2-4 (y): S2 4 µg boost immune sera of young adult mice, S2-4 + adj (y): S2 4 µg + adjuvant boost immune sera of young adult mice, S-0.8 + adj (y, x3): S 0.8 µg + adjuvant 2nd boost immune sera of young adult mice, S-0.8 + adj (y, x3, 19W): S 0.8 µg + adjuvant immune sera collected at week 19 post 2nd boost of young adult mice, S-4 + adj (a, x3): S 4 µg + adjuvant 2nd boost sera of old aged mice. IV-0.8-10 + adj (y, x3): inactivated adjuvanted SARS-CoV-2 vaccination in young age mice (prime 0.8 µg of heat-inactivated and gamma-irradiated virus, 2 times boost with 10 µg inactivated adjuvanted SARS-CoV-2 of heat-inactivated and gamma-irradiated virus). Adj: adjuvants (MPL + QS-21, 1 µg + 10 µg). Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. ( D – F ) ACE2 receptor binding inhibition titers in pooled immune sera ( n = 6–8) with triplicate wells. Inhibition percentage (%) of hACE2 binding to RBD was measured after incubation with serially diluted immune sera in the plate precoated with hACE2 protein. Immune sera of groups are the same as in ( A – C ). Statistical significance was calculated using two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. **; p

    Techniques Used: Neutralization, Binding Assay, Inhibition, Luciferase, Activity Assay, Infection, Expressing, Mouse Assay, Irradiation, Incubation

    B cell and T cell immune responses to SARS-CoV-2 S vaccination in young adult and old aged mice. To determine cellular immunity, spleen cells were prepared from immunized young adult ( n = 6) and old aged mice ( n = 8). ( A ) Antibody-secreting cells (ASCs) specific for full-length S protein were determined on the ELISpot plate precoated with full-length S protein. ( B ) IFN-γ-secreting cells were analyzed by in vitro stimulation with pooled S peptides or full-length S protein using ELISpot assay. ( C , D ). IFN-γ + CD4 and IFN-γ + CD8 T cells were determined by flow cytometry after in vitro stimulation with pooled S peptides and intracellular cytokine antibi staining. S-0.8 (y): S 0.8 µg vaccination of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant vaccination of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant vaccination of old aged mice. Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. Statistical significance was calculated using one-way ANOVA and a Dunnett’s multiple-comparison test. Error bars indicate the mean ± SEM. *; p
    Figure Legend Snippet: B cell and T cell immune responses to SARS-CoV-2 S vaccination in young adult and old aged mice. To determine cellular immunity, spleen cells were prepared from immunized young adult ( n = 6) and old aged mice ( n = 8). ( A ) Antibody-secreting cells (ASCs) specific for full-length S protein were determined on the ELISpot plate precoated with full-length S protein. ( B ) IFN-γ-secreting cells were analyzed by in vitro stimulation with pooled S peptides or full-length S protein using ELISpot assay. ( C , D ). IFN-γ + CD4 and IFN-γ + CD8 T cells were determined by flow cytometry after in vitro stimulation with pooled S peptides and intracellular cytokine antibi staining. S-0.8 (y): S 0.8 µg vaccination of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant vaccination of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant vaccination of old aged mice. Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. Statistical significance was calculated using one-way ANOVA and a Dunnett’s multiple-comparison test. Error bars indicate the mean ± SEM. *; p

    Techniques Used: Mouse Assay, Enzyme-linked Immunospot, In Vitro, Flow Cytometry, Staining

    SARS-CoV-2 full-length spike (S) ectodomain and subunit proteins and receptor binding activities. ( A ) Full-length S (S1–S2) ectodomain contains aa residues 16-1213, S1 subunit aa 16-685 (green), and S2 subunit aa 686-1213. NTD: N-terminal domain (blue), RBD: receptor binding domain. ( B , C ) The receptor binding properties were determined using serially diluted soluble hACE2-Fc (0.5–2 µg/mL) on the 96-well plates precoated with 0.8 µg ( B ) or 2 µg ( C ) of S (S1–S2) and S1 subunit proteins. Due to different molecular masses of S and S1 proteins despite the same concentration, molarity in nanomoles (nM) is indicated for each protein coated.
    Figure Legend Snippet: SARS-CoV-2 full-length spike (S) ectodomain and subunit proteins and receptor binding activities. ( A ) Full-length S (S1–S2) ectodomain contains aa residues 16-1213, S1 subunit aa 16-685 (green), and S2 subunit aa 686-1213. NTD: N-terminal domain (blue), RBD: receptor binding domain. ( B , C ) The receptor binding properties were determined using serially diluted soluble hACE2-Fc (0.5–2 µg/mL) on the 96-well plates precoated with 0.8 µg ( B ) or 2 µg ( C ) of S (S1–S2) and S1 subunit proteins. Due to different molecular masses of S and S1 proteins despite the same concentration, molarity in nanomoles (nM) is indicated for each protein coated.

    Techniques Used: Binding Assay, Concentration Assay

    2) Product Images from "Original antigenic sin responses to heterologous Betacoronavirus spike proteins are observed in mice following intramuscular administration, but are not apparent in children following SARS-CoV-2 infection"

    Article Title: Original antigenic sin responses to heterologous Betacoronavirus spike proteins are observed in mice following intramuscular administration, but are not apparent in children following SARS-CoV-2 infection

    Journal: medRxiv

    doi: 10.1101/2021.04.29.21256344

    HKU1 antibodies are prevalent in healthy children and children with acute COVID-19 and MIS-C. SARS-CoV-2 (A) and HKU1 (B) spike IgG antibody titers and FRNT neutralization titers (C) in healthy pediatric controls compared to children hospitalized with acute COVID-19 and MIS-C. * P
    Figure Legend Snippet: HKU1 antibodies are prevalent in healthy children and children with acute COVID-19 and MIS-C. SARS-CoV-2 (A) and HKU1 (B) spike IgG antibody titers and FRNT neutralization titers (C) in healthy pediatric controls compared to children hospitalized with acute COVID-19 and MIS-C. * P

    Techniques Used: Neutralization

    Schematic of intramuscular spike protein administrations in groups of five BALB/c mice. Group 1 received prime and boost with SARS-CoV-2 spike, followed by prime and boost with HKU1 spike. Group 2 received a reciprocal administration regimen, with prime and boost with HKU1 spike, followed by prime and boost by HKU1 spike. D, days post-administration; S, spike; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. *These mice were immunized with nucleocapsid protein 21 and 42 days prior to utilization for this study.
    Figure Legend Snippet: Schematic of intramuscular spike protein administrations in groups of five BALB/c mice. Group 1 received prime and boost with SARS-CoV-2 spike, followed by prime and boost with HKU1 spike. Group 2 received a reciprocal administration regimen, with prime and boost with HKU1 spike, followed by prime and boost by HKU1 spike. D, days post-administration; S, spike; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. *These mice were immunized with nucleocapsid protein 21 and 42 days prior to utilization for this study.

    Techniques Used: Mouse Assay

    HKU1 spike IgG antibodies correlated positively with both SAR-CoV-2 spike IgG and SARS-CoV-2 neutralizing antibodies in children with acute COVID-19 and MIS-C. Linear regression analyses compared the log-transformed antibody titers of (A) SARS-CoV-2 spike IgG vs. HKU1 spike IgG; (B) HKU1 spike IgG vs. SARS-CoV-2 neutralization titers; and (C) SARS-CoV-2 spike IgG vs. SARS-CoV-2 neutralization titers among children with acute COVID-19 or MIS-C. Spearman’s correlation coefficients (r) and P-values are shown.
    Figure Legend Snippet: HKU1 spike IgG antibodies correlated positively with both SAR-CoV-2 spike IgG and SARS-CoV-2 neutralizing antibodies in children with acute COVID-19 and MIS-C. Linear regression analyses compared the log-transformed antibody titers of (A) SARS-CoV-2 spike IgG vs. HKU1 spike IgG; (B) HKU1 spike IgG vs. SARS-CoV-2 neutralization titers; and (C) SARS-CoV-2 spike IgG vs. SARS-CoV-2 neutralization titers among children with acute COVID-19 or MIS-C. Spearman’s correlation coefficients (r) and P-values are shown.

    Techniques Used: Transformation Assay, Neutralization

    Priming mice with HKU1 spike protein prior to boosting with SARS-CoV-2 spike protein completely impeded the development of SARS-CoV-2 neutralizing antibodies. SARS-CoV-2 (A,B) and HKU1 (C,D) full-length spike IgG binding and SARS-CoV-2 neutralizing (E, F) antibodies in mice are shown as log(end-point titer). Group 1 was primed with two doses of alum-adjuvanted SARS-CoV-2 spike and boosted with two doses of alum-adjuvanted HKU1 spike (A, C, E). Group 2 received the reciprocal regimen of HKU1 spike prime and SARS-CoV-2 spike boost (B, D, F). * P
    Figure Legend Snippet: Priming mice with HKU1 spike protein prior to boosting with SARS-CoV-2 spike protein completely impeded the development of SARS-CoV-2 neutralizing antibodies. SARS-CoV-2 (A,B) and HKU1 (C,D) full-length spike IgG binding and SARS-CoV-2 neutralizing (E, F) antibodies in mice are shown as log(end-point titer). Group 1 was primed with two doses of alum-adjuvanted SARS-CoV-2 spike and boosted with two doses of alum-adjuvanted HKU1 spike (A, C, E). Group 2 received the reciprocal regimen of HKU1 spike prime and SARS-CoV-2 spike boost (B, D, F). * P

    Techniques Used: Mouse Assay, Binding Assay

    3) Product Images from "A serological assay to detect human SARS-CoV-2 antibodies"

    Article Title: A serological assay to detect human SARS-CoV-2 antibodies

    Journal: Journal of Taibah University Medical Sciences

    doi: 10.1016/j.jtumed.2020.11.011

    Optimization of the SARS-CoV-2-S antigen concentration. The sera from four COVID-19 patients and negative controls (NCs) at 1:100 dilution were tested against decreasing concentrations of the SARS-CoV-2 spike protein (4 μg/ml to 0.5 μg/ml) for IgG and IgM reactivity.
    Figure Legend Snippet: Optimization of the SARS-CoV-2-S antigen concentration. The sera from four COVID-19 patients and negative controls (NCs) at 1:100 dilution were tested against decreasing concentrations of the SARS-CoV-2 spike protein (4 μg/ml to 0.5 μg/ml) for IgG and IgM reactivity.

    Techniques Used: Concentration Assay

    4) 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

    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

    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

    5) Product Images from "Immunogenicity of a DNA vaccine candidate for COVID-19"

    Article Title: Immunogenicity of a DNA vaccine candidate for COVID-19

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16505-0

    T cell epitope mapping after INO-4800 administration to BALB/c mice. Splenocytes were stimulated for 20 h with SARS-CoV-2 peptide matrix pools. a T cell responses following stimulation with matrix mapping SARS-CoV-2 peptide pools. Bars represent the mean + SD of five mice. b Map of the SARS-CoV-2 Spike protein and identification of immunodominant peptides in BALB/c mice. A known immunodominant SARS-CoV HLA-A2 epitope is included for comparison.
    Figure Legend Snippet: T cell epitope mapping after INO-4800 administration to BALB/c mice. Splenocytes were stimulated for 20 h with SARS-CoV-2 peptide matrix pools. a T cell responses following stimulation with matrix mapping SARS-CoV-2 peptide pools. Bars represent the mean + SD of five mice. b Map of the SARS-CoV-2 Spike protein and identification of immunodominant peptides in BALB/c mice. A known immunodominant SARS-CoV HLA-A2 epitope is included for comparison.

    Techniques Used: Mouse Assay

    Humoral responses to SARS-CoV-2 in Hartley guinea pigs after a single dose of INO-4800. Hartley guinea pigs were immunized on Day 0 with 100 µg INO-4800 or pVAX-empty vector as described in the methods. a SARS-CoV-2 S protein antigen binding of IgG in serial serum dilutions at day 0 and 14. Data shown represent mean OD450 nm values (mean + SD) for the five guinea pigs. b Serum IgG binding titers (mean ± SD) to SARS-CoV-2 S protein at day 14. Values depicted are mean ± SD. P values determined by Mann–Whitney test.
    Figure Legend Snippet: Humoral responses to SARS-CoV-2 in Hartley guinea pigs after a single dose of INO-4800. Hartley guinea pigs were immunized on Day 0 with 100 µg INO-4800 or pVAX-empty vector as described in the methods. a SARS-CoV-2 S protein antigen binding of IgG in serial serum dilutions at day 0 and 14. Data shown represent mean OD450 nm values (mean + SD) for the five guinea pigs. b Serum IgG binding titers (mean ± SD) to SARS-CoV-2 S protein at day 14. Values depicted are mean ± SD. P values determined by Mann–Whitney test.

    Techniques Used: Plasmid Preparation, Binding Assay, MANN-WHITNEY

    Induction of T cell responses in BALB/c mice post-administration of INO-4800. BALB/c mice ( n = 5/group) were immunized with 2.5 or 10 µg INO-4800. T cell responses were analyzed in the animals on days 4, 7, 10 for plots a b, and day 14 for plot c. T cell responses were measured by IFN-γ ELISpot in splenocytes stimulated for 20h with overlapping peptide pools spanning the SARS-CoV-2 ( a ), SARS-CoV ( b ), or MERS-CoV ( c ) Spike proteins. Bars represent the mean + SD. Data from individual mice is shown in Supplementary Data 2 .
    Figure Legend Snippet: Induction of T cell responses in BALB/c mice post-administration of INO-4800. BALB/c mice ( n = 5/group) were immunized with 2.5 or 10 µg INO-4800. T cell responses were analyzed in the animals on days 4, 7, 10 for plots a b, and day 14 for plot c. T cell responses were measured by IFN-γ ELISpot in splenocytes stimulated for 20h with overlapping peptide pools spanning the SARS-CoV-2 ( a ), SARS-CoV ( b ), or MERS-CoV ( c ) Spike proteins. Bars represent the mean + SD. Data from individual mice is shown in Supplementary Data 2 .

    Techniques Used: Mouse Assay, Enzyme-linked Immunospot

    INO-4800 immunized mouse and guinea pig sera compete with ACE2 receptor for SARS-CoV-2 Spike protein binding. a Soluble ACE2 receptor binds to CoV-2 full-length spike with an EC 50 of 0.025 µg/ml. b Purified serum IgG from BALB/c mice ( n of 5 per group) after second immunization with INO-4800 yields significant competition against ACE2 receptor. Serum IgG samples from the animals were run in triplicate. c IgGs purified from n = 5 mice day 7 post second immunization with INO-4800 show significant competition against ACE2 receptor binding to SARS-CoV-2 S 1 + 2 protein. The soluble ACE2 concentration for the competition assay is ~0.1 µg ml −1 . Bars represent the mean and standard deviation of AUC for curves displayed in Supplementary Fig. 1 . d Hartley guinea pigs were immunized on Day 0 and 14 with 100 µg INO-4800 or pVAX-empty vector as described in the methods. Day 28 collected sera (diluted 1:20) was added SARS-CoV-2 coated wells prior to the addition of serial dilutions of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 5 INO-4800-treated and 3 pVAX-treated animals were used in this experiment. e Serial dilutions of guinea pig sera collected on day 21 were added to SARS-CoV-2 coated wells prior to the addition of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 4 INO-4800-treated and 5 pVAX-treated guinea pigs were used in this experiment.
    Figure Legend Snippet: INO-4800 immunized mouse and guinea pig sera compete with ACE2 receptor for SARS-CoV-2 Spike protein binding. a Soluble ACE2 receptor binds to CoV-2 full-length spike with an EC 50 of 0.025 µg/ml. b Purified serum IgG from BALB/c mice ( n of 5 per group) after second immunization with INO-4800 yields significant competition against ACE2 receptor. Serum IgG samples from the animals were run in triplicate. c IgGs purified from n = 5 mice day 7 post second immunization with INO-4800 show significant competition against ACE2 receptor binding to SARS-CoV-2 S 1 + 2 protein. The soluble ACE2 concentration for the competition assay is ~0.1 µg ml −1 . Bars represent the mean and standard deviation of AUC for curves displayed in Supplementary Fig. 1 . d Hartley guinea pigs were immunized on Day 0 and 14 with 100 µg INO-4800 or pVAX-empty vector as described in the methods. Day 28 collected sera (diluted 1:20) was added SARS-CoV-2 coated wells prior to the addition of serial dilutions of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 5 INO-4800-treated and 3 pVAX-treated animals were used in this experiment. e Serial dilutions of guinea pig sera collected on day 21 were added to SARS-CoV-2 coated wells prior to the addition of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 4 INO-4800-treated and 5 pVAX-treated guinea pigs were used in this experiment.

    Techniques Used: Protein Binding, Purification, Mouse Assay, Binding Assay, Concentration Assay, Competitive Binding Assay, Standard Deviation, Plasmid Preparation

    Detection of SARS-CoV-2 S protein-reactive antibodies in the BAL of INO-4800 immunized animals. BALB/c mice (n of 5 per group) were immunized on days 0 and 14 with INO-4800 or pVAX and BAL collected at day 21 ( a , b ). Hartley guinea pigs ( n of 5 per group) were immunized on days 0, 14 and 21 with INO-4800 or pVAX and BAL collected at day 42 ( c , d ). Bronchoalveolar lavage fluid was assayed in duplicate for SARS-CoV-2 Spike protein-specific IgG antibodies by ELISA. Data are presented as endpoint titers ( a , c ), and BAL dilution curves with raw OD 450 nm values ( b , d ). a , c Bars represent the average of each group and error bars the standard deviation. ** p
    Figure Legend Snippet: Detection of SARS-CoV-2 S protein-reactive antibodies in the BAL of INO-4800 immunized animals. BALB/c mice (n of 5 per group) were immunized on days 0 and 14 with INO-4800 or pVAX and BAL collected at day 21 ( a , b ). Hartley guinea pigs ( n of 5 per group) were immunized on days 0, 14 and 21 with INO-4800 or pVAX and BAL collected at day 42 ( c , d ). Bronchoalveolar lavage fluid was assayed in duplicate for SARS-CoV-2 Spike protein-specific IgG antibodies by ELISA. Data are presented as endpoint titers ( a , c ), and BAL dilution curves with raw OD 450 nm values ( b , d ). a , c Bars represent the average of each group and error bars the standard deviation. ** p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Comparison of SARS-CoV-2, SARS-CoV and MERS-CoV spike glycoproteins. a Amino acid alignment of coronavirus spike proteins including 11 SARS-CoV-2 sequences with mutations (GISAID). Gray bars indicates identical amino acids and colored bars represent mutations relative to Wuhan-Hu-1. RBD, Cleavage Site, Fusion Peptide and Transmembrane domains are indicated in red. b Structural models for SARS-CoV-2, SARS and MERS spike glycoproteins with one chain represented as cartoon and two chains represented as surface. RBD of SARS-CoV-2 is colored yellow.
    Figure Legend Snippet: Comparison of SARS-CoV-2, SARS-CoV and MERS-CoV spike glycoproteins. a Amino acid alignment of coronavirus spike proteins including 11 SARS-CoV-2 sequences with mutations (GISAID). Gray bars indicates identical amino acids and colored bars represent mutations relative to Wuhan-Hu-1. RBD, Cleavage Site, Fusion Peptide and Transmembrane domains are indicated in red. b Structural models for SARS-CoV-2, SARS and MERS spike glycoproteins with one chain represented as cartoon and two chains represented as surface. RBD of SARS-CoV-2 is colored yellow.

    Techniques Used:

    Design and expression of COVID-19 synthetic DNA vaccine constructs. a Schematic diagram of COVID-19 synthetic DNA vaccine constructs, pGX9501 (matched) and pGX9503 (outlier (OL)) containing the IgE leader sequence and SARS-CoV-2 spike protein insert. b RT-PCR assay of RNA extracts from COS-7 cells transfected in duplicate with pGX9501 and pGX9503. Extracted RNA was analyzed by RT-PCR using PCR assays designed for each target and for COS-7 β-Actin mRNA, used as an internal expression normalization gene. Delta C T (∆ C T ) was calculated as the C T of the target minus the C T of β-Actin for each transfection concentration and is plotted against the log of the mass of pDNA transfected (Plotted as mean ± SD). c Analysis of in vitro expression of Spike protein after transfection of 293T cells with pGX9501, pGX9503 or MOCK plasmid by Western blot. 293T cell lysates were resolved on a gel and probed with a polyclonal anti-SARS Spike Protein. Blots were stripped then probed with an anti-β-actin loading control. d In vitro immunofluorescent staining of 293T cells transfected with 3 µg/well of pGX9501, pGX9503 or pVax (empty control vector). Expression of Spike protein was measured with polyclonal anti-SARS Spike Protein IgG and anti-IgG secondary (green). Cell nuclei were counterstained with DAPI (blue). Images were captured using ImageXpress Pico automated cell imaging system. Scale bars are 80.15 µm (left), 66.8 µm (middle) and 77.31 µm (right).
    Figure Legend Snippet: Design and expression of COVID-19 synthetic DNA vaccine constructs. a Schematic diagram of COVID-19 synthetic DNA vaccine constructs, pGX9501 (matched) and pGX9503 (outlier (OL)) containing the IgE leader sequence and SARS-CoV-2 spike protein insert. b RT-PCR assay of RNA extracts from COS-7 cells transfected in duplicate with pGX9501 and pGX9503. Extracted RNA was analyzed by RT-PCR using PCR assays designed for each target and for COS-7 β-Actin mRNA, used as an internal expression normalization gene. Delta C T (∆ C T ) was calculated as the C T of the target minus the C T of β-Actin for each transfection concentration and is plotted against the log of the mass of pDNA transfected (Plotted as mean ± SD). c Analysis of in vitro expression of Spike protein after transfection of 293T cells with pGX9501, pGX9503 or MOCK plasmid by Western blot. 293T cell lysates were resolved on a gel and probed with a polyclonal anti-SARS Spike Protein. Blots were stripped then probed with an anti-β-actin loading control. d In vitro immunofluorescent staining of 293T cells transfected with 3 µg/well of pGX9501, pGX9503 or pVax (empty control vector). Expression of Spike protein was measured with polyclonal anti-SARS Spike Protein IgG and anti-IgG secondary (green). Cell nuclei were counterstained with DAPI (blue). Images were captured using ImageXpress Pico automated cell imaging system. Scale bars are 80.15 µm (left), 66.8 µm (middle) and 77.31 µm (right).

    Techniques Used: Expressing, Construct, Sequencing, Reverse Transcription Polymerase Chain Reaction, Transfection, Polymerase Chain Reaction, Concentration Assay, In Vitro, Plasmid Preparation, Western Blot, Staining, Imaging

    Neutralizing antibody responses after immunization of INO-4800. BALB/c mice ( n of 5 per group) were immunized twice on days 0 and 14 with 10 µg of INO-4800. Sera was collected on day 7 post-second immunization and serial dilutions were incubated with a pseudovirus displaying the SARS-CoV-2 Spike and co-incubated with ACE2–293T cells. a Neutralization ID50 (mean ± SD) in naïve and INO-4800 immunized mice and b relative luminescence units (RLU) for sera from naive mice (green) and mice vaccinated with INO-4800 (red) as described in “Methods”.
    Figure Legend Snippet: Neutralizing antibody responses after immunization of INO-4800. BALB/c mice ( n of 5 per group) were immunized twice on days 0 and 14 with 10 µg of INO-4800. Sera was collected on day 7 post-second immunization and serial dilutions were incubated with a pseudovirus displaying the SARS-CoV-2 Spike and co-incubated with ACE2–293T cells. a Neutralization ID50 (mean ± SD) in naïve and INO-4800 immunized mice and b relative luminescence units (RLU) for sera from naive mice (green) and mice vaccinated with INO-4800 (red) as described in “Methods”.

    Techniques Used: Mouse Assay, Incubation, Neutralization

    Humoral responses to SARS-CoV-2 and SARS-CoV antigens in BALB/c mice after a single dose of INO-4800. BALB/c mice were immunized on day 0 with indicated doses of INO-4800 or pVAX-empty vector as described in the methods. a Protein antigen binding of IgG at 1:50 and 1:250 serum dilutions from mice at day 14 immunized with 25 µg of INO-4800 or pVAX. Data shown represent mean OD450 nm values (mean + SD) for each group of 3 mice. b SARS-CoV-2 S1 + 2 or c SARS-CoV-2 RBD protein antigen binding of IgG in serial serum dilutions from mice at day 14. Data shown represent mean OD450 nm values (mean + SD) for each group of eight mice ( b , c ) and five mice ( d , e ). Serum IgG binding endpoint titers to ( c ) SARS-CoV-2 S1 + 2 and ( e ) SARS-CoV-2 RBD protein. Data representative of two independent experiments. Values depicted are mean +/− SD. P values determined by Mann–Whitney test.
    Figure Legend Snippet: Humoral responses to SARS-CoV-2 and SARS-CoV antigens in BALB/c mice after a single dose of INO-4800. BALB/c mice were immunized on day 0 with indicated doses of INO-4800 or pVAX-empty vector as described in the methods. a Protein antigen binding of IgG at 1:50 and 1:250 serum dilutions from mice at day 14 immunized with 25 µg of INO-4800 or pVAX. Data shown represent mean OD450 nm values (mean + SD) for each group of 3 mice. b SARS-CoV-2 S1 + 2 or c SARS-CoV-2 RBD protein antigen binding of IgG in serial serum dilutions from mice at day 14. Data shown represent mean OD450 nm values (mean + SD) for each group of eight mice ( b , c ) and five mice ( d , e ). Serum IgG binding endpoint titers to ( c ) SARS-CoV-2 S1 + 2 and ( e ) SARS-CoV-2 RBD protein. Data representative of two independent experiments. Values depicted are mean +/− SD. P values determined by Mann–Whitney test.

    Techniques Used: Mouse Assay, Plasmid Preparation, Binding Assay, MANN-WHITNEY

    6) Product Images from "mRNA-based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection in rodents"

    Article Title: mRNA-based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection in rodents

    Journal: NPJ Vaccines

    doi: 10.1038/s41541-021-00311-w

    CVnCoV elicits high levels of humoral and cellular immune responses. Female Balb/c mice ( n = 8/group) were vaccinated IM on day 0, day 7, day 14 or day 21 with 2 µg of CVnCoV. All animals received a second immunisation on day 28 and 1.5 µg of Alum-adjuvanted SARS-CoV-2 S ectodomain (S ECD ) protein and 0.9% NaCl (buffer) administered on day 0 and day 28 served as positive and negative control, respectively. A Cytokine induction in sera of vaccinated mice was assessed 14 h post injection. Dotted lines represent the lower limit of quantification. B S ECD protein-specific binding antibodies, displayed as endpoint titres for IgG1 and IgG2a and C CPE-based virus-neutralising titres in serum upon one (day 28) or two vaccinations (day 35 and day 49). D Multifunctional IFN-γ/TNF-positive CD4 + T and CD8 + T cells analysed in splenocytes isolated on day 49. Cells were stimulated with a specific SARS-CoV-2 spike protein peptide library (15mers 11 overlapping) for 24 h followed by intracellular cytokines staining and detection by flow cytometry. Each dot represents an individual animal, bars depict the median. Statistical analysis was performed using Mann–Whitney testing.
    Figure Legend Snippet: CVnCoV elicits high levels of humoral and cellular immune responses. Female Balb/c mice ( n = 8/group) were vaccinated IM on day 0, day 7, day 14 or day 21 with 2 µg of CVnCoV. All animals received a second immunisation on day 28 and 1.5 µg of Alum-adjuvanted SARS-CoV-2 S ectodomain (S ECD ) protein and 0.9% NaCl (buffer) administered on day 0 and day 28 served as positive and negative control, respectively. A Cytokine induction in sera of vaccinated mice was assessed 14 h post injection. Dotted lines represent the lower limit of quantification. B S ECD protein-specific binding antibodies, displayed as endpoint titres for IgG1 and IgG2a and C CPE-based virus-neutralising titres in serum upon one (day 28) or two vaccinations (day 35 and day 49). D Multifunctional IFN-γ/TNF-positive CD4 + T and CD8 + T cells analysed in splenocytes isolated on day 49. Cells were stimulated with a specific SARS-CoV-2 spike protein peptide library (15mers 11 overlapping) for 24 h followed by intracellular cytokines staining and detection by flow cytometry. Each dot represents an individual animal, bars depict the median. Statistical analysis was performed using Mann–Whitney testing.

    Techniques Used: Mouse Assay, Negative Control, Injection, Binding Assay, Isolation, Staining, Flow Cytometry, MANN-WHITNEY

    CVnCoV protects hamsters from SARS-CoV-2 challenge infection. Female Syrian golden hamsters ( n = 5/group) were vaccinated with 10 or 2 µg of CVnCoV, 1.5 µg of Alum-adjuvanted S ECD protein or buffer on day 0 and d28. As additional controls, animals were either left untreated or infected intranasally (IN) with 10 2 TCID 50 /dose of SARS-CoV-2 on day 0 of the experiment. A Total IgG antibodies binding to S ECD displayed as ELISA endpoint titres of all groups except for infected animals that were not analysed or B VNTs determined via CPE-based assay upon one (day 28) or two vaccinations (day 42 and day 56). On day 56, all animals except for the untreated group were challenged by IN infection with 10 2 TCID 50 /dose of SARS-CoV-2 in a total dose volume of 0.1 ml. Animals in the untreated groups were mock infected with buffer as a negative control. Animals were followed for 4 days post challenge (p.c.) and euthanised on day 60 of the experiment. Detectable levels of replication-competent virus in C throat swabs on days 56 to day 60, D nasal turbinate on day 60 and E lung tissues on day 60 were analysed. Each dot represents an individual animal, bars depict the median. Statistical analysis was performed using Mann–Whitney testing.
    Figure Legend Snippet: CVnCoV protects hamsters from SARS-CoV-2 challenge infection. Female Syrian golden hamsters ( n = 5/group) were vaccinated with 10 or 2 µg of CVnCoV, 1.5 µg of Alum-adjuvanted S ECD protein or buffer on day 0 and d28. As additional controls, animals were either left untreated or infected intranasally (IN) with 10 2 TCID 50 /dose of SARS-CoV-2 on day 0 of the experiment. A Total IgG antibodies binding to S ECD displayed as ELISA endpoint titres of all groups except for infected animals that were not analysed or B VNTs determined via CPE-based assay upon one (day 28) or two vaccinations (day 42 and day 56). On day 56, all animals except for the untreated group were challenged by IN infection with 10 2 TCID 50 /dose of SARS-CoV-2 in a total dose volume of 0.1 ml. Animals in the untreated groups were mock infected with buffer as a negative control. Animals were followed for 4 days post challenge (p.c.) and euthanised on day 60 of the experiment. Detectable levels of replication-competent virus in C throat swabs on days 56 to day 60, D nasal turbinate on day 60 and E lung tissues on day 60 were analysed. Each dot represents an individual animal, bars depict the median. Statistical analysis was performed using Mann–Whitney testing.

    Techniques Used: Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control, MANN-WHITNEY

    CVnCoV elicits high titres of functional antibodies against SARS-CoV-2. Female Balb/c mice ( n = 7/group) were vaccinated IM with 0.25, 1 and 4 µg of CVnCoV on day 0 and day 21. Animals ( n = 5/group) vaccinated with NaCl (Buffer) served as negative controls. A Spike protein-specific binding antibodies binding to S ECD , RBD, S trimer and NTD, displayed as ELISA endpoint titres for IgG1 and IgG2a for day 21 and day 42. B CPE-based VNTs in serum samples taken on day 21 and day 42. C Per cent of S ECD -binding IgG1 and IgG2a antibodies remaining using serum from animals vaccinated with 2 µg of CVnCoV or 1.5 µg Alum-adjuvanted S ECD upon 30 min wash in 8% urea compared to buffer wash. D S-specific signal detectable on the surface of HeLa cells expressing full-length SARS-CoV-2 S upon incubation with fluorescently labelled monoclonal neutralising antibody in the absence or presence of mouse serum. Serum employed was either from mice vaccinated with 2 µg of CVnCoV, 1.5 µg of Alum-adjuvanted protein or buffer. Each dot represents an individual animal, bars depict the median. S ECD S ectodomain, RBD receptor-binding domain of S, NTD N-terminal domain of S, nAb neutralising antibody.
    Figure Legend Snippet: CVnCoV elicits high titres of functional antibodies against SARS-CoV-2. Female Balb/c mice ( n = 7/group) were vaccinated IM with 0.25, 1 and 4 µg of CVnCoV on day 0 and day 21. Animals ( n = 5/group) vaccinated with NaCl (Buffer) served as negative controls. A Spike protein-specific binding antibodies binding to S ECD , RBD, S trimer and NTD, displayed as ELISA endpoint titres for IgG1 and IgG2a for day 21 and day 42. B CPE-based VNTs in serum samples taken on day 21 and day 42. C Per cent of S ECD -binding IgG1 and IgG2a antibodies remaining using serum from animals vaccinated with 2 µg of CVnCoV or 1.5 µg Alum-adjuvanted S ECD upon 30 min wash in 8% urea compared to buffer wash. D S-specific signal detectable on the surface of HeLa cells expressing full-length SARS-CoV-2 S upon incubation with fluorescently labelled monoclonal neutralising antibody in the absence or presence of mouse serum. Serum employed was either from mice vaccinated with 2 µg of CVnCoV, 1.5 µg of Alum-adjuvanted protein or buffer. Each dot represents an individual animal, bars depict the median. S ECD S ectodomain, RBD receptor-binding domain of S, NTD N-terminal domain of S, nAb neutralising antibody.

    Techniques Used: Functional Assay, Mouse Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Incubation

    Protein translated from CVnCoV is cleaved, post-translationally modified and presented on the cell surface. A Schematic drawing of SARS-CoV-2 S-2P encoded by CVnCoV. B In vitro translation of the mRNA component of CVnCoV in a rabbit reticulocyte lysate system. Translation of nascent proteins was detected via western blotting. Water and luciferase control mRNA were employed as negative and positive controls, respectively. HeLa cells were transfected with 2 µg of the mRNA component of CVnCoV. Twenty-four hours post transfection, cells were analysed for S expression via C western blotting using an S-specific antibody and D flow cytometric analyses using an S-specific antibody either with or without membrane permeabilisation allowing detection of total (intracellular) or cell-surface bound (cell surface) S protein. Relative protein expression in western blotting was quantified using the Image Studio Lite Ver 5.2 software. Geometric mean fluorescence intensity (GMFI) of transfected HeLa cells are expressed as mean + standard deviation (SD) of duplicate samples. Blots shown in C derive from the same experiment and were processed in parallel. NTD N-terminal domain, RBD receptor-binding domain, IVT in vitro translation, TM transmembrane domain, Tub Tubulin.
    Figure Legend Snippet: Protein translated from CVnCoV is cleaved, post-translationally modified and presented on the cell surface. A Schematic drawing of SARS-CoV-2 S-2P encoded by CVnCoV. B In vitro translation of the mRNA component of CVnCoV in a rabbit reticulocyte lysate system. Translation of nascent proteins was detected via western blotting. Water and luciferase control mRNA were employed as negative and positive controls, respectively. HeLa cells were transfected with 2 µg of the mRNA component of CVnCoV. Twenty-four hours post transfection, cells were analysed for S expression via C western blotting using an S-specific antibody and D flow cytometric analyses using an S-specific antibody either with or without membrane permeabilisation allowing detection of total (intracellular) or cell-surface bound (cell surface) S protein. Relative protein expression in western blotting was quantified using the Image Studio Lite Ver 5.2 software. Geometric mean fluorescence intensity (GMFI) of transfected HeLa cells are expressed as mean + standard deviation (SD) of duplicate samples. Blots shown in C derive from the same experiment and were processed in parallel. NTD N-terminal domain, RBD receptor-binding domain, IVT in vitro translation, TM transmembrane domain, Tub Tubulin.

    Techniques Used: Modification, In Vitro, Western Blot, Luciferase, Transfection, Expressing, Software, Fluorescence, Standard Deviation, Binding Assay

    CVnCoV protects the respiratory tract from challenge infection with no signs of vaccine-enhanced disease. Histopathological analyses of hamsters vaccinated with 10 or 2 µg of CVnCoV and 1.5 µg of Alum-adjuvanted S ECD protein or buffer on day 0 and d28, left untreated and mock-infected or infected intranasally (IN) with 10 2 TCID 50 /dose of SARS-CoV-2 on day 0 of the experiment followed by of SARS-CoV-2 challenge infection on d56. Histopathological analysis was performed on day 60, 4 days post challenge infection, on formalin-fixed, paraffin-embedded tissues sections sampled on day 4 post challenge. Histopathological assessment scoring was performed according to severity of inspected parameter. Each dot represents an individual animal, bars depict the median. Statistical analysis was performed using Mann–Whitney testing.
    Figure Legend Snippet: CVnCoV protects the respiratory tract from challenge infection with no signs of vaccine-enhanced disease. Histopathological analyses of hamsters vaccinated with 10 or 2 µg of CVnCoV and 1.5 µg of Alum-adjuvanted S ECD protein or buffer on day 0 and d28, left untreated and mock-infected or infected intranasally (IN) with 10 2 TCID 50 /dose of SARS-CoV-2 on day 0 of the experiment followed by of SARS-CoV-2 challenge infection on d56. Histopathological analysis was performed on day 60, 4 days post challenge infection, on formalin-fixed, paraffin-embedded tissues sections sampled on day 4 post challenge. Histopathological assessment scoring was performed according to severity of inspected parameter. Each dot represents an individual animal, bars depict the median. Statistical analysis was performed using Mann–Whitney testing.

    Techniques Used: Infection, Formalin-fixed Paraffin-Embedded, MANN-WHITNEY

    7) Product Images from "SARS-CoV-2 infection induces autoimmune antibody secretion more in lean than in obese COVID-19 patients"

    Article Title: SARS-CoV-2 infection induces autoimmune antibody secretion more in lean than in obese COVID-19 patients

    Journal: medRxiv

    doi: 10.1101/2021.05.05.21256686

    Evaluation of Spike-specific IgG antibodies in serum samples of lean and obese COVID-19 patients, as compared to uninfected controls. SARS-CoV-2 Spike-specific IgG antibodies were measured by ELISA. *p
    Figure Legend Snippet: Evaluation of Spike-specific IgG antibodies in serum samples of lean and obese COVID-19 patients, as compared to uninfected controls. SARS-CoV-2 Spike-specific IgG antibodies were measured by ELISA. *p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    8) 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

    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

    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

    9) Product Images from "Soluble Spike DNA vaccine provides long-term protective immunity against SAR-CoV-2 in mice and nonhuman primates"

    Article Title: Soluble Spike DNA vaccine provides long-term protective immunity against SAR-CoV-2 in mice and nonhuman primates

    Journal: bioRxiv

    doi: 10.1101/2020.10.09.334136

    Diagram and immunogenicity of SARS-CoV-2 DNA vaccines. Schematic diagram of COVID-19 DNA vaccine expressing soluble SARS-CoV-2 S protein (S ΔTM ) or full-length SARS-CoV-2 S protein (S) (A) . BALB/c mice (n=4-10/group) were immunized at week 0 and 2 with pGX27-S ΔTM , pGX27-S or pGX27 (empty control vector) as described in the methods. Sera were collected 2 weeks post-prime (blue) and 2 weeks post-boost (red) and evaluated for SARS-CoV-2 S-specific IgG antibodies (B) .
    Figure Legend Snippet: Diagram and immunogenicity of SARS-CoV-2 DNA vaccines. Schematic diagram of COVID-19 DNA vaccine expressing soluble SARS-CoV-2 S protein (S ΔTM ) or full-length SARS-CoV-2 S protein (S) (A) . BALB/c mice (n=4-10/group) were immunized at week 0 and 2 with pGX27-S ΔTM , pGX27-S or pGX27 (empty control vector) as described in the methods. Sera were collected 2 weeks post-prime (blue) and 2 weeks post-boost (red) and evaluated for SARS-CoV-2 S-specific IgG antibodies (B) .

    Techniques Used: Expressing, Mouse Assay, Plasmid Preparation

    10) Product Images from "Intradermal-delivered DNA vaccine provides anamnestic protection in a rhesus macaque SARS-CoV-2 challenge model"

    Article Title: Intradermal-delivered DNA vaccine provides anamnestic protection in a rhesus macaque SARS-CoV-2 challenge model

    Journal: bioRxiv

    doi: 10.1101/2020.07.28.225649

    Humoral and cellular immune responses in rhesus macaques. ( A ) The study outline showing the vaccination regimen and sample collection timepoints. ( B ) Schematic of SARS-CoV-2 spike protein. ( C ) SARS-CoV-2 S1+S2 ECD, S1, RBD and S2 protein antigen binding of IgG in serially diluted NHP sera collected. Data represents the mean endpoint titers for each individual NHP. (D E) Pseudoneutralization assay using NHP sera, showing the presence of SARS-CoV-2 specific neutralizing antibodies against the D614 ( D ) and G614 ( E ) variants of SARS-CoV-2. ( F G ) Serum collected at Week 6 from INO-4800 vaccinated NHPs inhibited ACE2 binding to SARS-CoV-2 Spike protein. A plate-based ACE2 competition assay ( F ) and a flow-based ACE2 competition assay ( G ) showing inhibition of ACE2 binding by NHP sera. ( H ) T cell responses were measured by IFN-γ ELISpot in PBMCs stimulated for 20h with overlapping peptide pools spanning the SARS-CoV-2 Spike protein. Bars represent the mean + SD.
    Figure Legend Snippet: Humoral and cellular immune responses in rhesus macaques. ( A ) The study outline showing the vaccination regimen and sample collection timepoints. ( B ) Schematic of SARS-CoV-2 spike protein. ( C ) SARS-CoV-2 S1+S2 ECD, S1, RBD and S2 protein antigen binding of IgG in serially diluted NHP sera collected. Data represents the mean endpoint titers for each individual NHP. (D E) Pseudoneutralization assay using NHP sera, showing the presence of SARS-CoV-2 specific neutralizing antibodies against the D614 ( D ) and G614 ( E ) variants of SARS-CoV-2. ( F G ) Serum collected at Week 6 from INO-4800 vaccinated NHPs inhibited ACE2 binding to SARS-CoV-2 Spike protein. A plate-based ACE2 competition assay ( F ) and a flow-based ACE2 competition assay ( G ) showing inhibition of ACE2 binding by NHP sera. ( H ) T cell responses were measured by IFN-γ ELISpot in PBMCs stimulated for 20h with overlapping peptide pools spanning the SARS-CoV-2 Spike protein. Bars represent the mean + SD.

    Techniques Used: Binding Assay, Competitive Binding Assay, Inhibition, Enzyme-linked Immunospot

    Recall of humoral immune responses after viral challenge. ( A ) Study outline. ( B ) IgG binding ELISA. SARS-CoV-2 S1+S2 and SARS-CoV-2 RBD protein antigen binding of IgG in diluted NHP sera collected prior to challenge and post challenge in INO-4800 vaccinated (right panels) and naïve animals (left panels). ( C ) Pseudo-neutralization assay showing the presence of SARS-CoV-2 specific neutralizing antibodies against the D614 and G614 variants of SARS-CoV-2 before and after viral challenge in unvaccinated (left panels) and INO-4800 vaccinated (right panels).
    Figure Legend Snippet: Recall of humoral immune responses after viral challenge. ( A ) Study outline. ( B ) IgG binding ELISA. SARS-CoV-2 S1+S2 and SARS-CoV-2 RBD protein antigen binding of IgG in diluted NHP sera collected prior to challenge and post challenge in INO-4800 vaccinated (right panels) and naïve animals (left panels). ( C ) Pseudo-neutralization assay showing the presence of SARS-CoV-2 specific neutralizing antibodies against the D614 and G614 variants of SARS-CoV-2 before and after viral challenge in unvaccinated (left panels) and INO-4800 vaccinated (right panels).

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

    11) Product Images from "Clonal dissection of immunodominance and cross-reactivity of the CD4+ T cell response to SARS-CoV-2"

    Article Title: Clonal dissection of immunodominance and cross-reactivity of the CD4+ T cell response to SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2021.03.23.436642

    Sorting of T cell subsets and identification of SARS-CoV-2 Spike- and Nucleoprotein-reactive CD4 + T cells in COVID-19 and pre-pandemic samples. ( A ) Sorting strategy to isolate CD4 + total memory T cells and Tcm, Tem and cTfh subsets. ( B, C ) Characterization of antigen-specific T cells by CFSE dilution combined with CD25 and ICOS co-expression at day 7 following stimulation with Spike or Nucleoprotein in the presence of autologous monocytes. Negative controls of T cells cultured with monocytes alone are reported as dashed lines. Shown are data from patient P2 and from a pre-pandemic healthy donor sample (HD1).
    Figure Legend Snippet: Sorting of T cell subsets and identification of SARS-CoV-2 Spike- and Nucleoprotein-reactive CD4 + T cells in COVID-19 and pre-pandemic samples. ( A ) Sorting strategy to isolate CD4 + total memory T cells and Tcm, Tem and cTfh subsets. ( B, C ) Characterization of antigen-specific T cells by CFSE dilution combined with CD25 and ICOS co-expression at day 7 following stimulation with Spike or Nucleoprotein in the presence of autologous monocytes. Negative controls of T cells cultured with monocytes alone are reported as dashed lines. Shown are data from patient P2 and from a pre-pandemic healthy donor sample (HD1).

    Techniques Used: Transmission Electron Microscopy, Expressing, Cell Culture

    12) Product Images from "Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice"

    Article Title: Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice

    Journal: Vaccines

    doi: 10.3390/vaccines9040316

    Adjuvanted S and S1 immune sera exhibit high titers of SARS-CoV-2 pseudovirus neutralization and receptor binding inhibition activities. ( A – C ) Reduction percentage (%) in relative luminometer units (RLU) as a measure of luciferase activity for SARS-CoV-2 spike pseudotyped lentivirus infection in HEK293 cells expressing human ACE2 receptor. Data were obtained from pooled sera ( n = 6 to 8) with triplicate wells. S-0.8 (y): S 0.8 µg boost sera of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant boost sera of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant boost sera of old aged mice, S1-4 (y): S1 4 µg boost immune sera of young adult mice, S1-4 + adj (y): S1 4 µg + adjuvant boost immune sera of young adult mice, S2-4 (y): S2 4 µg boost immune sera of young adult mice, S2-4 + adj (y): S2 4 µg + adjuvant boost immune sera of young adult mice, S-0.8 + adj (y, x3): S 0.8 µg + adjuvant 2nd boost immune sera of young adult mice, S-0.8 + adj (y, x3, 19W): S 0.8 µg + adjuvant immune sera collected at week 19 post 2nd boost of young adult mice, S-4 + adj (a, x3): S 4 µg + adjuvant 2nd boost sera of old aged mice. IV-0.8-10 + adj (y, x3): inactivated adjuvanted SARS-CoV-2 vaccination in young age mice (prime 0.8 µg of heat-inactivated and gamma-irradiated virus, 2 times boost with 10 µg inactivated adjuvanted SARS-CoV-2 of heat-inactivated and gamma-irradiated virus). Adj: adjuvants (MPL + QS-21, 1 µg + 10 µg). Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. ( D – F ) ACE2 receptor binding inhibition titers in pooled immune sera ( n = 6–8) with triplicate wells. Inhibition percentage (%) of hACE2 binding to RBD was measured after incubation with serially diluted immune sera in the plate precoated with hACE2 protein. Immune sera of groups are the same as in ( A – C ). Statistical significance was calculated using two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. **; p
    Figure Legend Snippet: Adjuvanted S and S1 immune sera exhibit high titers of SARS-CoV-2 pseudovirus neutralization and receptor binding inhibition activities. ( A – C ) Reduction percentage (%) in relative luminometer units (RLU) as a measure of luciferase activity for SARS-CoV-2 spike pseudotyped lentivirus infection in HEK293 cells expressing human ACE2 receptor. Data were obtained from pooled sera ( n = 6 to 8) with triplicate wells. S-0.8 (y): S 0.8 µg boost sera of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant boost sera of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant boost sera of old aged mice, S1-4 (y): S1 4 µg boost immune sera of young adult mice, S1-4 + adj (y): S1 4 µg + adjuvant boost immune sera of young adult mice, S2-4 (y): S2 4 µg boost immune sera of young adult mice, S2-4 + adj (y): S2 4 µg + adjuvant boost immune sera of young adult mice, S-0.8 + adj (y, x3): S 0.8 µg + adjuvant 2nd boost immune sera of young adult mice, S-0.8 + adj (y, x3, 19W): S 0.8 µg + adjuvant immune sera collected at week 19 post 2nd boost of young adult mice, S-4 + adj (a, x3): S 4 µg + adjuvant 2nd boost sera of old aged mice. IV-0.8-10 + adj (y, x3): inactivated adjuvanted SARS-CoV-2 vaccination in young age mice (prime 0.8 µg of heat-inactivated and gamma-irradiated virus, 2 times boost with 10 µg inactivated adjuvanted SARS-CoV-2 of heat-inactivated and gamma-irradiated virus). Adj: adjuvants (MPL + QS-21, 1 µg + 10 µg). Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. ( D – F ) ACE2 receptor binding inhibition titers in pooled immune sera ( n = 6–8) with triplicate wells. Inhibition percentage (%) of hACE2 binding to RBD was measured after incubation with serially diluted immune sera in the plate precoated with hACE2 protein. Immune sera of groups are the same as in ( A – C ). Statistical significance was calculated using two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. **; p

    Techniques Used: Neutralization, Binding Assay, Inhibition, Luciferase, Activity Assay, Infection, Expressing, Mouse Assay, Irradiation, Incubation

    B cell and T cell immune responses to SARS-CoV-2 S vaccination in young adult and old aged mice. To determine cellular immunity, spleen cells were prepared from immunized young adult ( n = 6) and old aged mice ( n = 8). ( A ) Antibody-secreting cells (ASCs) specific for full-length S protein were determined on the ELISpot plate precoated with full-length S protein. ( B ) IFN-γ-secreting cells were analyzed by in vitro stimulation with pooled S peptides or full-length S protein using ELISpot assay. ( C , D ). IFN-γ + CD4 and IFN-γ + CD8 T cells were determined by flow cytometry after in vitro stimulation with pooled S peptides and intracellular cytokine antibi staining. S-0.8 (y): S 0.8 µg vaccination of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant vaccination of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant vaccination of old aged mice. Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. Statistical significance was calculated using one-way ANOVA and a Dunnett’s multiple-comparison test. Error bars indicate the mean ± SEM. *; p
    Figure Legend Snippet: B cell and T cell immune responses to SARS-CoV-2 S vaccination in young adult and old aged mice. To determine cellular immunity, spleen cells were prepared from immunized young adult ( n = 6) and old aged mice ( n = 8). ( A ) Antibody-secreting cells (ASCs) specific for full-length S protein were determined on the ELISpot plate precoated with full-length S protein. ( B ) IFN-γ-secreting cells were analyzed by in vitro stimulation with pooled S peptides or full-length S protein using ELISpot assay. ( C , D ). IFN-γ + CD4 and IFN-γ + CD8 T cells were determined by flow cytometry after in vitro stimulation with pooled S peptides and intracellular cytokine antibi staining. S-0.8 (y): S 0.8 µg vaccination of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant vaccination of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant vaccination of old aged mice. Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. Statistical significance was calculated using one-way ANOVA and a Dunnett’s multiple-comparison test. Error bars indicate the mean ± SEM. *; p

    Techniques Used: Mouse Assay, Enzyme-linked Immunospot, In Vitro, Flow Cytometry, Staining

    SARS-CoV-2 full-length spike (S) ectodomain and subunit proteins and receptor binding activities. ( A ) Full-length S (S1–S2) ectodomain contains aa residues 16-1213, S1 subunit aa 16-685 (green), and S2 subunit aa 686-1213. NTD: N-terminal domain (blue), RBD: receptor binding domain. ( B , C ) The receptor binding properties were determined using serially diluted soluble hACE2-Fc (0.5–2 µg/mL) on the 96-well plates precoated with 0.8 µg ( B ) or 2 µg ( C ) of S (S1–S2) and S1 subunit proteins. Due to different molecular masses of S and S1 proteins despite the same concentration, molarity in nanomoles (nM) is indicated for each protein coated.
    Figure Legend Snippet: SARS-CoV-2 full-length spike (S) ectodomain and subunit proteins and receptor binding activities. ( A ) Full-length S (S1–S2) ectodomain contains aa residues 16-1213, S1 subunit aa 16-685 (green), and S2 subunit aa 686-1213. NTD: N-terminal domain (blue), RBD: receptor binding domain. ( B , C ) The receptor binding properties were determined using serially diluted soluble hACE2-Fc (0.5–2 µg/mL) on the 96-well plates precoated with 0.8 µg ( B ) or 2 µg ( C ) of S (S1–S2) and S1 subunit proteins. Due to different molecular masses of S and S1 proteins despite the same concentration, molarity in nanomoles (nM) is indicated for each protein coated.

    Techniques Used: Binding Assay, Concentration Assay

    13) Product Images from "Prime-boost vaccination of mice and rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates"

    Article Title: Prime-boost vaccination of mice and rhesus macaques with two novel adenovirus vectored COVID-19 vaccine candidates

    Journal: Emerging Microbes & Infections

    doi: 10.1080/22221751.2021.1931466

    Characterization of Sad23L-nCoV-S and Ad49L-nCoV-S vaccines. (A) Recombinant adenovirus constructs Sad23L-nCoV-S and Ad49L-nCoV-S carrying the full-length S gene of SARS-CoV-2 under CMV promotor regulation within the deleted E1 region of Sad23L or Ad49L vector. (B) Western blot analysis for the expression of S protein from Sad23L-nCoV-S or Ad49L-nCoV-S infected HEK-293A cell lysates by rabbit polyclonal antibody to RBD. Sad23L-GFP or Ad49L-GFP virus infected cells were used as mock controls. (C) Expression of S protein in HEK-293A cells detected by immunofluorescence staining. (D) Seroprevalence of neutralizing antibody (AdNAb) to Ad5, Ad49L or Sad23L vector in 600 healthy blood donors.
    Figure Legend Snippet: Characterization of Sad23L-nCoV-S and Ad49L-nCoV-S vaccines. (A) Recombinant adenovirus constructs Sad23L-nCoV-S and Ad49L-nCoV-S carrying the full-length S gene of SARS-CoV-2 under CMV promotor regulation within the deleted E1 region of Sad23L or Ad49L vector. (B) Western blot analysis for the expression of S protein from Sad23L-nCoV-S or Ad49L-nCoV-S infected HEK-293A cell lysates by rabbit polyclonal antibody to RBD. Sad23L-GFP or Ad49L-GFP virus infected cells were used as mock controls. (C) Expression of S protein in HEK-293A cells detected by immunofluorescence staining. (D) Seroprevalence of neutralizing antibody (AdNAb) to Ad5, Ad49L or Sad23L vector in 600 healthy blood donors.

    Techniques Used: Recombinant, Construct, Plasmid Preparation, Western Blot, Expressing, Infection, Immunofluorescence, Staining

    14) Product Images from "A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes"

    Article Title: A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes

    Journal: Nature Communications

    doi: 10.1038/s41467-021-22926-2

    Structural analysis of P4A1 Fab and SARS-CoV-2 RBD complex. a The overall P4A1-Fab-RBD complex structure superimposed with the hACE2-RBD complex. The P4A1 heavy chain (colored slate blue), light chain (colored salmon red), and hACE (colored pale green) are displayed in cartoon representation. The SARS-CoV-2 RBD is colored in gray and displayed in surface representation. b The epitope of P4A1 shown in surface representation. The CDR loops of heavy chain (HCDR) and light chain (LCDR) are colored in purple and magenta, respectively. The epitopes from the heavy chain and light chain are colored in slate blue and salmon red, respectively. The only residue K417, which contacts with both heavy chain and light chain, is colored in pink. The light-chain frame region 3 (LFR3) is colored in orange. The identical residues on RBD shared in P4A1 and hACE2 binding are labeled in red. The residues are numbered according to SARS-CoV-2 RBD. c The detailed interactions between SARS-CoV-2 RBD with HCDR, LCDR, and LFR3. The residues are shown in sticks with identical colors to ( b ).
    Figure Legend Snippet: Structural analysis of P4A1 Fab and SARS-CoV-2 RBD complex. a The overall P4A1-Fab-RBD complex structure superimposed with the hACE2-RBD complex. The P4A1 heavy chain (colored slate blue), light chain (colored salmon red), and hACE (colored pale green) are displayed in cartoon representation. The SARS-CoV-2 RBD is colored in gray and displayed in surface representation. b The epitope of P4A1 shown in surface representation. The CDR loops of heavy chain (HCDR) and light chain (LCDR) are colored in purple and magenta, respectively. The epitopes from the heavy chain and light chain are colored in slate blue and salmon red, respectively. The only residue K417, which contacts with both heavy chain and light chain, is colored in pink. The light-chain frame region 3 (LFR3) is colored in orange. The identical residues on RBD shared in P4A1 and hACE2 binding are labeled in red. The residues are numbered according to SARS-CoV-2 RBD. c The detailed interactions between SARS-CoV-2 RBD with HCDR, LCDR, and LFR3. The residues are shown in sticks with identical colors to ( b ).

    Techniques Used: Binding Assay, Labeling

    Therapeutic efficacy of in the rhesus macaque model of SARS-CoV-2 infection. a Experimental design for therapeutic testing of P4A1–2A in the rhesus macaque ( n = 3/group). b Viral load in oropharyngeal swabs tested by RT-qPCR was monitored for 7 days. c Viral load in the respiratory tissues (including trachea, left and right bronchus, and all six lung lobes) collected at necropsy on 7 days post infection (d.p.i., n = 1/group) was tested by RT-qPCR. d Representative images of histopathology in lung tissue from isotype control or P4A1–2A 50 mg/kg treated animals (collected at 7 d.p.i., n = 1/group).
    Figure Legend Snippet: Therapeutic efficacy of in the rhesus macaque model of SARS-CoV-2 infection. a Experimental design for therapeutic testing of P4A1–2A in the rhesus macaque ( n = 3/group). b Viral load in oropharyngeal swabs tested by RT-qPCR was monitored for 7 days. c Viral load in the respiratory tissues (including trachea, left and right bronchus, and all six lung lobes) collected at necropsy on 7 days post infection (d.p.i., n = 1/group) was tested by RT-qPCR. d Representative images of histopathology in lung tissue from isotype control or P4A1–2A 50 mg/kg treated animals (collected at 7 d.p.i., n = 1/group).

    Techniques Used: Infection, Quantitative RT-PCR, Histopathology

    The activities of IgG4 antibody P4A1–2A to different SARS-CoV-2 S protein mutants, FcRs, and C1q. a Binding of antibody P4A1 to SARS-CoV-2 S protein N354D/D364Y, R408I, W436R, V367F, or D614G mutants determined by surface plasmon resonance (SPR). b Pseudovirus neutralization assay in hACE2-overexpressing HEK293 cells. Experiment performed in triplicates with symbols represent each of the triplicates. c The binding affinity of P4A1 and P4A1–2A for different human FcRs and complement C1q.
    Figure Legend Snippet: The activities of IgG4 antibody P4A1–2A to different SARS-CoV-2 S protein mutants, FcRs, and C1q. a Binding of antibody P4A1 to SARS-CoV-2 S protein N354D/D364Y, R408I, W436R, V367F, or D614G mutants determined by surface plasmon resonance (SPR). b Pseudovirus neutralization assay in hACE2-overexpressing HEK293 cells. Experiment performed in triplicates with symbols represent each of the triplicates. c The binding affinity of P4A1 and P4A1–2A for different human FcRs and complement C1q.

    Techniques Used: Binding Assay, SPR Assay, Neutralization

    Characterization of neutralizing antibodies from convalescent patients. a Characterization of SARS-CoV-2 S protein-specific antibodies. Upper panels: binding of antibodies to the full-length S protein, S1 protein, and S2 protein was evaluated by ELISA (in duplicates with symbols show each of the replicates). Lower left panel: blockage of the binding of SARS-CoV-2 Spike S1 protein to Vero E6 cells by antibodies evaluated by flow cytometry (data in singleton). Lower middle panel: pseudovirus neutralization assay in Huh-7 cells (data in singleton). Lower right panel: in triplicates with symbols show each of the triplicates and SARS-CoV-2 live virus neutralization assay. All experiments were repeated at least two more times (except S2 binding that was repeated one more time) with similar results. b Images of Vero E6 cell-infected SARS-CoV-2 treated with antibodies of different concentrations. Green (stained with SARS-CoV-2 nucleocapsid protein (NP) antibody) indicates viral infected cells and blue (Hoechst 33258) represents cell nuclei. Experiment was performed in triplicates and repeated two more times with similar results.
    Figure Legend Snippet: Characterization of neutralizing antibodies from convalescent patients. a Characterization of SARS-CoV-2 S protein-specific antibodies. Upper panels: binding of antibodies to the full-length S protein, S1 protein, and S2 protein was evaluated by ELISA (in duplicates with symbols show each of the replicates). Lower left panel: blockage of the binding of SARS-CoV-2 Spike S1 protein to Vero E6 cells by antibodies evaluated by flow cytometry (data in singleton). Lower middle panel: pseudovirus neutralization assay in Huh-7 cells (data in singleton). Lower right panel: in triplicates with symbols show each of the triplicates and SARS-CoV-2 live virus neutralization assay. All experiments were repeated at least two more times (except S2 binding that was repeated one more time) with similar results. b Images of Vero E6 cell-infected SARS-CoV-2 treated with antibodies of different concentrations. Green (stained with SARS-CoV-2 nucleocapsid protein (NP) antibody) indicates viral infected cells and blue (Hoechst 33258) represents cell nuclei. Experiment was performed in triplicates and repeated two more times with similar results.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Neutralization, Infection, Staining

    15) Product Images from "Intradermal-delivered DNA vaccine provides anamnestic protection in a rhesus macaque SARS-CoV-2 challenge model"

    Article Title: Intradermal-delivered DNA vaccine provides anamnestic protection in a rhesus macaque SARS-CoV-2 challenge model

    Journal: bioRxiv

    doi: 10.1101/2020.07.28.225649

    Humoral and cellular immune responses in rhesus macaques. ( A ) The study outline showing the vaccination regimen and sample collection timepoints. ( B ) Schematic of SARS-CoV-2 spike protein. ( C ) SARS-CoV-2 S1+S2 ECD, S1, RBD and S2 protein antigen binding of IgG in serially diluted NHP sera collected. Data represents the mean endpoint titers for each individual NHP. (D E) Pseudoneutralization assay using NHP sera, showing the presence of SARS-CoV-2 specific neutralizing antibodies against the D614 ( D ) and G614 ( E ) variants of SARS-CoV-2. ( F G ) Serum collected at Week 6 from INO-4800 vaccinated NHPs inhibited ACE2 binding to SARS-CoV-2 Spike protein. A plate-based ACE2 competition assay ( F ) and a flow-based ACE2 competition assay ( G ) showing inhibition of ACE2 binding by NHP sera. ( H ) T cell responses were measured by IFN-γ ELISpot in PBMCs stimulated for 20h with overlapping peptide pools spanning the SARS-CoV-2 Spike protein. Bars represent the mean + SD.
    Figure Legend Snippet: Humoral and cellular immune responses in rhesus macaques. ( A ) The study outline showing the vaccination regimen and sample collection timepoints. ( B ) Schematic of SARS-CoV-2 spike protein. ( C ) SARS-CoV-2 S1+S2 ECD, S1, RBD and S2 protein antigen binding of IgG in serially diluted NHP sera collected. Data represents the mean endpoint titers for each individual NHP. (D E) Pseudoneutralization assay using NHP sera, showing the presence of SARS-CoV-2 specific neutralizing antibodies against the D614 ( D ) and G614 ( E ) variants of SARS-CoV-2. ( F G ) Serum collected at Week 6 from INO-4800 vaccinated NHPs inhibited ACE2 binding to SARS-CoV-2 Spike protein. A plate-based ACE2 competition assay ( F ) and a flow-based ACE2 competition assay ( G ) showing inhibition of ACE2 binding by NHP sera. ( H ) T cell responses were measured by IFN-γ ELISpot in PBMCs stimulated for 20h with overlapping peptide pools spanning the SARS-CoV-2 Spike protein. Bars represent the mean + SD.

    Techniques Used: Binding Assay, Competitive Binding Assay, Inhibition, Enzyme-linked Immunospot

    Recall of humoral immune responses after viral challenge. ( A ) Study outline. ( B ) IgG binding ELISA. SARS-CoV-2 S1+S2 and SARS-CoV-2 RBD protein antigen binding of IgG in diluted NHP sera collected prior to challenge and post challenge in INO-4800 vaccinated (right panels) and naïve animals (left panels). ( C ) Pseudo-neutralization assay showing the presence of SARS-CoV-2 specific neutralizing antibodies against the D614 and G614 variants of SARS-CoV-2 before and after viral challenge in unvaccinated (left panels) and INO-4800 vaccinated (right panels).
    Figure Legend Snippet: Recall of humoral immune responses after viral challenge. ( A ) Study outline. ( B ) IgG binding ELISA. SARS-CoV-2 S1+S2 and SARS-CoV-2 RBD protein antigen binding of IgG in diluted NHP sera collected prior to challenge and post challenge in INO-4800 vaccinated (right panels) and naïve animals (left panels). ( C ) Pseudo-neutralization assay showing the presence of SARS-CoV-2 specific neutralizing antibodies against the D614 and G614 variants of SARS-CoV-2 before and after viral challenge in unvaccinated (left panels) and INO-4800 vaccinated (right panels).

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

    16) Product Images from "IgA2 Antibodies against SARS-CoV-2 Correlate with NET Formation and Fatal Outcome in Severely Diseased COVID-19 Patients"

    Article Title: IgA2 Antibodies against SARS-CoV-2 Correlate with NET Formation and Fatal Outcome in Severely Diseased COVID-19 Patients

    Journal: Cells

    doi: 10.3390/cells9122676

    SARS-CoV-2-specific IgA2 levels correlate with disease severity. ( a ) Levels of IgG, IgA1 and IgA2 directed against S1+S2 protein of SARS-CoV-2 in the plasma of healthy control subjects (N = 15) as well as SARS-CoV-2-infected patients with no or mild disease symptoms (N = 34), moderate symptoms (N = 31) or severe symptoms requiring intensive care treatment (N = 17). Dotted lines represent mean + standard deviation (SD) of the healthy control group. ( b ) Levels of IgG, IgA1 and IgA2 directed against S1+S2 protein of SARS-CoV-2 in the plasma of COVID-19 patients with severe disease who recovered (N = 13) or died (N = 4). Dotted lines represent mean + SD of the healthy control group from ( a ). Significances were tested with the Kruskal–Wallis test followed by Dunn’s multiple comparison test for all groups vs. the control group ( a ) and a two-sided Mann–Whitney U test ( b ). * p
    Figure Legend Snippet: SARS-CoV-2-specific IgA2 levels correlate with disease severity. ( a ) Levels of IgG, IgA1 and IgA2 directed against S1+S2 protein of SARS-CoV-2 in the plasma of healthy control subjects (N = 15) as well as SARS-CoV-2-infected patients with no or mild disease symptoms (N = 34), moderate symptoms (N = 31) or severe symptoms requiring intensive care treatment (N = 17). Dotted lines represent mean + standard deviation (SD) of the healthy control group. ( b ) Levels of IgG, IgA1 and IgA2 directed against S1+S2 protein of SARS-CoV-2 in the plasma of COVID-19 patients with severe disease who recovered (N = 13) or died (N = 4). Dotted lines represent mean + SD of the healthy control group from ( a ). Significances were tested with the Kruskal–Wallis test followed by Dunn’s multiple comparison test for all groups vs. the control group ( a ) and a two-sided Mann–Whitney U test ( b ). * p

    Techniques Used: Infection, Standard Deviation, MANN-WHITNEY

    SARS-CoV-2-specific IgA2 levels correlate with ecDNA in severely diseased patients. ( a , b ) Correlation of levels of IgG, IgA1 and IgA2 directed against S1+S2 protein of SARS-CoV-2 with CRP in the plasma of SARS-CoV-2-infected patients with ( a ) severe disease (N = 10) or ( b ) moderate disease (N = 31). Filled squares represent patients who died. ( c ) Correlation of levels of IgG, IgA1 and IgA2 directed against S1+S2 protein of SARS-CoV-2 in the plasma of SARS-CoV-2-infected subjects with severe disease (N = 10) with the amount of ecDNA. Filled squares represent patients who died. Significance was tested with Spearman’s correlation coefficient. p
    Figure Legend Snippet: SARS-CoV-2-specific IgA2 levels correlate with ecDNA in severely diseased patients. ( a , b ) Correlation of levels of IgG, IgA1 and IgA2 directed against S1+S2 protein of SARS-CoV-2 with CRP in the plasma of SARS-CoV-2-infected patients with ( a ) severe disease (N = 10) or ( b ) moderate disease (N = 31). Filled squares represent patients who died. ( c ) Correlation of levels of IgG, IgA1 and IgA2 directed against S1+S2 protein of SARS-CoV-2 in the plasma of SARS-CoV-2-infected subjects with severe disease (N = 10) with the amount of ecDNA. Filled squares represent patients who died. Significance was tested with Spearman’s correlation coefficient. p

    Techniques Used: Infection

    17) 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

    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

    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

    18) Product Images from "Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2"

    Article Title: Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2

    Journal: Communications Biology

    doi: 10.1038/s42003-021-02029-w

    Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.
    Figure Legend Snippet: Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.

    Techniques Used: Concentration Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

    CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.
    Figure Legend Snippet: CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.

    Techniques Used: Infection, In Vitro, In Vivo, Expressing, Activity Assay, Neutralization, Mouse Assay, Real-time Polymerase Chain Reaction

    CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.
    Figure Legend Snippet: CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.

    Techniques Used: Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Staining, Concentration Assay, Flow Cytometry

    19) Product Images from "Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies"

    Article Title: Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies

    Journal: Cell

    doi: 10.1016/j.cell.2021.04.042

    Characterization of glycan-dependent binding of FDG Abs to recombinant SARS-CoV-2 spike protein, related to Figure 6 (A) FDG, SARS-CoV-1 RBD (D001), and influenza HA (CH65) mAbs were tested in ELISA for binding to recombinant SARS-CoV-2 Spike (S) protein. mAb binding was assessed in the absence (-) or presence (+) of D-mannose [1M] to determine if free high mannose can outcompete glycans on the S protein for binding. Binding Ab titers reported as Log AUC were shown in Figure 1 . (B) FDG mAbs were tested in ELISA for binding to a set of commercially available constructs expressing the SARS-CoV-2 S1 and S2 extracellular domain (left), S2 domain (middle), and the receptor binding domain (right). Black and white bars show binding in the absence and presence of D-mannose [1M], respectively. Binding Ab titers were reported Log AUC. (C) We tested 2G12 mAb for binding to (from top to bottom) soluble stabilized recombinant HIV-1 Env trimer (CH505TF SOSIP), Man 9 -V3 and non-glycosylated aglycone V3 peptide, and recombinant SARS-CoV-2 spike ectodomain. HIV-1 CH505TF SOSIP was captured using mouse anti-AVI-tag mAb, whereas SARS-CoV-2 ectodomain and peptides (Man 9 -V3 and Aglycone) were captured using streptavidin. Blue and red symbols indicated binding in the absence and presence of D-mannose [1M], respectively. Binding was measured at OD 450nm . All ELISAs (A-C) were done using BSA-based buffers (see STAR Methods ). Data shown are from a representative assay. (D) Size-exclusion chromatogram of protein A affinity purified 2G12 IgG. (E) NSEM 2D class averages of (top) 2G12 IgG dimer, (bottom) 2G12 IgG monomer and (right) 2G12 Fab obtained by digesting 2G12 IgG monomer with papain. (F) SPR sensorgrams showing binding of 2G12 IgG dimer (red line) and 2G12 IgG monomer (black line) to the SARS-CoV-2 S protein. (G) We tested FDG mAbs for binding to the unmutated SARS-CoV-2 S and the N709-glycan deleted variant. Binding was assessed by SPR by capturing the unmutated spike and the N709-glycan deleted spike on flow cells 2 and 4 of a streptavidin coated (SA) chip, and flowing over a 200 nM solution of each Ab simultaneously over all four flow cells. Flow cells 1 and 3 were used as reference flow cells for flow cells 2 and 4, respectively. Buffer blanks were run in a similar manner and the sensorgrams were double-referenced by first subtracting the signal from the reference flow cell and then subtracting the reference-corrected buffer blank. CR3022 IgG and ACE-2 tagged with a mouse-Fc region were used as controls. (H) Binding of 2G12 to spike proteins of SARS-CoV-2 (red), SARS-CoV (blue) and MERS-CoV (green). The SARS-CoV-2/2G12 complex is shown with the spike in red and the bound 2G12 as a transparent pink surface, with the glycans contacting 2G12 shown as sticks and the respective Asn residues as spheres. Similar glycosylation was observed for the SARS-CoV and MERS-CoV S proteins (Asn residues shown as spheres). SPR binding data shown for SARS-CoV-2 (red), SARS-CoV (blue) and MERS-CoV (green) S proteins. The data shown are representative of three independent experiments, and for each dataset the graph showed three technical replicates. IgGs were captured on flow cells of a CM5 chip immobilized with human Anti-Fc antibody (8000RU). 200 nM solution of the SARS-CoV-2 spike was flowed over the flow cells. The surface was regenerated between injections by flowing over 3M MgCl2 solution for 10 s with flow rate of 100μl/min.
    Figure Legend Snippet: Characterization of glycan-dependent binding of FDG Abs to recombinant SARS-CoV-2 spike protein, related to Figure 6 (A) FDG, SARS-CoV-1 RBD (D001), and influenza HA (CH65) mAbs were tested in ELISA for binding to recombinant SARS-CoV-2 Spike (S) protein. mAb binding was assessed in the absence (-) or presence (+) of D-mannose [1M] to determine if free high mannose can outcompete glycans on the S protein for binding. Binding Ab titers reported as Log AUC were shown in Figure 1 . (B) FDG mAbs were tested in ELISA for binding to a set of commercially available constructs expressing the SARS-CoV-2 S1 and S2 extracellular domain (left), S2 domain (middle), and the receptor binding domain (right). Black and white bars show binding in the absence and presence of D-mannose [1M], respectively. Binding Ab titers were reported Log AUC. (C) We tested 2G12 mAb for binding to (from top to bottom) soluble stabilized recombinant HIV-1 Env trimer (CH505TF SOSIP), Man 9 -V3 and non-glycosylated aglycone V3 peptide, and recombinant SARS-CoV-2 spike ectodomain. HIV-1 CH505TF SOSIP was captured using mouse anti-AVI-tag mAb, whereas SARS-CoV-2 ectodomain and peptides (Man 9 -V3 and Aglycone) were captured using streptavidin. Blue and red symbols indicated binding in the absence and presence of D-mannose [1M], respectively. Binding was measured at OD 450nm . All ELISAs (A-C) were done using BSA-based buffers (see STAR Methods ). Data shown are from a representative assay. (D) Size-exclusion chromatogram of protein A affinity purified 2G12 IgG. (E) NSEM 2D class averages of (top) 2G12 IgG dimer, (bottom) 2G12 IgG monomer and (right) 2G12 Fab obtained by digesting 2G12 IgG monomer with papain. (F) SPR sensorgrams showing binding of 2G12 IgG dimer (red line) and 2G12 IgG monomer (black line) to the SARS-CoV-2 S protein. (G) We tested FDG mAbs for binding to the unmutated SARS-CoV-2 S and the N709-glycan deleted variant. Binding was assessed by SPR by capturing the unmutated spike and the N709-glycan deleted spike on flow cells 2 and 4 of a streptavidin coated (SA) chip, and flowing over a 200 nM solution of each Ab simultaneously over all four flow cells. Flow cells 1 and 3 were used as reference flow cells for flow cells 2 and 4, respectively. Buffer blanks were run in a similar manner and the sensorgrams were double-referenced by first subtracting the signal from the reference flow cell and then subtracting the reference-corrected buffer blank. CR3022 IgG and ACE-2 tagged with a mouse-Fc region were used as controls. (H) Binding of 2G12 to spike proteins of SARS-CoV-2 (red), SARS-CoV (blue) and MERS-CoV (green). The SARS-CoV-2/2G12 complex is shown with the spike in red and the bound 2G12 as a transparent pink surface, with the glycans contacting 2G12 shown as sticks and the respective Asn residues as spheres. Similar glycosylation was observed for the SARS-CoV and MERS-CoV S proteins (Asn residues shown as spheres). SPR binding data shown for SARS-CoV-2 (red), SARS-CoV (blue) and MERS-CoV (green) S proteins. The data shown are representative of three independent experiments, and for each dataset the graph showed three technical replicates. IgGs were captured on flow cells of a CM5 chip immobilized with human Anti-Fc antibody (8000RU). 200 nM solution of the SARS-CoV-2 spike was flowed over the flow cells. The surface was regenerated between injections by flowing over 3M MgCl2 solution for 10 s with flow rate of 100μl/min.

    Techniques Used: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Construct, Expressing, Affinity Purification, SPR Assay, Variant Assay, Chromatin Immunoprecipitation

    Cryo-EM data processing details for SARS-CoV-2 S protein complex with 2G12, related to Figure 6 (A) Representative micrograph. (B) CTF fit (C) Representative 2D class averages. (D) Maps for (left) unliganded and (right) 2G12-bound S obtained after 3 D classification. (E-G) Refined maps for SARS-CoV-2 S protein bound to (E) 1-2G12, (F) 2-2G12-, and (G) 2-2G12 (with partial occupancy at the third binding site) Fab2 molecules. Red arrow in (G) points to disordered 2G12 Fab2 bound at the third binding site. (H) (Left) Map combining all particles and focusing refinement on the region within the masks that is shown as a gray mesh overlaid on the final refined map shown as a gray surface. (Right) Fourier shell correlation curves. (I) (Left) Cryo-EM reconstruction of 2G12 bound to the SARS-CoV-2 spike colored by local resolution. (Right) Zoomed-in view showing the cryo-EM reconstruction of the bound 2G12 Fab. (J) (Left) Two distinct states were resolved from the cryo-EM data by heterogeneous classification. Density for the two observed states were shown in green and gray. (Right) Cartoon representation of the SARS-CoV-2 S-protein (bright green, bright orange, blue) and the two 2G12 orientations. The axis of rotation hinged around glycan 709 is represented by a gray cylinder. (K) Zoomed-in view of (from left to right) a region in the S2 domain with map shown as blue mesh and fitted model shown as sticks; glycan 709; glycan 801; glycan 717 bound to 2G12. While not in direct contact with the bound antibody, the HR1 helix may play an indirect role in the binding by stabilizing glycan 717 via a stacking interaction with residues N925 and Q926.
    Figure Legend Snippet: Cryo-EM data processing details for SARS-CoV-2 S protein complex with 2G12, related to Figure 6 (A) Representative micrograph. (B) CTF fit (C) Representative 2D class averages. (D) Maps for (left) unliganded and (right) 2G12-bound S obtained after 3 D classification. (E-G) Refined maps for SARS-CoV-2 S protein bound to (E) 1-2G12, (F) 2-2G12-, and (G) 2-2G12 (with partial occupancy at the third binding site) Fab2 molecules. Red arrow in (G) points to disordered 2G12 Fab2 bound at the third binding site. (H) (Left) Map combining all particles and focusing refinement on the region within the masks that is shown as a gray mesh overlaid on the final refined map shown as a gray surface. (Right) Fourier shell correlation curves. (I) (Left) Cryo-EM reconstruction of 2G12 bound to the SARS-CoV-2 spike colored by local resolution. (Right) Zoomed-in view showing the cryo-EM reconstruction of the bound 2G12 Fab. (J) (Left) Two distinct states were resolved from the cryo-EM data by heterogeneous classification. Density for the two observed states were shown in green and gray. (Right) Cartoon representation of the SARS-CoV-2 S-protein (bright green, bright orange, blue) and the two 2G12 orientations. The axis of rotation hinged around glycan 709 is represented by a gray cylinder. (K) Zoomed-in view of (from left to right) a region in the S2 domain with map shown as blue mesh and fitted model shown as sticks; glycan 709; glycan 801; glycan 717 bound to 2G12. While not in direct contact with the bound antibody, the HR1 helix may play an indirect role in the binding by stabilizing glycan 717 via a stacking interaction with residues N925 and Q926.

    Techniques Used: Cryo-EM Sample Prep, Binding Assay

    Glycan-dependent binding of FDG Abs to recombinant SARS-CoV-2 spike (A) FDG mAbs were tested for binding recombinant SARS-CoV-2 Spike (S) in ELISA. Ab binding was assessed in the absence (−) or presence (+) of 1M D-mannose. Binding Ab titers were reported as Log AUC. Controls were SARS-CoV-1 RBD (D001) and influenza HA (CH65) reactive mAbs. Data shown are from a representative assay. (B) Cryo-EM reconstruction of 2G12 in complex with recombinant SARS-CoV-2 S. The cryo-EM map was colored by chain. SARS-CoV-2 S chains were colored salmon, green, and blue. The 2G12 chains are colored dark gray and orange for the heavy chains (HC), and yellow and dark pink for the light chains (LC). (C) Top: schematic showing domain organization of SARS-CoV-2 S, with positions of N-linked glycosylation sequons numbered and shown as branches. Bottom: zoomed-in view of domain-swapped, dimerized 2G12 Fab interacting with SARS-CoV-2 S. The structure was colored by chain as in (B), with 2G12 and SARS-CoV-2 S shown in cartoon representation and the interacting glycans in surface representation. (D) Binding of 2G12 Fab dimer to (top) unmutated and (bottom) N709A mutant recombinant S proteins, measured by SPR using single-cycle kinetics. The black lines show the data and the red lines show the fit of the data to a 1:1 Langmuir binding model. (E) Binding of the unmutated and N709A mutant S proteins to a panel of FDG Abs measured by SPR; data shown as a heatmap for Log AUC binding. See also Figures S6 and S7 and Data S4 .
    Figure Legend Snippet: Glycan-dependent binding of FDG Abs to recombinant SARS-CoV-2 spike (A) FDG mAbs were tested for binding recombinant SARS-CoV-2 Spike (S) in ELISA. Ab binding was assessed in the absence (−) or presence (+) of 1M D-mannose. Binding Ab titers were reported as Log AUC. Controls were SARS-CoV-1 RBD (D001) and influenza HA (CH65) reactive mAbs. Data shown are from a representative assay. (B) Cryo-EM reconstruction of 2G12 in complex with recombinant SARS-CoV-2 S. The cryo-EM map was colored by chain. SARS-CoV-2 S chains were colored salmon, green, and blue. The 2G12 chains are colored dark gray and orange for the heavy chains (HC), and yellow and dark pink for the light chains (LC). (C) Top: schematic showing domain organization of SARS-CoV-2 S, with positions of N-linked glycosylation sequons numbered and shown as branches. Bottom: zoomed-in view of domain-swapped, dimerized 2G12 Fab interacting with SARS-CoV-2 S. The structure was colored by chain as in (B), with 2G12 and SARS-CoV-2 S shown in cartoon representation and the interacting glycans in surface representation. (D) Binding of 2G12 Fab dimer to (top) unmutated and (bottom) N709A mutant recombinant S proteins, measured by SPR using single-cycle kinetics. The black lines show the data and the red lines show the fit of the data to a 1:1 Langmuir binding model. (E) Binding of the unmutated and N709A mutant S proteins to a panel of FDG Abs measured by SPR; data shown as a heatmap for Log AUC binding. See also Figures S6 and S7 and Data S4 .

    Techniques Used: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Cryo-EM Sample Prep, Mutagenesis, SPR Assay

    20) Product Images from "Insight into Vaccine Development for Alphacoronaviruses Based on Structural and Immunological Analyses of Spike Proteins"

    Article Title: Insight into Vaccine Development for Alphacoronaviruses Based on Structural and Immunological Analyses of Spike Proteins

    Journal: Journal of Virology

    doi: 10.1128/JVI.02284-20

    Structure-based B-cell epitope predictions of Beta-CoV (SARS-CoV and SARS-CoV-2) and Alpha-CoV (HCoV-229E). (A, C, and E) The predicted B-cell epitopes of SARS-CoV, SARS-CoV-2, and HCoV-229E are shown. The linear (red cartoon) and conformational (yellow sphere) B-cell epitopes were predicted using Bepipred 2.0 or DiscoTope 2.0 and labeled into the corresponding structure via PyMOL. (B, D, and F) The complex structures of the RBDs of SARS-CoV (PDB ID: 2AJF ), SARS-CoV-2 (PDB ID: 6M0J ), and HCoV-229E (PDB ID: 6ATK ) with the receptors (hACE2 and hAPN) are shown. The interface area and the surface area were calculated via PDBePISA. The RBM region of the RBD and the receptors (hACE2 and hAPN) are shown in red and cyan, respectively.
    Figure Legend Snippet: Structure-based B-cell epitope predictions of Beta-CoV (SARS-CoV and SARS-CoV-2) and Alpha-CoV (HCoV-229E). (A, C, and E) The predicted B-cell epitopes of SARS-CoV, SARS-CoV-2, and HCoV-229E are shown. The linear (red cartoon) and conformational (yellow sphere) B-cell epitopes were predicted using Bepipred 2.0 or DiscoTope 2.0 and labeled into the corresponding structure via PyMOL. (B, D, and F) The complex structures of the RBDs of SARS-CoV (PDB ID: 2AJF ), SARS-CoV-2 (PDB ID: 6M0J ), and HCoV-229E (PDB ID: 6ATK ) with the receptors (hACE2 and hAPN) are shown. The interface area and the surface area were calculated via PDBePISA. The RBM region of the RBD and the receptors (hACE2 and hAPN) are shown in red and cyan, respectively.

    Techniques Used: Labeling

    Immunological analysis of Beta-CoV (SARS-CoV and SARS-CoV-2) and Alpha-CoV (HCoV-229E) spike proteins. (A and B) Cross-reactivity of the SARS-CoV S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for SARS-CoV S-trimer (red) and SARS-CoV RBD (blue) were 2-fold serially diluted and reacted with the S-trimer (A) or RBD (B), respectively. (C and D) Cross-reactivity of the SARS-CoV-2 S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for SARS-CoV-2 S-trimer (magenta) and SARS-CoV-2 RBD (cyan) were 10-fold diluted and reacted with SARS-CoV-2 S-trimer (C) and RBD (D). (E and F) Cross-reactivity of the HCoV-229E S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for HCoV-229E S-trimer (orange) and HCoV-229E RBD (green) were 2-fold diluted and reacted with HCoV-229E S-trimer (E) and RBD (F). (G and H) The antibody titers of sera from mice immunized with the HCoV-229E RBD (10 μg, brown; 50 μg, purple). Mouse sera were reacted with the HCoV-229E RBD (G) or S-trimer (H). Mouse sera were serially diluted from a 500-fold dilution. All the data are presented as the mean OD 450 ± SD ( n = 3), and the IgG antibody titers of each serum were calculated as the maximum endpoint dilution that remained positive. (I, J, and K) The neutralization assay of mouse sera from the S-trimer and RBD against SARS-CoV, SARS-CoV-2, and HCoV-229E pseudoviruses. The limit of detection for the assay depends on the initial dilution and is represented by dotted lines; a reciprocal IC 90 titer of 40 was assigned. The data are presented as the mean reciprocal IC 90 titer ± SD ( n = 3). All differences between means with P
    Figure Legend Snippet: Immunological analysis of Beta-CoV (SARS-CoV and SARS-CoV-2) and Alpha-CoV (HCoV-229E) spike proteins. (A and B) Cross-reactivity of the SARS-CoV S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for SARS-CoV S-trimer (red) and SARS-CoV RBD (blue) were 2-fold serially diluted and reacted with the S-trimer (A) or RBD (B), respectively. (C and D) Cross-reactivity of the SARS-CoV-2 S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for SARS-CoV-2 S-trimer (magenta) and SARS-CoV-2 RBD (cyan) were 10-fold diluted and reacted with SARS-CoV-2 S-trimer (C) and RBD (D). (E and F) Cross-reactivity of the HCoV-229E S-trimer and RBD-specific sera is measured using ELISA. Mouse sera for HCoV-229E S-trimer (orange) and HCoV-229E RBD (green) were 2-fold diluted and reacted with HCoV-229E S-trimer (E) and RBD (F). (G and H) The antibody titers of sera from mice immunized with the HCoV-229E RBD (10 μg, brown; 50 μg, purple). Mouse sera were reacted with the HCoV-229E RBD (G) or S-trimer (H). Mouse sera were serially diluted from a 500-fold dilution. All the data are presented as the mean OD 450 ± SD ( n = 3), and the IgG antibody titers of each serum were calculated as the maximum endpoint dilution that remained positive. (I, J, and K) The neutralization assay of mouse sera from the S-trimer and RBD against SARS-CoV, SARS-CoV-2, and HCoV-229E pseudoviruses. The limit of detection for the assay depends on the initial dilution and is represented by dotted lines; a reciprocal IC 90 titer of 40 was assigned. The data are presented as the mean reciprocal IC 90 titer ± SD ( n = 3). All differences between means with P

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

    Structural analysis of S1-RBDs from coronavirus S-trimers. (A) Schematic diagram of coronavirus spike protein organization. S1, receptor-binding subunit; S2, membrane fusion subunit; NTD, N-terminal domain; RBD, receptor-binding domain; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic tail. (B) Overall structure comparison of coronavirus S-trimers. The S-trimer structures of HCoV-229E (PDB ID: 6U7H ), HCoV-NL63 (PDB ID: 5SZS ), PEDV (PDB ID: 6U7K ), FIPV (PDB ID: 6JX7 ), PDCoV (PDB ID: 6BFU ), IBV (PDB ID: 6CV0 ), SARS-CoV (PDB ID: 5X5B ), SARS-CoV-2 (PDB ID: 6VSB ), MERS-CoV (PDB ID: 5X5F ), HCoV-HKU1 (PDB ID: 5I08 ), HCoV-OC43 (PDB ID: 6OHW ), and mouse hepatitis virus (MHV) (PDB ID: 3JCL ) are shown. The S1-RBDs are colored in magenta. The lengths of the coronavirus S-trimers are shown as previously reported.
    Figure Legend Snippet: Structural analysis of S1-RBDs from coronavirus S-trimers. (A) Schematic diagram of coronavirus spike protein organization. S1, receptor-binding subunit; S2, membrane fusion subunit; NTD, N-terminal domain; RBD, receptor-binding domain; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic tail. (B) Overall structure comparison of coronavirus S-trimers. The S-trimer structures of HCoV-229E (PDB ID: 6U7H ), HCoV-NL63 (PDB ID: 5SZS ), PEDV (PDB ID: 6U7K ), FIPV (PDB ID: 6JX7 ), PDCoV (PDB ID: 6BFU ), IBV (PDB ID: 6CV0 ), SARS-CoV (PDB ID: 5X5B ), SARS-CoV-2 (PDB ID: 6VSB ), MERS-CoV (PDB ID: 5X5F ), HCoV-HKU1 (PDB ID: 5I08 ), HCoV-OC43 (PDB ID: 6OHW ), and mouse hepatitis virus (MHV) (PDB ID: 3JCL ) are shown. The S1-RBDs are colored in magenta. The lengths of the coronavirus S-trimers are shown as previously reported.

    Techniques Used: Binding Assay

    21) Product Images from "Single-cell sequencing of plasma cells from COVID-19 patients reveals highly expanded clonal lineages produce specific and neutralizing antibodies to SARS-CoV-2"

    Article Title: Single-cell sequencing of plasma cells from COVID-19 patients reveals highly expanded clonal lineages produce specific and neutralizing antibodies to SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2021.02.12.430940

    An integrated workflow for interrogating the antibody specificity of PCs from COVID-19 patients. Serum and peripheral blood mononuclear cells (PBMCs) are collected from convalescent COVID-19 patients (with confirmed PCR positive test). Serum is assayed with IgA and IgG ELISAs as well as POCTs. From a subset of 16 patients PCs are isolated from PBMCs by magnetic cell sorting and to then undergo gel encapsulation and barcoding for single-cell sequencing of their antibody heavy and light chain transcripts. Antibody repertoire analysis is performed to identify expanded plasma cell clonal lineages, which are then reformatted into single ORF full-length synthetic antibody genes including homology arms, to allow for single step cloning-free genome editing. The resulting mammalian display library then undergoes high-throughput screening for SARS-CoV-2 binding by flow cytometry and deep sequencing to recover the identity of the corresponding clonal lineages. Supernatant is used to determine cross-reactivity of antibodies within the library with coronavirus antigens.
    Figure Legend Snippet: An integrated workflow for interrogating the antibody specificity of PCs from COVID-19 patients. Serum and peripheral blood mononuclear cells (PBMCs) are collected from convalescent COVID-19 patients (with confirmed PCR positive test). Serum is assayed with IgA and IgG ELISAs as well as POCTs. From a subset of 16 patients PCs are isolated from PBMCs by magnetic cell sorting and to then undergo gel encapsulation and barcoding for single-cell sequencing of their antibody heavy and light chain transcripts. Antibody repertoire analysis is performed to identify expanded plasma cell clonal lineages, which are then reformatted into single ORF full-length synthetic antibody genes including homology arms, to allow for single step cloning-free genome editing. The resulting mammalian display library then undergoes high-throughput screening for SARS-CoV-2 binding by flow cytometry and deep sequencing to recover the identity of the corresponding clonal lineages. Supernatant is used to determine cross-reactivity of antibodies within the library with coronavirus antigens.

    Techniques Used: Polymerase Chain Reaction, Isolation, FACS, Sequencing, Clone Assay, High Throughput Screening Assay, Binding Assay, Flow Cytometry

    22) 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

    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

    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

    23) Product Images from "Quantum Dot-Conjugated SARS-CoV-2 Spike Pseudo-Virions Enable Tracking of Angiotensin Converting Enzyme 2 Binding and Endocytosis"

    Article Title: Quantum Dot-Conjugated SARS-CoV-2 Spike Pseudo-Virions Enable Tracking of Angiotensin Converting Enzyme 2 Binding and Endocytosis

    Journal: ACS Nano

    doi: 10.1021/acsnano.0c05975

    NP-based inhibition assay. (a) Left: The structure of neutralizing antibody (top) bound to SARS-CoV-2 Spike RBD (bottom, green). Right: Schematic diagram of the inhibition assay depicting blocking of the interaction between RBD and ACE2 and the resulting inhibition of energy transfer from QD to AuNP. (b) PL recovery of QD 514 -RBD in the presence of neutralizing antibody Ab1. (c) Inhibition test using anti-Spike antibody without neutralizing ability, showing almost no PL recovery of QD 514 -RBD. (d) Calculated EC 50 s for neutralizing antibodies Ab1 and Ab2 were 60 nM and 125 nM with R 2 > 99%, respectively.
    Figure Legend Snippet: NP-based inhibition assay. (a) Left: The structure of neutralizing antibody (top) bound to SARS-CoV-2 Spike RBD (bottom, green). Right: Schematic diagram of the inhibition assay depicting blocking of the interaction between RBD and ACE2 and the resulting inhibition of energy transfer from QD to AuNP. (b) PL recovery of QD 514 -RBD in the presence of neutralizing antibody Ab1. (c) Inhibition test using anti-Spike antibody without neutralizing ability, showing almost no PL recovery of QD 514 -RBD. (d) Calculated EC 50 s for neutralizing antibodies Ab1 and Ab2 were 60 nM and 125 nM with R 2 > 99%, respectively.

    Techniques Used: Inhibition, Blocking Assay

    24) Product Images from "SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface"

    Article Title: SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

    Journal: Nature Communications

    doi: 10.1038/s41467-021-21609-2

    Characterization of the neutralizing antibody STE73-2E9 in IgG format. A Neutralization of 20–30 pfu SARS-CoV-2 by STE73-2E9, -9G3, and -2G8. Palivizumab was used as isotype control. B Validation of neutralization potency of STE73-2E9 using 100 pfu. Neutralization assays were performed in triplicates, mean ± s.e.m. are given. C Titration ELISA on the indicated antigens. ELISA shows single titration of two representative experiments (see also Supplementary Fig. 7 ). D Cross-reactivity to other coronavirus spike proteins analzyed by ELISA. S1-HIS SARS-CoV-2 Hi5 was produced in house. S1-HIS SARS-CoV-2 HEK and all other coronavirus S1 domain proteins were obtained commercially. ELISA experiments were performed in duplicate and the mean values are given. E , F Kinetic parameter determination through single-cycle kinetic titration SPR of STE73-2E9 IgG on HEK cell produced RBD-SD1 and S1-S2, respectively (concentrations: 200, 100, 50, 25, 12.5, 6.25 nM).
    Figure Legend Snippet: Characterization of the neutralizing antibody STE73-2E9 in IgG format. A Neutralization of 20–30 pfu SARS-CoV-2 by STE73-2E9, -9G3, and -2G8. Palivizumab was used as isotype control. B Validation of neutralization potency of STE73-2E9 using 100 pfu. Neutralization assays were performed in triplicates, mean ± s.e.m. are given. C Titration ELISA on the indicated antigens. ELISA shows single titration of two representative experiments (see also Supplementary Fig. 7 ). D Cross-reactivity to other coronavirus spike proteins analzyed by ELISA. S1-HIS SARS-CoV-2 Hi5 was produced in house. S1-HIS SARS-CoV-2 HEK and all other coronavirus S1 domain proteins were obtained commercially. ELISA experiments were performed in duplicate and the mean values are given. E , F Kinetic parameter determination through single-cycle kinetic titration SPR of STE73-2E9 IgG on HEK cell produced RBD-SD1 and S1-S2, respectively (concentrations: 200, 100, 50, 25, 12.5, 6.25 nM).

    Techniques Used: Neutralization, Titration, Enzyme-linked Immunosorbent Assay, Produced, SPR Assay

    Inhibition of SARS-CoV-2 spike protein binding to cell (flow cytometry). A Inhibition prescreen of 109 scFv-Fc antibodies on ACE2-positive cells using 1500 nM antibody and 50 nM spike protein (30:1 ratio). The antibodies selected for detailed analysis are marked in colors. Data show single measurements. B IC50 determination by flow cytometry using 50 nM S1-S2 trimer and 4.7–1500 nM scFv-Fc. C IC50 determination by flow cytometry using 10 nM RBD and 0.03–1000 nM scFv-Fc. The inhibition assays were made as single titrations. Logistic5 fit of Origin was used to determine the IC50.
    Figure Legend Snippet: Inhibition of SARS-CoV-2 spike protein binding to cell (flow cytometry). A Inhibition prescreen of 109 scFv-Fc antibodies on ACE2-positive cells using 1500 nM antibody and 50 nM spike protein (30:1 ratio). The antibodies selected for detailed analysis are marked in colors. Data show single measurements. B IC50 determination by flow cytometry using 50 nM S1-S2 trimer and 4.7–1500 nM scFv-Fc. C IC50 determination by flow cytometry using 10 nM RBD and 0.03–1000 nM scFv-Fc. The inhibition assays were made as single titrations. Logistic5 fit of Origin was used to determine the IC50.

    Techniques Used: Inhibition, Protein Binding, Flow Cytometry

    Determination of EC50 on RBD. Binding in titration ELISA of the 17 best inhibiting scFv-Fc on RBD (fusion protein with murine Fc part), S1 (fusion protein with murine Fc part), or S1-S2 (fusion protein with His tag). Sequence SARS-CoV-2 (Gene bank QHD43416). An unrelated antibody with murine Fc part (TUN219-2C1), human HEK293 cell lysate, BSA, or lysozyme were used as controls. Experiments were performed in duplicate and mean values are given. EC50 were calculated with GraphPad Prism Version 6.1, fitting to a four-parameter logistic curve.
    Figure Legend Snippet: Determination of EC50 on RBD. Binding in titration ELISA of the 17 best inhibiting scFv-Fc on RBD (fusion protein with murine Fc part), S1 (fusion protein with murine Fc part), or S1-S2 (fusion protein with His tag). Sequence SARS-CoV-2 (Gene bank QHD43416). An unrelated antibody with murine Fc part (TUN219-2C1), human HEK293 cell lysate, BSA, or lysozyme were used as controls. Experiments were performed in duplicate and mean values are given. EC50 were calculated with GraphPad Prism Version 6.1, fitting to a four-parameter logistic curve.

    Techniques Used: Binding Assay, Titration, Enzyme-linked Immunosorbent Assay, Sequencing

    25) Product Images from "Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2"

    Article Title: Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2

    Journal: Communications Biology

    doi: 10.1038/s42003-021-02029-w

    Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.
    Figure Legend Snippet: Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.

    Techniques Used: Concentration Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

    CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.
    Figure Legend Snippet: CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.

    Techniques Used: Infection, In Vitro, In Vivo, Expressing, Activity Assay, Neutralization, Mouse Assay, Real-time Polymerase Chain Reaction

    CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.
    Figure Legend Snippet: CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.

    Techniques Used: Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Staining, Concentration Assay, Flow Cytometry

    26) Product Images from "Original antigenic sin responses to heterologous Betacoronavirus spike proteins are observed in mice following intramuscular administration, but are not apparent in children following SARS-CoV-2 infection"

    Article Title: Original antigenic sin responses to heterologous Betacoronavirus spike proteins are observed in mice following intramuscular administration, but are not apparent in children following SARS-CoV-2 infection

    Journal: medRxiv

    doi: 10.1101/2021.04.29.21256344

    HKU1 antibodies are prevalent in healthy children and children with acute COVID-19 and MIS-C. SARS-CoV-2 (A) and HKU1 (B) spike IgG antibody titers and FRNT neutralization titers (C) in healthy pediatric controls compared to children hospitalized with acute COVID-19 and MIS-C. * P
    Figure Legend Snippet: HKU1 antibodies are prevalent in healthy children and children with acute COVID-19 and MIS-C. SARS-CoV-2 (A) and HKU1 (B) spike IgG antibody titers and FRNT neutralization titers (C) in healthy pediatric controls compared to children hospitalized with acute COVID-19 and MIS-C. * P

    Techniques Used: Neutralization

    Schematic of intramuscular spike protein administrations in groups of five BALB/c mice. Group 1 received prime and boost with SARS-CoV-2 spike, followed by prime and boost with HKU1 spike. Group 2 received a reciprocal administration regimen, with prime and boost with HKU1 spike, followed by prime and boost by HKU1 spike. D, days post-administration; S, spike; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. *These mice were immunized with nucleocapsid protein 21 and 42 days prior to utilization for this study.
    Figure Legend Snippet: Schematic of intramuscular spike protein administrations in groups of five BALB/c mice. Group 1 received prime and boost with SARS-CoV-2 spike, followed by prime and boost with HKU1 spike. Group 2 received a reciprocal administration regimen, with prime and boost with HKU1 spike, followed by prime and boost by HKU1 spike. D, days post-administration; S, spike; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. *These mice were immunized with nucleocapsid protein 21 and 42 days prior to utilization for this study.

    Techniques Used: Mouse Assay

    HKU1 spike IgG antibodies correlated positively with both SAR-CoV-2 spike IgG and SARS-CoV-2 neutralizing antibodies in children with acute COVID-19 and MIS-C. Linear regression analyses compared the log-transformed antibody titers of (A) SARS-CoV-2 spike IgG vs. HKU1 spike IgG; (B) HKU1 spike IgG vs. SARS-CoV-2 neutralization titers; and (C) SARS-CoV-2 spike IgG vs. SARS-CoV-2 neutralization titers among children with acute COVID-19 or MIS-C. Spearman’s correlation coefficients (r) and P-values are shown.
    Figure Legend Snippet: HKU1 spike IgG antibodies correlated positively with both SAR-CoV-2 spike IgG and SARS-CoV-2 neutralizing antibodies in children with acute COVID-19 and MIS-C. Linear regression analyses compared the log-transformed antibody titers of (A) SARS-CoV-2 spike IgG vs. HKU1 spike IgG; (B) HKU1 spike IgG vs. SARS-CoV-2 neutralization titers; and (C) SARS-CoV-2 spike IgG vs. SARS-CoV-2 neutralization titers among children with acute COVID-19 or MIS-C. Spearman’s correlation coefficients (r) and P-values are shown.

    Techniques Used: Transformation Assay, Neutralization

    Priming mice with HKU1 spike protein prior to boosting with SARS-CoV-2 spike protein completely impeded the development of SARS-CoV-2 neutralizing antibodies. SARS-CoV-2 (A,B) and HKU1 (C,D) full-length spike IgG binding and SARS-CoV-2 neutralizing (E, F) antibodies in mice are shown as log(end-point titer). Group 1 was primed with two doses of alum-adjuvanted SARS-CoV-2 spike and boosted with two doses of alum-adjuvanted HKU1 spike (A, C, E). Group 2 received the reciprocal regimen of HKU1 spike prime and SARS-CoV-2 spike boost (B, D, F). * P
    Figure Legend Snippet: Priming mice with HKU1 spike protein prior to boosting with SARS-CoV-2 spike protein completely impeded the development of SARS-CoV-2 neutralizing antibodies. SARS-CoV-2 (A,B) and HKU1 (C,D) full-length spike IgG binding and SARS-CoV-2 neutralizing (E, F) antibodies in mice are shown as log(end-point titer). Group 1 was primed with two doses of alum-adjuvanted SARS-CoV-2 spike and boosted with two doses of alum-adjuvanted HKU1 spike (A, C, E). Group 2 received the reciprocal regimen of HKU1 spike prime and SARS-CoV-2 spike boost (B, D, F). * P

    Techniques Used: Mouse Assay, Binding Assay

    27) Product Images from "mRNA-based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection in rodents"

    Article Title: mRNA-based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection in rodents

    Journal: NPJ Vaccines

    doi: 10.1038/s41541-021-00311-w

    CVnCoV elicits high levels of humoral and cellular immune responses. Female Balb/c mice ( n = 8/group) were vaccinated IM on day 0, day 7, day 14 or day 21 with 2 µg of CVnCoV. All animals received a second immunisation on day 28 and 1.5 µg of Alum-adjuvanted SARS-CoV-2 S ectodomain (S ECD ) protein and 0.9% NaCl (buffer) administered on day 0 and day 28 served as positive and negative control, respectively. A Cytokine induction in sera of vaccinated mice was assessed 14 h post injection. Dotted lines represent the lower limit of quantification. B S ECD protein-specific binding antibodies, displayed as endpoint titres for IgG1 and IgG2a and C CPE-based virus-neutralising titres in serum upon one (day 28) or two vaccinations (day 35 and day 49). D Multifunctional IFN-γ/TNF-positive CD4 + T and CD8 + T cells analysed in splenocytes isolated on day 49. Cells were stimulated with a specific SARS-CoV-2 spike protein peptide library (15mers 11 overlapping) for 24 h followed by intracellular cytokines staining and detection by flow cytometry. Each dot represents an individual animal, bars depict the median. Statistical analysis was performed using Mann–Whitney testing.
    Figure Legend Snippet: CVnCoV elicits high levels of humoral and cellular immune responses. Female Balb/c mice ( n = 8/group) were vaccinated IM on day 0, day 7, day 14 or day 21 with 2 µg of CVnCoV. All animals received a second immunisation on day 28 and 1.5 µg of Alum-adjuvanted SARS-CoV-2 S ectodomain (S ECD ) protein and 0.9% NaCl (buffer) administered on day 0 and day 28 served as positive and negative control, respectively. A Cytokine induction in sera of vaccinated mice was assessed 14 h post injection. Dotted lines represent the lower limit of quantification. B S ECD protein-specific binding antibodies, displayed as endpoint titres for IgG1 and IgG2a and C CPE-based virus-neutralising titres in serum upon one (day 28) or two vaccinations (day 35 and day 49). D Multifunctional IFN-γ/TNF-positive CD4 + T and CD8 + T cells analysed in splenocytes isolated on day 49. Cells were stimulated with a specific SARS-CoV-2 spike protein peptide library (15mers 11 overlapping) for 24 h followed by intracellular cytokines staining and detection by flow cytometry. Each dot represents an individual animal, bars depict the median. Statistical analysis was performed using Mann–Whitney testing.

    Techniques Used: Mouse Assay, Negative Control, Injection, Binding Assay, Isolation, Staining, Flow Cytometry, MANN-WHITNEY

    CVnCoV protects hamsters from SARS-CoV-2 challenge infection. Female Syrian golden hamsters ( n = 5/group) were vaccinated with 10 or 2 µg of CVnCoV, 1.5 µg of Alum-adjuvanted S ECD protein or buffer on day 0 and d28. As additional controls, animals were either left untreated or infected intranasally (IN) with 10 2 TCID 50 /dose of SARS-CoV-2 on day 0 of the experiment. A Total IgG antibodies binding to S ECD displayed as ELISA endpoint titres of all groups except for infected animals that were not analysed or B VNTs determined via CPE-based assay upon one (day 28) or two vaccinations (day 42 and day 56). On day 56, all animals except for the untreated group were challenged by IN infection with 10 2 TCID 50 /dose of SARS-CoV-2 in a total dose volume of 0.1 ml. Animals in the untreated groups were mock infected with buffer as a negative control. Animals were followed for 4 days post challenge (p.c.) and euthanised on day 60 of the experiment. Detectable levels of replication-competent virus in C throat swabs on days 56 to day 60, D nasal turbinate on day 60 and E lung tissues on day 60 were analysed. Each dot represents an individual animal, bars depict the median. Statistical analysis was performed using Mann–Whitney testing.
    Figure Legend Snippet: CVnCoV protects hamsters from SARS-CoV-2 challenge infection. Female Syrian golden hamsters ( n = 5/group) were vaccinated with 10 or 2 µg of CVnCoV, 1.5 µg of Alum-adjuvanted S ECD protein or buffer on day 0 and d28. As additional controls, animals were either left untreated or infected intranasally (IN) with 10 2 TCID 50 /dose of SARS-CoV-2 on day 0 of the experiment. A Total IgG antibodies binding to S ECD displayed as ELISA endpoint titres of all groups except for infected animals that were not analysed or B VNTs determined via CPE-based assay upon one (day 28) or two vaccinations (day 42 and day 56). On day 56, all animals except for the untreated group were challenged by IN infection with 10 2 TCID 50 /dose of SARS-CoV-2 in a total dose volume of 0.1 ml. Animals in the untreated groups were mock infected with buffer as a negative control. Animals were followed for 4 days post challenge (p.c.) and euthanised on day 60 of the experiment. Detectable levels of replication-competent virus in C throat swabs on days 56 to day 60, D nasal turbinate on day 60 and E lung tissues on day 60 were analysed. Each dot represents an individual animal, bars depict the median. Statistical analysis was performed using Mann–Whitney testing.

    Techniques Used: Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, Negative Control, MANN-WHITNEY

    CVnCoV elicits high titres of functional antibodies against SARS-CoV-2. Female Balb/c mice ( n = 7/group) were vaccinated IM with 0.25, 1 and 4 µg of CVnCoV on day 0 and day 21. Animals ( n = 5/group) vaccinated with NaCl (Buffer) served as negative controls. A Spike protein-specific binding antibodies binding to S ECD , RBD, S trimer and NTD, displayed as ELISA endpoint titres for IgG1 and IgG2a for day 21 and day 42. B CPE-based VNTs in serum samples taken on day 21 and day 42. C Per cent of S ECD -binding IgG1 and IgG2a antibodies remaining using serum from animals vaccinated with 2 µg of CVnCoV or 1.5 µg Alum-adjuvanted S ECD upon 30 min wash in 8% urea compared to buffer wash. D S-specific signal detectable on the surface of HeLa cells expressing full-length SARS-CoV-2 S upon incubation with fluorescently labelled monoclonal neutralising antibody in the absence or presence of mouse serum. Serum employed was either from mice vaccinated with 2 µg of CVnCoV, 1.5 µg of Alum-adjuvanted protein or buffer. Each dot represents an individual animal, bars depict the median. S ECD S ectodomain, RBD receptor-binding domain of S, NTD N-terminal domain of S, nAb neutralising antibody.
    Figure Legend Snippet: CVnCoV elicits high titres of functional antibodies against SARS-CoV-2. Female Balb/c mice ( n = 7/group) were vaccinated IM with 0.25, 1 and 4 µg of CVnCoV on day 0 and day 21. Animals ( n = 5/group) vaccinated with NaCl (Buffer) served as negative controls. A Spike protein-specific binding antibodies binding to S ECD , RBD, S trimer and NTD, displayed as ELISA endpoint titres for IgG1 and IgG2a for day 21 and day 42. B CPE-based VNTs in serum samples taken on day 21 and day 42. C Per cent of S ECD -binding IgG1 and IgG2a antibodies remaining using serum from animals vaccinated with 2 µg of CVnCoV or 1.5 µg Alum-adjuvanted S ECD upon 30 min wash in 8% urea compared to buffer wash. D S-specific signal detectable on the surface of HeLa cells expressing full-length SARS-CoV-2 S upon incubation with fluorescently labelled monoclonal neutralising antibody in the absence or presence of mouse serum. Serum employed was either from mice vaccinated with 2 µg of CVnCoV, 1.5 µg of Alum-adjuvanted protein or buffer. Each dot represents an individual animal, bars depict the median. S ECD S ectodomain, RBD receptor-binding domain of S, NTD N-terminal domain of S, nAb neutralising antibody.

    Techniques Used: Functional Assay, Mouse Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Incubation

    Protein translated from CVnCoV is cleaved, post-translationally modified and presented on the cell surface. A Schematic drawing of SARS-CoV-2 S-2P encoded by CVnCoV. B In vitro translation of the mRNA component of CVnCoV in a rabbit reticulocyte lysate system. Translation of nascent proteins was detected via western blotting. Water and luciferase control mRNA were employed as negative and positive controls, respectively. HeLa cells were transfected with 2 µg of the mRNA component of CVnCoV. Twenty-four hours post transfection, cells were analysed for S expression via C western blotting using an S-specific antibody and D flow cytometric analyses using an S-specific antibody either with or without membrane permeabilisation allowing detection of total (intracellular) or cell-surface bound (cell surface) S protein. Relative protein expression in western blotting was quantified using the Image Studio Lite Ver 5.2 software. Geometric mean fluorescence intensity (GMFI) of transfected HeLa cells are expressed as mean + standard deviation (SD) of duplicate samples. Blots shown in C derive from the same experiment and were processed in parallel. NTD N-terminal domain, RBD receptor-binding domain, IVT in vitro translation, TM transmembrane domain, Tub Tubulin.
    Figure Legend Snippet: Protein translated from CVnCoV is cleaved, post-translationally modified and presented on the cell surface. A Schematic drawing of SARS-CoV-2 S-2P encoded by CVnCoV. B In vitro translation of the mRNA component of CVnCoV in a rabbit reticulocyte lysate system. Translation of nascent proteins was detected via western blotting. Water and luciferase control mRNA were employed as negative and positive controls, respectively. HeLa cells were transfected with 2 µg of the mRNA component of CVnCoV. Twenty-four hours post transfection, cells were analysed for S expression via C western blotting using an S-specific antibody and D flow cytometric analyses using an S-specific antibody either with or without membrane permeabilisation allowing detection of total (intracellular) or cell-surface bound (cell surface) S protein. Relative protein expression in western blotting was quantified using the Image Studio Lite Ver 5.2 software. Geometric mean fluorescence intensity (GMFI) of transfected HeLa cells are expressed as mean + standard deviation (SD) of duplicate samples. Blots shown in C derive from the same experiment and were processed in parallel. NTD N-terminal domain, RBD receptor-binding domain, IVT in vitro translation, TM transmembrane domain, Tub Tubulin.

    Techniques Used: Modification, In Vitro, Western Blot, Luciferase, Transfection, Expressing, Software, Fluorescence, Standard Deviation, Binding Assay

    CVnCoV protects the respiratory tract from challenge infection with no signs of vaccine-enhanced disease. Histopathological analyses of hamsters vaccinated with 10 or 2 µg of CVnCoV and 1.5 µg of Alum-adjuvanted S ECD protein or buffer on day 0 and d28, left untreated and mock-infected or infected intranasally (IN) with 10 2 TCID 50 /dose of SARS-CoV-2 on day 0 of the experiment followed by of SARS-CoV-2 challenge infection on d56. Histopathological analysis was performed on day 60, 4 days post challenge infection, on formalin-fixed, paraffin-embedded tissues sections sampled on day 4 post challenge. Histopathological assessment scoring was performed according to severity of inspected parameter. Each dot represents an individual animal, bars depict the median. Statistical analysis was performed using Mann–Whitney testing.
    Figure Legend Snippet: CVnCoV protects the respiratory tract from challenge infection with no signs of vaccine-enhanced disease. Histopathological analyses of hamsters vaccinated with 10 or 2 µg of CVnCoV and 1.5 µg of Alum-adjuvanted S ECD protein or buffer on day 0 and d28, left untreated and mock-infected or infected intranasally (IN) with 10 2 TCID 50 /dose of SARS-CoV-2 on day 0 of the experiment followed by of SARS-CoV-2 challenge infection on d56. Histopathological analysis was performed on day 60, 4 days post challenge infection, on formalin-fixed, paraffin-embedded tissues sections sampled on day 4 post challenge. Histopathological assessment scoring was performed according to severity of inspected parameter. Each dot represents an individual animal, bars depict the median. Statistical analysis was performed using Mann–Whitney testing.

    Techniques Used: Infection, Formalin-fixed Paraffin-Embedded, MANN-WHITNEY

    28) Product Images from "Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry"

    Article Title: Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1009392

    Ability of ACE2 orthologs and their humanized mutants to mediate entry of SARS-CoV-2 and SARS-CoV pseudoparticles. A549 cells transduced with mouse ACE2, koala ACE2 or their humanized mutants were infected with SARS-CoV-2 (A-B) or SARS-CoV (C) pseudoparticles. Two days after infection, cells were lysed and luciferase activity determined. VSV pseudoparticles were used as the control. All infections were performed in triplicate, and the data are representative of two independent experiments (mean ± standard deviation). ns, no significance; *, P
    Figure Legend Snippet: Ability of ACE2 orthologs and their humanized mutants to mediate entry of SARS-CoV-2 and SARS-CoV pseudoparticles. A549 cells transduced with mouse ACE2, koala ACE2 or their humanized mutants were infected with SARS-CoV-2 (A-B) or SARS-CoV (C) pseudoparticles. Two days after infection, cells were lysed and luciferase activity determined. VSV pseudoparticles were used as the control. All infections were performed in triplicate, and the data are representative of two independent experiments (mean ± standard deviation). ns, no significance; *, P

    Techniques Used: Transduction, Infection, Luciferase, Activity Assay, Standard Deviation

    The genetic determinants of ACE2 required for SARS-CoV-2 entry. Mouse, koala, and New World monkey ACE2 cannot serve as functional receptors to support SARS-CoV-2 entry, as determined by different genetic restrictions. Position 31 in koala ACE2 is Thr whereas that in human is Lys. Substitution of Thr with Lys in koala ACE2 allowed for binding to the SARS-CoV-2 spike and viral entry. Different from koala ACE2, the genetic restriction of mouse ACE2 His353. Lys353 in human ACE2 can hydrogen bond with Gly502 of the SARS-CoV-2 spike protein, stabilizing the ACE2-spike complex. The presence of His at this position in mouse ACE2 disrupts this interaction. However, humanization of mouse ACE2 at position 353 renders the protein supportive of SARS-CoV-2 entry. The genetic determinants of New World monkey ACE2 were localized at positons 41 and 42 as we previously reported[ 20 ]. Thus, three genetic determinants of the ability of ACE2 to serve as the SARS-CoV-2 receptor were identified by comparative analysis of ACE2 orthologs, and the receptor activities of normally non-susceptible ACE2 orthologs could be rescued by genetic modification.
    Figure Legend Snippet: The genetic determinants of ACE2 required for SARS-CoV-2 entry. Mouse, koala, and New World monkey ACE2 cannot serve as functional receptors to support SARS-CoV-2 entry, as determined by different genetic restrictions. Position 31 in koala ACE2 is Thr whereas that in human is Lys. Substitution of Thr with Lys in koala ACE2 allowed for binding to the SARS-CoV-2 spike and viral entry. Different from koala ACE2, the genetic restriction of mouse ACE2 His353. Lys353 in human ACE2 can hydrogen bond with Gly502 of the SARS-CoV-2 spike protein, stabilizing the ACE2-spike complex. The presence of His at this position in mouse ACE2 disrupts this interaction. However, humanization of mouse ACE2 at position 353 renders the protein supportive of SARS-CoV-2 entry. The genetic determinants of New World monkey ACE2 were localized at positons 41 and 42 as we previously reported[ 20 ]. Thus, three genetic determinants of the ability of ACE2 to serve as the SARS-CoV-2 receptor were identified by comparative analysis of ACE2 orthologs, and the receptor activities of normally non-susceptible ACE2 orthologs could be rescued by genetic modification.

    Techniques Used: Functional Assay, Binding Assay, Modification

    The potential residues in ACE2 that restrict SARS-CoV-2 entry. (A) A phylogenetic tree was constructed based on the protein sequences of ACE2 orthologs by using the Neighbor-joining method conducted in program MEGA7[ 40 ]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The ACE2 sequences of these species were downloaded from NCBI, and accession numbers are shown in S1 Fig . (B) Alignment of the residues of human, koala and mouse ACE2 at the interface of ACE2 with the SARS-CoV-2 spike protein (first three rows). The restrictive residues of koala or mouse ACE2 are highlighted in red. The favorable residues of human ACE2 are highlighted in green. A series of mutant ACE2 orthologs bearing restrictive or favorable residues were constructed in this study (remaining rows). (C) Cartoon of the binding interface between human ACE2 and the SARS-CoV-2 receptor-binding domain (RBD) (PDB code: 6M0J). ACE2 and the SARS-CoV-2 RBD colored in green and cyan, respectively. Key residues (K31, Y83, and K353) discussed in this study and their interacting residues are shown as ball-and-stick representations.
    Figure Legend Snippet: The potential residues in ACE2 that restrict SARS-CoV-2 entry. (A) A phylogenetic tree was constructed based on the protein sequences of ACE2 orthologs by using the Neighbor-joining method conducted in program MEGA7[ 40 ]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The ACE2 sequences of these species were downloaded from NCBI, and accession numbers are shown in S1 Fig . (B) Alignment of the residues of human, koala and mouse ACE2 at the interface of ACE2 with the SARS-CoV-2 spike protein (first three rows). The restrictive residues of koala or mouse ACE2 are highlighted in red. The favorable residues of human ACE2 are highlighted in green. A series of mutant ACE2 orthologs bearing restrictive or favorable residues were constructed in this study (remaining rows). (C) Cartoon of the binding interface between human ACE2 and the SARS-CoV-2 receptor-binding domain (RBD) (PDB code: 6M0J). ACE2 and the SARS-CoV-2 RBD colored in green and cyan, respectively. Key residues (K31, Y83, and K353) discussed in this study and their interacting residues are shown as ball-and-stick representations.

    Techniques Used: Construct, Mutagenesis, Binding Assay

    The capability of ACE2 mutants to facilitate viral entry in vitro . (A-B) A549 cells transduced with lentiviruses expressing ACE2 orthologs or humanized mutants were infected with SARS-CoV-2 virus (MOI = 1). Expression of the viral nucleocapsid protein was visualized by immunofluorescence microscopy. Viral nucleocapsid (N) (red), and ACE2 (green) are shown. (C) The infection was quantified by a high-content imaging system. (D) The cell culture medium was collected and then virus titer was determined by focus-forming assay. The graph shows the mean and SD (mean ± standard deviation) from two independent experiments performed in triplicate. ns, no significance; *, P
    Figure Legend Snippet: The capability of ACE2 mutants to facilitate viral entry in vitro . (A-B) A549 cells transduced with lentiviruses expressing ACE2 orthologs or humanized mutants were infected with SARS-CoV-2 virus (MOI = 1). Expression of the viral nucleocapsid protein was visualized by immunofluorescence microscopy. Viral nucleocapsid (N) (red), and ACE2 (green) are shown. (C) The infection was quantified by a high-content imaging system. (D) The cell culture medium was collected and then virus titer was determined by focus-forming assay. The graph shows the mean and SD (mean ± standard deviation) from two independent experiments performed in triplicate. ns, no significance; *, P

    Techniques Used: In Vitro, Transduction, Expressing, Infection, Immunofluorescence, Microscopy, Imaging, Cell Culture, Focus Forming Assay, Standard Deviation

    The humanized mouse ACE2 mediate viral entry in vivo. (A) Schematic representation of the experiment timeline; (B-D) The wild-type mice were transduced by recombinant adenovirus expressing ACE2 variants for 3 days, followed by SARS-CoV-2 challenge. Mice were sacrificed at day 3 post infection (n = 5 mice per group) and lung tissues were collected for H E staining (B), immunostaining (C), and viral load titration (D). The ACE2 and viral N antigen expression were detected with anti-FLAG and anti-N serum, respectively. Representative images are shown from n = 5 mice. Scale bar, 100μm (B and C). Viral load was determined by focus-forming assay. ns, no significance; *, P
    Figure Legend Snippet: The humanized mouse ACE2 mediate viral entry in vivo. (A) Schematic representation of the experiment timeline; (B-D) The wild-type mice were transduced by recombinant adenovirus expressing ACE2 variants for 3 days, followed by SARS-CoV-2 challenge. Mice were sacrificed at day 3 post infection (n = 5 mice per group) and lung tissues were collected for H E staining (B), immunostaining (C), and viral load titration (D). The ACE2 and viral N antigen expression were detected with anti-FLAG and anti-N serum, respectively. Representative images are shown from n = 5 mice. Scale bar, 100μm (B and C). Viral load was determined by focus-forming assay. ns, no significance; *, P

    Techniques Used: In Vivo, Mouse Assay, Recombinant, Expressing, Infection, Staining, Immunostaining, Titration, Focus Forming Assay

    ACE2 mutants bind viral spike protein. (A-B) A549 cells were transduced with ACE2 orthologs or their mutants as indicated, incubated with the recombinant S1 domain of the SARS-CoV-2 or SARS-CoV spike protein C-terminally fused with Fc, and then stained with goat anti-human IgG (H + L) conjugated to Alexa Fluor 647 for flow cytometry analysis. Values are expressed as the percent of cells positive for S1-Fc among the ACE2-expressing cells (zsGreen1+ cells) and shown as the means ± SD from 3 biological replicates. This experiments were independently performed three times with similar results. (C) Representative immunoblots of A549 cells transduced with lentiviruses expressing FLAG-tagged ACE2 orthologs and humanized mutants were subjected to immunoblotting. Tubulin served as the loading control. This experiments were independently performed twice with similar results. (D) The structure of SARS-CoV RBD/ACE2 complex[ 31 ] (PDB: 2AJF) and SARS-CoV-2 RBD/ACE2[ 24 ] (PDB: 6M0J) were selected for comparison. SARS-CoV RBD, SARS-CoV-2 RBD and ACE2 are colored salmon, cyan and green, respectively. K31 of ACE2 and its adjacent residues on SARS-CoV RBD or SARS-CoV2 RBD are shown as sticks.
    Figure Legend Snippet: ACE2 mutants bind viral spike protein. (A-B) A549 cells were transduced with ACE2 orthologs or their mutants as indicated, incubated with the recombinant S1 domain of the SARS-CoV-2 or SARS-CoV spike protein C-terminally fused with Fc, and then stained with goat anti-human IgG (H + L) conjugated to Alexa Fluor 647 for flow cytometry analysis. Values are expressed as the percent of cells positive for S1-Fc among the ACE2-expressing cells (zsGreen1+ cells) and shown as the means ± SD from 3 biological replicates. This experiments were independently performed three times with similar results. (C) Representative immunoblots of A549 cells transduced with lentiviruses expressing FLAG-tagged ACE2 orthologs and humanized mutants were subjected to immunoblotting. Tubulin served as the loading control. This experiments were independently performed twice with similar results. (D) The structure of SARS-CoV RBD/ACE2 complex[ 31 ] (PDB: 2AJF) and SARS-CoV-2 RBD/ACE2[ 24 ] (PDB: 6M0J) were selected for comparison. SARS-CoV RBD, SARS-CoV-2 RBD and ACE2 are colored salmon, cyan and green, respectively. K31 of ACE2 and its adjacent residues on SARS-CoV RBD or SARS-CoV2 RBD are shown as sticks.

    Techniques Used: Transduction, Incubation, Recombinant, Staining, Flow Cytometry, Expressing, Western Blot

    29) Product Images from "Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2"

    Article Title: Structure and function analysis of a potent human neutralizing antibody CA521FALA against SARS-CoV-2

    Journal: Communications Biology

    doi: 10.1038/s42003-021-02029-w

    Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.
    Figure Legend Snippet: Serum concentration vs. time profiles of CA521 FALA in mice and rhesus monkeys. a Four mice were administered intravenously at a dose of 10 mg/kg with CA521 FALA . Antibody concentrations in serum were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. b Three healthy rhesus monkeys were administered intravenously at a dose of 50 mg/kg with CA521 FALA . The antibody concentration in serum at different time points were determined in Elisa with SARS-CoV-2 (2019-nCoV) spike protein as the capture reagent. The main PK kinetic parameters were calculated using Phoenix WinNonlin.

    Techniques Used: Concentration Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay

    CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.
    Figure Legend Snippet: CA521 FALA inhibited SARS-CoV-2 infection in vitro and in vivo. a CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into Huh7 cells. b CA521 FALA inhibits SARS-CoV-2 pseudovirus infection into hACE2 expressing HEK293T cells. c CA521 FALA inhibits an authentic SARS-CoV-2 strain (BetaCoV/Beijing/IMEBJ01/2020) infection into Vero cells in vitro. Neutralizing activity of mAbs was measured using a standard plaque reduction neutralization with Vero cells. PRNT50 values were determined using non-linear regression analysis. d , e CA521 FALA exited therapeutic efficacy in SARS-CoV-2 susceptible mice. BALB/c mice who received a SARS-CoV-2 mouse-adapted strain (MASCp6) challenge were administered intraperitoneally with a single dose of 20 mg/kg of CA521 FALA ( n = 4) or PBS ( n = 6) in a therapeutic setting. The level of viral RNA was detected in the lung ( d ) and trachea ( e ) at 3 days post infection (3dpi) with a Quantitative PCR assay. f , g Histopathological analysis of lung samples from PBS group or CA521 FALA group at 3 dpi. Scale bar: 100 μm.

    Techniques Used: Infection, In Vitro, In Vivo, Expressing, Activity Assay, Neutralization, Mouse Assay, Real-time Polymerase Chain Reaction

    CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.
    Figure Legend Snippet: CA521 FALA can block the binding of SARS-CoV-2-RBD to hACE2 receptor and specifically bind Spike of SARS-CoV-2. a CA521 FALA can effectively block RBD binding to ACE2 receptor in ELISA. CA521 FALA and hACE2 protein can block the binding of SARS-CoV-2 RBD and hACE2 with IC50 of 0.343 and 8.887 nM, respectively. Experiments were performed in duplicate, value = mean ± SD. b CA521 FALA could specifically bind to CHO-K1 cells expressing SARS-CoV-2 Spike. SARS-CoV-2 Spike protein transfected CHO-K1 cells were stained with isotype control, CA521 FALA at a concentration of 0.74 μg/mL. FITC-anti-HuFc secondary antibody was used for flow cytometry. Irrelevant mAb with the same constant region of CA521 FALA was used as an isotype. Experiments were performed in triplicate and one representative data was displayed. c – e CA521 FALA could specifically bind to SARS-CoV-2 Spike protein, but does not cross-react with SARS-CoV Spike or MERS-CoV Spike protein in Elisa. CA521 FALA binds SARS-CoV-2 Spike protein with EC50 of 0.014 nM. CA13, which is an anti- SARS-CoV-2 S2 domain mAb, can bind Spike of SARS-CoV-2 and SARS-CoV with EC50 of 0.015 and 0.019 nM. Experiments were performed in triplicate, value = Mean ± SD. f – h The binding kinetics of CA521 FALA were assessed by biolayer Interferometry (BLI) assay using the Octet RED96 system (FortéBio). Trimer protein is from Shuimu BioSciences. Experiments were performed three times and one representative data was displayed.

    Techniques Used: Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Staining, Concentration Assay, Flow Cytometry

    30) 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

    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

    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

    31) Product Images from "Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates"

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

    Journal: Vaccines

    doi: 10.3390/vaccines9040307

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

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

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

    Techniques Used: Staining, MANN-WHITNEY

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

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

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

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

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

    Techniques Used: Expressing, Mouse Assay, Plasmid Preparation

    32) Product Images from "Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients"

    Article Title: Array-based analysis of SARS-CoV-2, other coronaviruses, and influenza antibodies in convalescent COVID-19 patients

    Journal: bioRxiv

    doi: 10.1101/2020.06.15.153064

    Results from the Adarza Ziva system for pre-COVID-19 serum samples and single-donor samples from convalescent COVID-19 (PCR-positive) subjects. Pre-COVID-19 single-donor results were averaged (blue bars). Black bars indicate threshold positive values, calculated as two standard deviations above the average negative (pre-COVID-19) signal. Red bars indicate PCR+ individuals yielding signals below the threshold on all SARS-CoV-2 antigens, while green bars indicate signals from single-donor convalescent COVID-19 samples with at least one SARS-CoV-2 antigen response above threshold.
    Figure Legend Snippet: Results from the Adarza Ziva system for pre-COVID-19 serum samples and single-donor samples from convalescent COVID-19 (PCR-positive) subjects. Pre-COVID-19 single-donor results were averaged (blue bars). Black bars indicate threshold positive values, calculated as two standard deviations above the average negative (pre-COVID-19) signal. Red bars indicate PCR+ individuals yielding signals below the threshold on all SARS-CoV-2 antigens, while green bars indicate signals from single-donor convalescent COVID-19 samples with at least one SARS-CoV-2 antigen response above threshold.

    Techniques Used: Polymerase Chain Reaction

    AIR assay for antibodies to respiratory viruses. For each antigen, six replicate spots are printed in two different locations on the chip. Each group of six spots is surrounded by negative control reference spots (anti-FITC). Blank (background) areas are included as additional negative controls. Key: 1: human coronavirus (HKU isolate) spike glycoprotein, aa 1-760; 2: MERS-CoV spike glycoprotein, S1 domain; 3: MERS-CoV spike glycoprotein, receptor binding domain (RBD); 4: SARS-CoV spike glycoprotein, S1 domain; 5: SARS-CoV spike glycoprotein, RBD; 6: SARS-CoV-2 spike glycoprotein, S1+S2 ECD; 7: SARS-CoV-2 spike glycoprotein, S2 ECD; 8: SARS-CoV-2 spike glycoprotein, S1 domain; 9: SARS-CoV-2 spike glycoprotein, RBD; 10: human coronavirus (HCoV-229E isolate) spike glycoprotein, S1+S2 ECD; 11: human coronavirus (HCoV-OC43 isolate) spike glycoprotein, S1+S2 ECD; 12: influenza B/Brisbane/2008 hemagglutinin; 13: influenza A/California/2009 (H1N1) hemagglutinin; 14: influenza A/Wisconsin/2005 (H3N2) influenza. F1 , F2 , and F3 are derived from spotting three different dilutions of anti-FITC. The image at right is a representative array exposed to Pooled Normal Human Serum (PNHS) at a 1:4 dilution.
    Figure Legend Snippet: AIR assay for antibodies to respiratory viruses. For each antigen, six replicate spots are printed in two different locations on the chip. Each group of six spots is surrounded by negative control reference spots (anti-FITC). Blank (background) areas are included as additional negative controls. Key: 1: human coronavirus (HKU isolate) spike glycoprotein, aa 1-760; 2: MERS-CoV spike glycoprotein, S1 domain; 3: MERS-CoV spike glycoprotein, receptor binding domain (RBD); 4: SARS-CoV spike glycoprotein, S1 domain; 5: SARS-CoV spike glycoprotein, RBD; 6: SARS-CoV-2 spike glycoprotein, S1+S2 ECD; 7: SARS-CoV-2 spike glycoprotein, S2 ECD; 8: SARS-CoV-2 spike glycoprotein, S1 domain; 9: SARS-CoV-2 spike glycoprotein, RBD; 10: human coronavirus (HCoV-229E isolate) spike glycoprotein, S1+S2 ECD; 11: human coronavirus (HCoV-OC43 isolate) spike glycoprotein, S1+S2 ECD; 12: influenza B/Brisbane/2008 hemagglutinin; 13: influenza A/California/2009 (H1N1) hemagglutinin; 14: influenza A/Wisconsin/2005 (H3N2) influenza. F1 , F2 , and F3 are derived from spotting three different dilutions of anti-FITC. The image at right is a representative array exposed to Pooled Normal Human Serum (PNHS) at a 1:4 dilution.

    Techniques Used: Chromatin Immunoprecipitation, Negative Control, Binding Assay, Derivative Assay

    Correlation of AIR and ELISA data for SARS-CoV-2 S1+S2 ECD (left) and RBD (right). Exponential trend lines and associated R 2 values are indicated.
    Figure Legend Snippet: Correlation of AIR and ELISA data for SARS-CoV-2 S1+S2 ECD (left) and RBD (right). Exponential trend lines and associated R 2 values are indicated.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Response of a commercial anti-SARS-CoV-2 rabbit polyclonal antibody (pAb) on the array. (A) array exposed to array exposed to 20% FBS + 10% PNHS; (B) array exposed to 1 μg/mL anti-SARS-CoV-2 pAb in 20% FBS + 10% PNHS. Strong responses to SARS-CoV-2 S1+S2 ECD, S1, and RBD are observed, as well as smaller cross-reactive responses to HCoV-229E, HCoV-OC43, and MERS spike proteins; (C) quantitative data for the titration. Concentrations of pAb are provided at the top of each column in ng/mL; response values at each concentration for each antigen are provided in Angstroms of build. (D) Titration curves for the four SARS-CoV-2 antigens with standard deviation of replicate probe spots at each concentration.
    Figure Legend Snippet: Response of a commercial anti-SARS-CoV-2 rabbit polyclonal antibody (pAb) on the array. (A) array exposed to array exposed to 20% FBS + 10% PNHS; (B) array exposed to 1 μg/mL anti-SARS-CoV-2 pAb in 20% FBS + 10% PNHS. Strong responses to SARS-CoV-2 S1+S2 ECD, S1, and RBD are observed, as well as smaller cross-reactive responses to HCoV-229E, HCoV-OC43, and MERS spike proteins; (C) quantitative data for the titration. Concentrations of pAb are provided at the top of each column in ng/mL; response values at each concentration for each antigen are provided in Angstroms of build. (D) Titration curves for the four SARS-CoV-2 antigens with standard deviation of replicate probe spots at each concentration.

    Techniques Used: Titration, Concentration Assay, Standard Deviation

    33) Product Images from "Clonal dissection of immunodominance and cross-reactivity of the CD4+ T cell response to SARS-CoV-2"

    Article Title: Clonal dissection of immunodominance and cross-reactivity of the CD4+ T cell response to SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2021.03.23.436642

    Sorting of T cell subsets and identification of SARS-CoV-2 Spike- and Nucleoprotein-reactive CD4 + T cells in COVID-19 and pre-pandemic samples. ( A ) Sorting strategy to isolate CD4 + total memory T cells and Tcm, Tem and cTfh subsets. ( B, C ) Characterization of antigen-specific T cells by CFSE dilution combined with CD25 and ICOS co-expression at day 7 following stimulation with Spike or Nucleoprotein in the presence of autologous monocytes. Negative controls of T cells cultured with monocytes alone are reported as dashed lines. Shown are data from patient P2 and from a pre-pandemic healthy donor sample (HD1).
    Figure Legend Snippet: Sorting of T cell subsets and identification of SARS-CoV-2 Spike- and Nucleoprotein-reactive CD4 + T cells in COVID-19 and pre-pandemic samples. ( A ) Sorting strategy to isolate CD4 + total memory T cells and Tcm, Tem and cTfh subsets. ( B, C ) Characterization of antigen-specific T cells by CFSE dilution combined with CD25 and ICOS co-expression at day 7 following stimulation with Spike or Nucleoprotein in the presence of autologous monocytes. Negative controls of T cells cultured with monocytes alone are reported as dashed lines. Shown are data from patient P2 and from a pre-pandemic healthy donor sample (HD1).

    Techniques Used: Transmission Electron Microscopy, Expressing, Cell Culture

    34) Product Images from "A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes"

    Article Title: A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes

    Journal: Nature Communications

    doi: 10.1038/s41467-021-22926-2

    Structural analysis of P4A1 Fab and SARS-CoV-2 RBD complex. a The overall P4A1-Fab-RBD complex structure superimposed with the hACE2-RBD complex. The P4A1 heavy chain (colored slate blue), light chain (colored salmon red), and hACE (colored pale green) are displayed in cartoon representation. The SARS-CoV-2 RBD is colored in gray and displayed in surface representation. b The epitope of P4A1 shown in surface representation. The CDR loops of heavy chain (HCDR) and light chain (LCDR) are colored in purple and magenta, respectively. The epitopes from the heavy chain and light chain are colored in slate blue and salmon red, respectively. The only residue K417, which contacts with both heavy chain and light chain, is colored in pink. The light-chain frame region 3 (LFR3) is colored in orange. The identical residues on RBD shared in P4A1 and hACE2 binding are labeled in red. The residues are numbered according to SARS-CoV-2 RBD. c The detailed interactions between SARS-CoV-2 RBD with HCDR, LCDR, and LFR3. The residues are shown in sticks with identical colors to ( b ).
    Figure Legend Snippet: Structural analysis of P4A1 Fab and SARS-CoV-2 RBD complex. a The overall P4A1-Fab-RBD complex structure superimposed with the hACE2-RBD complex. The P4A1 heavy chain (colored slate blue), light chain (colored salmon red), and hACE (colored pale green) are displayed in cartoon representation. The SARS-CoV-2 RBD is colored in gray and displayed in surface representation. b The epitope of P4A1 shown in surface representation. The CDR loops of heavy chain (HCDR) and light chain (LCDR) are colored in purple and magenta, respectively. The epitopes from the heavy chain and light chain are colored in slate blue and salmon red, respectively. The only residue K417, which contacts with both heavy chain and light chain, is colored in pink. The light-chain frame region 3 (LFR3) is colored in orange. The identical residues on RBD shared in P4A1 and hACE2 binding are labeled in red. The residues are numbered according to SARS-CoV-2 RBD. c The detailed interactions between SARS-CoV-2 RBD with HCDR, LCDR, and LFR3. The residues are shown in sticks with identical colors to ( b ).

    Techniques Used: Binding Assay, Labeling

    Therapeutic efficacy of in the rhesus macaque model of SARS-CoV-2 infection. a Experimental design for therapeutic testing of P4A1–2A in the rhesus macaque ( n = 3/group). b Viral load in oropharyngeal swabs tested by RT-qPCR was monitored for 7 days. c Viral load in the respiratory tissues (including trachea, left and right bronchus, and all six lung lobes) collected at necropsy on 7 days post infection (d.p.i., n = 1/group) was tested by RT-qPCR. d Representative images of histopathology in lung tissue from isotype control or P4A1–2A 50 mg/kg treated animals (collected at 7 d.p.i., n = 1/group).
    Figure Legend Snippet: Therapeutic efficacy of in the rhesus macaque model of SARS-CoV-2 infection. a Experimental design for therapeutic testing of P4A1–2A in the rhesus macaque ( n = 3/group). b Viral load in oropharyngeal swabs tested by RT-qPCR was monitored for 7 days. c Viral load in the respiratory tissues (including trachea, left and right bronchus, and all six lung lobes) collected at necropsy on 7 days post infection (d.p.i., n = 1/group) was tested by RT-qPCR. d Representative images of histopathology in lung tissue from isotype control or P4A1–2A 50 mg/kg treated animals (collected at 7 d.p.i., n = 1/group).

    Techniques Used: Infection, Quantitative RT-PCR, Histopathology

    The activities of IgG4 antibody P4A1–2A to different SARS-CoV-2 S protein mutants, FcRs, and C1q. a Binding of antibody P4A1 to SARS-CoV-2 S protein N354D/D364Y, R408I, W436R, V367F, or D614G mutants determined by surface plasmon resonance (SPR). b Pseudovirus neutralization assay in hACE2-overexpressing HEK293 cells. Experiment performed in triplicates with symbols represent each of the triplicates. c The binding affinity of P4A1 and P4A1–2A for different human FcRs and complement C1q.
    Figure Legend Snippet: The activities of IgG4 antibody P4A1–2A to different SARS-CoV-2 S protein mutants, FcRs, and C1q. a Binding of antibody P4A1 to SARS-CoV-2 S protein N354D/D364Y, R408I, W436R, V367F, or D614G mutants determined by surface plasmon resonance (SPR). b Pseudovirus neutralization assay in hACE2-overexpressing HEK293 cells. Experiment performed in triplicates with symbols represent each of the triplicates. c The binding affinity of P4A1 and P4A1–2A for different human FcRs and complement C1q.

    Techniques Used: Binding Assay, SPR Assay, Neutralization

    Characterization of neutralizing antibodies from convalescent patients. a Characterization of SARS-CoV-2 S protein-specific antibodies. Upper panels: binding of antibodies to the full-length S protein, S1 protein, and S2 protein was evaluated by ELISA (in duplicates with symbols show each of the replicates). Lower left panel: blockage of the binding of SARS-CoV-2 Spike S1 protein to Vero E6 cells by antibodies evaluated by flow cytometry (data in singleton). Lower middle panel: pseudovirus neutralization assay in Huh-7 cells (data in singleton). Lower right panel: in triplicates with symbols show each of the triplicates and SARS-CoV-2 live virus neutralization assay. All experiments were repeated at least two more times (except S2 binding that was repeated one more time) with similar results. b Images of Vero E6 cell-infected SARS-CoV-2 treated with antibodies of different concentrations. Green (stained with SARS-CoV-2 nucleocapsid protein (NP) antibody) indicates viral infected cells and blue (Hoechst 33258) represents cell nuclei. Experiment was performed in triplicates and repeated two more times with similar results.
    Figure Legend Snippet: Characterization of neutralizing antibodies from convalescent patients. a Characterization of SARS-CoV-2 S protein-specific antibodies. Upper panels: binding of antibodies to the full-length S protein, S1 protein, and S2 protein was evaluated by ELISA (in duplicates with symbols show each of the replicates). Lower left panel: blockage of the binding of SARS-CoV-2 Spike S1 protein to Vero E6 cells by antibodies evaluated by flow cytometry (data in singleton). Lower middle panel: pseudovirus neutralization assay in Huh-7 cells (data in singleton). Lower right panel: in triplicates with symbols show each of the triplicates and SARS-CoV-2 live virus neutralization assay. All experiments were repeated at least two more times (except S2 binding that was repeated one more time) with similar results. b Images of Vero E6 cell-infected SARS-CoV-2 treated with antibodies of different concentrations. Green (stained with SARS-CoV-2 nucleocapsid protein (NP) antibody) indicates viral infected cells and blue (Hoechst 33258) represents cell nuclei. Experiment was performed in triplicates and repeated two more times with similar results.

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Neutralization, Infection, Staining

    35) 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

    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

    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

    36) Product Images from "Immunogenicity of a DNA vaccine candidate for COVID-19"

    Article Title: Immunogenicity of a DNA vaccine candidate for COVID-19

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16505-0

    T cell epitope mapping after INO-4800 administration to BALB/c mice. Splenocytes were stimulated for 20 h with SARS-CoV-2 peptide matrix pools. a T cell responses following stimulation with matrix mapping SARS-CoV-2 peptide pools. Bars represent the mean + SD of five mice. b Map of the SARS-CoV-2 Spike protein and identification of immunodominant peptides in BALB/c mice. A known immunodominant SARS-CoV HLA-A2 epitope is included for comparison.
    Figure Legend Snippet: T cell epitope mapping after INO-4800 administration to BALB/c mice. Splenocytes were stimulated for 20 h with SARS-CoV-2 peptide matrix pools. a T cell responses following stimulation with matrix mapping SARS-CoV-2 peptide pools. Bars represent the mean + SD of five mice. b Map of the SARS-CoV-2 Spike protein and identification of immunodominant peptides in BALB/c mice. A known immunodominant SARS-CoV HLA-A2 epitope is included for comparison.

    Techniques Used: Mouse Assay

    Humoral responses to SARS-CoV-2 in Hartley guinea pigs after a single dose of INO-4800. Hartley guinea pigs were immunized on Day 0 with 100 µg INO-4800 or pVAX-empty vector as described in the methods. a SARS-CoV-2 S protein antigen binding of IgG in serial serum dilutions at day 0 and 14. Data shown represent mean OD450 nm values (mean + SD) for the five guinea pigs. b Serum IgG binding titers (mean ± SD) to SARS-CoV-2 S protein at day 14. Values depicted are mean ± SD. P values determined by Mann–Whitney test.
    Figure Legend Snippet: Humoral responses to SARS-CoV-2 in Hartley guinea pigs after a single dose of INO-4800. Hartley guinea pigs were immunized on Day 0 with 100 µg INO-4800 or pVAX-empty vector as described in the methods. a SARS-CoV-2 S protein antigen binding of IgG in serial serum dilutions at day 0 and 14. Data shown represent mean OD450 nm values (mean + SD) for the five guinea pigs. b Serum IgG binding titers (mean ± SD) to SARS-CoV-2 S protein at day 14. Values depicted are mean ± SD. P values determined by Mann–Whitney test.

    Techniques Used: Plasmid Preparation, Binding Assay, MANN-WHITNEY

    Induction of T cell responses in BALB/c mice post-administration of INO-4800. BALB/c mice ( n = 5/group) were immunized with 2.5 or 10 µg INO-4800. T cell responses were analyzed in the animals on days 4, 7, 10 for plots a b, and day 14 for plot c. T cell responses were measured by IFN-γ ELISpot in splenocytes stimulated for 20h with overlapping peptide pools spanning the SARS-CoV-2 ( a ), SARS-CoV ( b ), or MERS-CoV ( c ) Spike proteins. Bars represent the mean + SD. Data from individual mice is shown in Supplementary Data 2 .
    Figure Legend Snippet: Induction of T cell responses in BALB/c mice post-administration of INO-4800. BALB/c mice ( n = 5/group) were immunized with 2.5 or 10 µg INO-4800. T cell responses were analyzed in the animals on days 4, 7, 10 for plots a b, and day 14 for plot c. T cell responses were measured by IFN-γ ELISpot in splenocytes stimulated for 20h with overlapping peptide pools spanning the SARS-CoV-2 ( a ), SARS-CoV ( b ), or MERS-CoV ( c ) Spike proteins. Bars represent the mean + SD. Data from individual mice is shown in Supplementary Data 2 .

    Techniques Used: Mouse Assay, Enzyme-linked Immunospot

    INO-4800 immunized mouse and guinea pig sera compete with ACE2 receptor for SARS-CoV-2 Spike protein binding. a Soluble ACE2 receptor binds to CoV-2 full-length spike with an EC 50 of 0.025 µg/ml. b Purified serum IgG from BALB/c mice ( n of 5 per group) after second immunization with INO-4800 yields significant competition against ACE2 receptor. Serum IgG samples from the animals were run in triplicate. c IgGs purified from n = 5 mice day 7 post second immunization with INO-4800 show significant competition against ACE2 receptor binding to SARS-CoV-2 S 1 + 2 protein. The soluble ACE2 concentration for the competition assay is ~0.1 µg ml −1 . Bars represent the mean and standard deviation of AUC for curves displayed in Supplementary Fig. 1 . d Hartley guinea pigs were immunized on Day 0 and 14 with 100 µg INO-4800 or pVAX-empty vector as described in the methods. Day 28 collected sera (diluted 1:20) was added SARS-CoV-2 coated wells prior to the addition of serial dilutions of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 5 INO-4800-treated and 3 pVAX-treated animals were used in this experiment. e Serial dilutions of guinea pig sera collected on day 21 were added to SARS-CoV-2 coated wells prior to the addition of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 4 INO-4800-treated and 5 pVAX-treated guinea pigs were used in this experiment.
    Figure Legend Snippet: INO-4800 immunized mouse and guinea pig sera compete with ACE2 receptor for SARS-CoV-2 Spike protein binding. a Soluble ACE2 receptor binds to CoV-2 full-length spike with an EC 50 of 0.025 µg/ml. b Purified serum IgG from BALB/c mice ( n of 5 per group) after second immunization with INO-4800 yields significant competition against ACE2 receptor. Serum IgG samples from the animals were run in triplicate. c IgGs purified from n = 5 mice day 7 post second immunization with INO-4800 show significant competition against ACE2 receptor binding to SARS-CoV-2 S 1 + 2 protein. The soluble ACE2 concentration for the competition assay is ~0.1 µg ml −1 . Bars represent the mean and standard deviation of AUC for curves displayed in Supplementary Fig. 1 . d Hartley guinea pigs were immunized on Day 0 and 14 with 100 µg INO-4800 or pVAX-empty vector as described in the methods. Day 28 collected sera (diluted 1:20) was added SARS-CoV-2 coated wells prior to the addition of serial dilutions of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 5 INO-4800-treated and 3 pVAX-treated animals were used in this experiment. e Serial dilutions of guinea pig sera collected on day 21 were added to SARS-CoV-2 coated wells prior to the addition of ACE2 protein. Detection of ACE2 binding to SARS-CoV-2 S protein was measured. Sera collected from 4 INO-4800-treated and 5 pVAX-treated guinea pigs were used in this experiment.

    Techniques Used: Protein Binding, Purification, Mouse Assay, Binding Assay, Concentration Assay, Competitive Binding Assay, Standard Deviation, Plasmid Preparation

    Detection of SARS-CoV-2 S protein-reactive antibodies in the BAL of INO-4800 immunized animals. BALB/c mice (n of 5 per group) were immunized on days 0 and 14 with INO-4800 or pVAX and BAL collected at day 21 ( a , b ). Hartley guinea pigs ( n of 5 per group) were immunized on days 0, 14 and 21 with INO-4800 or pVAX and BAL collected at day 42 ( c , d ). Bronchoalveolar lavage fluid was assayed in duplicate for SARS-CoV-2 Spike protein-specific IgG antibodies by ELISA. Data are presented as endpoint titers ( a , c ), and BAL dilution curves with raw OD 450 nm values ( b , d ). a , c Bars represent the average of each group and error bars the standard deviation. ** p
    Figure Legend Snippet: Detection of SARS-CoV-2 S protein-reactive antibodies in the BAL of INO-4800 immunized animals. BALB/c mice (n of 5 per group) were immunized on days 0 and 14 with INO-4800 or pVAX and BAL collected at day 21 ( a , b ). Hartley guinea pigs ( n of 5 per group) were immunized on days 0, 14 and 21 with INO-4800 or pVAX and BAL collected at day 42 ( c , d ). Bronchoalveolar lavage fluid was assayed in duplicate for SARS-CoV-2 Spike protein-specific IgG antibodies by ELISA. Data are presented as endpoint titers ( a , c ), and BAL dilution curves with raw OD 450 nm values ( b , d ). a , c Bars represent the average of each group and error bars the standard deviation. ** p

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Comparison of SARS-CoV-2, SARS-CoV and MERS-CoV spike glycoproteins. a Amino acid alignment of coronavirus spike proteins including 11 SARS-CoV-2 sequences with mutations (GISAID). Gray bars indicates identical amino acids and colored bars represent mutations relative to Wuhan-Hu-1. RBD, Cleavage Site, Fusion Peptide and Transmembrane domains are indicated in red. b Structural models for SARS-CoV-2, SARS and MERS spike glycoproteins with one chain represented as cartoon and two chains represented as surface. RBD of SARS-CoV-2 is colored yellow.
    Figure Legend Snippet: Comparison of SARS-CoV-2, SARS-CoV and MERS-CoV spike glycoproteins. a Amino acid alignment of coronavirus spike proteins including 11 SARS-CoV-2 sequences with mutations (GISAID). Gray bars indicates identical amino acids and colored bars represent mutations relative to Wuhan-Hu-1. RBD, Cleavage Site, Fusion Peptide and Transmembrane domains are indicated in red. b Structural models for SARS-CoV-2, SARS and MERS spike glycoproteins with one chain represented as cartoon and two chains represented as surface. RBD of SARS-CoV-2 is colored yellow.

    Techniques Used:

    Design and expression of COVID-19 synthetic DNA vaccine constructs. a Schematic diagram of COVID-19 synthetic DNA vaccine constructs, pGX9501 (matched) and pGX9503 (outlier (OL)) containing the IgE leader sequence and SARS-CoV-2 spike protein insert. b RT-PCR assay of RNA extracts from COS-7 cells transfected in duplicate with pGX9501 and pGX9503. Extracted RNA was analyzed by RT-PCR using PCR assays designed for each target and for COS-7 β-Actin mRNA, used as an internal expression normalization gene. Delta C T (∆ C T ) was calculated as the C T of the target minus the C T of β-Actin for each transfection concentration and is plotted against the log of the mass of pDNA transfected (Plotted as mean ± SD). c Analysis of in vitro expression of Spike protein after transfection of 293T cells with pGX9501, pGX9503 or MOCK plasmid by Western blot. 293T cell lysates were resolved on a gel and probed with a polyclonal anti-SARS Spike Protein. Blots were stripped then probed with an anti-β-actin loading control. d In vitro immunofluorescent staining of 293T cells transfected with 3 µg/well of pGX9501, pGX9503 or pVax (empty control vector). Expression of Spike protein was measured with polyclonal anti-SARS Spike Protein IgG and anti-IgG secondary (green). Cell nuclei were counterstained with DAPI (blue). Images were captured using ImageXpress Pico automated cell imaging system. Scale bars are 80.15 µm (left), 66.8 µm (middle) and 77.31 µm (right).
    Figure Legend Snippet: Design and expression of COVID-19 synthetic DNA vaccine constructs. a Schematic diagram of COVID-19 synthetic DNA vaccine constructs, pGX9501 (matched) and pGX9503 (outlier (OL)) containing the IgE leader sequence and SARS-CoV-2 spike protein insert. b RT-PCR assay of RNA extracts from COS-7 cells transfected in duplicate with pGX9501 and pGX9503. Extracted RNA was analyzed by RT-PCR using PCR assays designed for each target and for COS-7 β-Actin mRNA, used as an internal expression normalization gene. Delta C T (∆ C T ) was calculated as the C T of the target minus the C T of β-Actin for each transfection concentration and is plotted against the log of the mass of pDNA transfected (Plotted as mean ± SD). c Analysis of in vitro expression of Spike protein after transfection of 293T cells with pGX9501, pGX9503 or MOCK plasmid by Western blot. 293T cell lysates were resolved on a gel and probed with a polyclonal anti-SARS Spike Protein. Blots were stripped then probed with an anti-β-actin loading control. d In vitro immunofluorescent staining of 293T cells transfected with 3 µg/well of pGX9501, pGX9503 or pVax (empty control vector). Expression of Spike protein was measured with polyclonal anti-SARS Spike Protein IgG and anti-IgG secondary (green). Cell nuclei were counterstained with DAPI (blue). Images were captured using ImageXpress Pico automated cell imaging system. Scale bars are 80.15 µm (left), 66.8 µm (middle) and 77.31 µm (right).

    Techniques Used: Expressing, Construct, Sequencing, Reverse Transcription Polymerase Chain Reaction, Transfection, Polymerase Chain Reaction, Concentration Assay, In Vitro, Plasmid Preparation, Western Blot, Staining, Imaging

    Neutralizing antibody responses after immunization of INO-4800. BALB/c mice ( n of 5 per group) were immunized twice on days 0 and 14 with 10 µg of INO-4800. Sera was collected on day 7 post-second immunization and serial dilutions were incubated with a pseudovirus displaying the SARS-CoV-2 Spike and co-incubated with ACE2–293T cells. a Neutralization ID50 (mean ± SD) in naïve and INO-4800 immunized mice and b relative luminescence units (RLU) for sera from naive mice (green) and mice vaccinated with INO-4800 (red) as described in “Methods”.
    Figure Legend Snippet: Neutralizing antibody responses after immunization of INO-4800. BALB/c mice ( n of 5 per group) were immunized twice on days 0 and 14 with 10 µg of INO-4800. Sera was collected on day 7 post-second immunization and serial dilutions were incubated with a pseudovirus displaying the SARS-CoV-2 Spike and co-incubated with ACE2–293T cells. a Neutralization ID50 (mean ± SD) in naïve and INO-4800 immunized mice and b relative luminescence units (RLU) for sera from naive mice (green) and mice vaccinated with INO-4800 (red) as described in “Methods”.

    Techniques Used: Mouse Assay, Incubation, Neutralization

    Humoral responses to SARS-CoV-2 and SARS-CoV antigens in BALB/c mice after a single dose of INO-4800. BALB/c mice were immunized on day 0 with indicated doses of INO-4800 or pVAX-empty vector as described in the methods. a Protein antigen binding of IgG at 1:50 and 1:250 serum dilutions from mice at day 14 immunized with 25 µg of INO-4800 or pVAX. Data shown represent mean OD450 nm values (mean + SD) for each group of 3 mice. b SARS-CoV-2 S1 + 2 or c SARS-CoV-2 RBD protein antigen binding of IgG in serial serum dilutions from mice at day 14. Data shown represent mean OD450 nm values (mean + SD) for each group of eight mice ( b , c ) and five mice ( d , e ). Serum IgG binding endpoint titers to ( c ) SARS-CoV-2 S1 + 2 and ( e ) SARS-CoV-2 RBD protein. Data representative of two independent experiments. Values depicted are mean +/− SD. P values determined by Mann–Whitney test.
    Figure Legend Snippet: Humoral responses to SARS-CoV-2 and SARS-CoV antigens in BALB/c mice after a single dose of INO-4800. BALB/c mice were immunized on day 0 with indicated doses of INO-4800 or pVAX-empty vector as described in the methods. a Protein antigen binding of IgG at 1:50 and 1:250 serum dilutions from mice at day 14 immunized with 25 µg of INO-4800 or pVAX. Data shown represent mean OD450 nm values (mean + SD) for each group of 3 mice. b SARS-CoV-2 S1 + 2 or c SARS-CoV-2 RBD protein antigen binding of IgG in serial serum dilutions from mice at day 14. Data shown represent mean OD450 nm values (mean + SD) for each group of eight mice ( b , c ) and five mice ( d , e ). Serum IgG binding endpoint titers to ( c ) SARS-CoV-2 S1 + 2 and ( e ) SARS-CoV-2 RBD protein. Data representative of two independent experiments. Values depicted are mean +/− SD. P values determined by Mann–Whitney test.

    Techniques Used: Mouse Assay, Plasmid Preparation, Binding Assay, MANN-WHITNEY

    37) Product Images from "Nucleocapsid and Spike Proteins of SARS-CoV-2 Drive Neutrophil Extracellular Trap Formation"

    Article Title: Nucleocapsid and Spike Proteins of SARS-CoV-2 Drive Neutrophil Extracellular Trap Formation

    Journal: Immune Network

    doi: 10.4110/in.2021.21.e16

    N and S proteins of SARS-CoV-2 induce NET formation from neutrophils. (A-C) Neutrophils were incubated with various concentrations (1, 10, and 100 nM) of the N protein, whole S protein, S1 subunits of the S protein, or S2 subunits of the S protein for 2 h. (A) NET formation in response to viral proteins was determined by Sytox Green staining. (B) Representative immunofluorescence images of NET formation in response to viral proteins of SARS-CoV-2. Representative images of more than 5 experiments are shown (scale bar, 10 μm). (C) ROS generation in response to viral proteins was determined by DCF-DA staining. (D, E) The effects of viral proteins on chemotaxis of neutrophils. One side of chamber was coated with either N, S, S1, or S2 protein and chemotaxis of neutrophils toward viral proteins was examined. (D) Neutrophil migration tracking analysis. The distances traveled by neutrophils were tracked for 45 min. Representative tracking results of thirty cells per each group are shown (n=3 per group). (E) Relative mean distance and relative mean velocity of neutrophils migrating toward viral proteins. Data are expressed as means±SEMs. Con, control; MPO, myeloperoxidase; H3Cit, citrullinated histone 3. * p
    Figure Legend Snippet: N and S proteins of SARS-CoV-2 induce NET formation from neutrophils. (A-C) Neutrophils were incubated with various concentrations (1, 10, and 100 nM) of the N protein, whole S protein, S1 subunits of the S protein, or S2 subunits of the S protein for 2 h. (A) NET formation in response to viral proteins was determined by Sytox Green staining. (B) Representative immunofluorescence images of NET formation in response to viral proteins of SARS-CoV-2. Representative images of more than 5 experiments are shown (scale bar, 10 μm). (C) ROS generation in response to viral proteins was determined by DCF-DA staining. (D, E) The effects of viral proteins on chemotaxis of neutrophils. One side of chamber was coated with either N, S, S1, or S2 protein and chemotaxis of neutrophils toward viral proteins was examined. (D) Neutrophil migration tracking analysis. The distances traveled by neutrophils were tracked for 45 min. Representative tracking results of thirty cells per each group are shown (n=3 per group). (E) Relative mean distance and relative mean velocity of neutrophils migrating toward viral proteins. Data are expressed as means±SEMs. Con, control; MPO, myeloperoxidase; H3Cit, citrullinated histone 3. * p

    Techniques Used: Incubation, Staining, Immunofluorescence, Chemotaxis Assay, Migration

    Related Articles

    Incubation:

    Article Title: Immunogenicity of a DNA vaccine candidate for COVID-19
    Article Snippet: Plates were blocked overnight at 4 °C with blocking buffer (1× PBS, 0.05% Tween 20, 5% evaporated milk and 1% FBS). .. Plates were washed four times with washing buffer then incubated with full length (S1 + S2) spike protein containing a C-terminal His tag (Sino Biologics, cat. 40589-V08B1) at 10 µg mL−1 for 1 h at RT. .. Plates were washed and then serial dilutions of purified mouse IgG mixed with 0.1 µg mL−1 recombinant human ACE2 with a human Fc tag (ACE2-IgHu) were incubated for 1–2 h at RT.

    Article Title: Multiple sites on SARS‐CoV‐2 spike protein are susceptible to proteolysis by cathepsins B, K, L, S, and V, et al. Multiple sites on SARS‐CoV‐2 spike protein are susceptible to proteolysis by cathepsins B, K, L, S, and V
    Article Snippet: The N terminal domain, receptor binding domain, fusion peptide, heptad repeat 1, central helix, and connector domains of each subunit of the SARS‐CoV‐2‐S protein were identified in SwissPBD by comparison of the SARS‐CoV‐2 PDB sequence and the domain sequences outlined by Wrapp et al. Each domain was colored in the SwissPBD model to visualize potential cleavage site locations in relation to these domains. .. A 5 p mol of recombinant human cathepsins B, K, L, S, and V (Enzo) was incubated with 5 ng/μl of SARS‐CoV‐2 (2019‐nCoV) Spike S1 + S2 ECD‐His Recombinant Protein (Sino Biological #40589‐V08B1) in phosphate buffer (pH 6.0), 2 mM DTT, 1 mM EDTA for at 37°C for defined time periods. .. Reducing SDS‐PAGE loading buffer was added and then heated at 95°C to terminate experiments.

    Mouse Assay:

    Article Title: Original antigenic sin responses to heterologous Betacoronavirus spike proteins are observed in mice following intramuscular administration, but are not apparent in children following SARS-CoV-2 infection
    Article Snippet: These mice were immunized 3 weeks later analogously with 10 µg alum-adjuvanted HCoV-HKU1 S1+S2 ECD-His (SinoBiological, 40606-V08B) IM, followed by an identical boost 21 days later. .. Three weeks later the same mice were immunized with 10 µg alum-adjuvanted SARS-CoV-2 S1+S2 ECD-His (SinoBiological, 40589-V08B1) IM, followed by a final boost 21 days later. ..

    Recombinant:

    Article Title: Multiple sites on SARS‐CoV‐2 spike protein are susceptible to proteolysis by cathepsins B, K, L, S, and V, et al. Multiple sites on SARS‐CoV‐2 spike protein are susceptible to proteolysis by cathepsins B, K, L, S, and V
    Article Snippet: The N terminal domain, receptor binding domain, fusion peptide, heptad repeat 1, central helix, and connector domains of each subunit of the SARS‐CoV‐2‐S protein were identified in SwissPBD by comparison of the SARS‐CoV‐2 PDB sequence and the domain sequences outlined by Wrapp et al. Each domain was colored in the SwissPBD model to visualize potential cleavage site locations in relation to these domains. .. A 5 p mol of recombinant human cathepsins B, K, L, S, and V (Enzo) was incubated with 5 ng/μl of SARS‐CoV‐2 (2019‐nCoV) Spike S1 + S2 ECD‐His Recombinant Protein (Sino Biological #40589‐V08B1) in phosphate buffer (pH 6.0), 2 mM DTT, 1 mM EDTA for at 37°C for defined time periods. .. Reducing SDS‐PAGE loading buffer was added and then heated at 95°C to terminate experiments.

    Article Title: mRNA-based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection in rodents
    Article Snippet: .. Immunisations Animals were injected intramuscularly with LNP-formulated SARS-CoV-2 mRNAs (CVnCoV), NaCl as negative control, 1 µg of formalin and heat-inactivated SARS-CoV-2 adjuvanted in Alhydrogel (Brenntag) 1% or 1.5 µg of recombinant SARS-CoV-2 spike protein (S1 + S2 ECD, His tag; Sino Biological, Cat. 40589-V08B1) adjuvanted in Alhydrogel (Brenntag) 2% as positive controls. .. As an additional positive control, hamsters were infected intranasally (IN) with 102 TCID50/dose of SARS-CoV-2 isolate BetaCoV/Munich/BavPat1/2020 in 0.1 ml on d0 of the experiment as indicated.

    Article Title: A serological assay to detect human SARS-CoV-2 antibodies
    Article Snippet: A total of two hundred samples were used: forty-one RT-PCR confirmed COVID-19 patients and one hundred and seventeen pre-pandemic serum samples used as negative controls (NCs). .. SARS-CoV-2 recombinant proteinsRecombinant SARS-CoV-2-S full length (cat no. 40589-V08B1), SARS-CoV-2-S1 (cat no. 40591-V08H), and SARS-CoV-2-S2 (cat no. 40590-V08B) proteins were all purchased from Sino Biological, USA. .. Indirect enzyme-linked immunosorbent assay (ELISA) The ELISA protocol was adapted from a previously established protocol with slight modifications., To establish, develop, and assess in-house ELISA with selected SARS-CoV-2 proteins, the assay was performed using serial dilutions of different serum samples (n = 200), categorised as follows: 1) qRT-PCR-confirmed hospitalised COVID-19 patients (n = 41); 2) convalescent COVID-19 patients (n = 42); and 3) archived samples from healthy volunteers taken 1 year prior to the pandemic (n = 117).

    Article Title: Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice
    Article Snippet: .. Recombinant Proteins and ReagentsSARS-CoV-2 different recombinant S and receptor proteins were obtained from Sino Biologicals (Wayne, PA, USA): Full-length S (S1–S2) ectodomain amino acid (aa) residues 16-1213 (40589-V08B1, 134.36 kDa, expressed in baculovirus-insect cells), S1 subunit (aa 16-685) with RBD domain (40591-V08H, 76.5 kDa, expressed in HEK293 cells); S2 subunit (aa 686-1213) with fusion domain (40589-V08B1, 59.36 kDa, expressed in baculovirus-insect cells); human angiotensin-converting enzyme 2 (hACE2) protein (aa 1-740) fused to Fc tag (10108-H02H, expressed in HEK293 cells). .. Heat-inactivated (65 °C, 30 min; NR-52286) and gamma-irradiated SARS-CoV-2 (NR-52287), and human embryonic kidney cells expressing hACE2 (HEK-293T-hACE2) were provided from BEI/ATCC resources.

    Injection:

    Article Title: mRNA-based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection in rodents
    Article Snippet: .. Immunisations Animals were injected intramuscularly with LNP-formulated SARS-CoV-2 mRNAs (CVnCoV), NaCl as negative control, 1 µg of formalin and heat-inactivated SARS-CoV-2 adjuvanted in Alhydrogel (Brenntag) 1% or 1.5 µg of recombinant SARS-CoV-2 spike protein (S1 + S2 ECD, His tag; Sino Biological, Cat. 40589-V08B1) adjuvanted in Alhydrogel (Brenntag) 2% as positive controls. .. As an additional positive control, hamsters were infected intranasally (IN) with 102 TCID50/dose of SARS-CoV-2 isolate BetaCoV/Munich/BavPat1/2020 in 0.1 ml on d0 of the experiment as indicated.

    Negative Control:

    Article Title: mRNA-based SARS-CoV-2 vaccine candidate CVnCoV induces high levels of virus-neutralising antibodies and mediates protection in rodents
    Article Snippet: .. Immunisations Animals were injected intramuscularly with LNP-formulated SARS-CoV-2 mRNAs (CVnCoV), NaCl as negative control, 1 µg of formalin and heat-inactivated SARS-CoV-2 adjuvanted in Alhydrogel (Brenntag) 1% or 1.5 µg of recombinant SARS-CoV-2 spike protein (S1 + S2 ECD, His tag; Sino Biological, Cat. 40589-V08B1) adjuvanted in Alhydrogel (Brenntag) 2% as positive controls. .. As an additional positive control, hamsters were infected intranasally (IN) with 102 TCID50/dose of SARS-CoV-2 isolate BetaCoV/Munich/BavPat1/2020 in 0.1 ml on d0 of the experiment as indicated.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 98
    Sino Biological sars cov 2 2019 ncov spike s1 s2 ecd his recombinant protein covid 19 spike research
    Adjuvanted S and S1 immune sera exhibit high titers of <t>SARS-CoV-2</t> pseudovirus neutralization and receptor binding inhibition activities. ( A – C ) Reduction percentage (%) in relative luminometer units (RLU) as a measure of luciferase activity for SARS-CoV-2 spike pseudotyped lentivirus infection in HEK293 cells expressing human ACE2 receptor. Data were obtained from pooled sera ( n = 6 to 8) with triplicate wells. S-0.8 (y): S 0.8 µg boost sera of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant boost sera of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant boost sera of old aged mice, S1-4 (y): S1 4 µg boost immune sera of young adult mice, S1-4 + adj (y): S1 4 µg + adjuvant boost immune sera of young adult mice, S2-4 (y): S2 4 µg boost immune sera of young adult mice, S2-4 + adj (y): S2 4 µg + adjuvant boost immune sera of young adult mice, S-0.8 + adj (y, x3): S 0.8 µg + adjuvant 2nd boost immune sera of young adult mice, S-0.8 + adj (y, x3, 19W): S 0.8 µg + adjuvant immune sera collected at week 19 post 2nd boost of young adult mice, S-4 + adj (a, x3): S 4 µg + adjuvant 2nd boost sera of old aged mice. IV-0.8-10 + adj (y, x3): inactivated adjuvanted SARS-CoV-2 vaccination in young age mice (prime 0.8 µg of heat-inactivated and gamma-irradiated virus, 2 times boost with 10 µg inactivated adjuvanted SARS-CoV-2 of heat-inactivated and gamma-irradiated virus). Adj: adjuvants (MPL + QS-21, 1 µg + 10 µg). Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. ( D – F ) ACE2 receptor binding inhibition titers in pooled immune sera ( n = 6–8) with triplicate wells. Inhibition percentage (%) of hACE2 binding to RBD was measured after incubation with serially diluted immune sera in the plate precoated with hACE2 protein. Immune sera of groups are the same as in ( A – C ). Statistical significance was calculated using two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. **; p
    Sars Cov 2 2019 Ncov Spike S1 S2 Ecd His Recombinant Protein Covid 19 Spike Research, supplied by Sino Biological, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sars cov 2 2019 ncov spike s1 s2 ecd his recombinant protein covid 19 spike research/product/Sino Biological
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sars cov 2 2019 ncov spike s1 s2 ecd his recombinant protein covid 19 spike research - by Bioz Stars, 2021-07
    98/100 stars
      Buy from Supplier

    Image Search Results


    Adjuvanted S and S1 immune sera exhibit high titers of SARS-CoV-2 pseudovirus neutralization and receptor binding inhibition activities. ( A – C ) Reduction percentage (%) in relative luminometer units (RLU) as a measure of luciferase activity for SARS-CoV-2 spike pseudotyped lentivirus infection in HEK293 cells expressing human ACE2 receptor. Data were obtained from pooled sera ( n = 6 to 8) with triplicate wells. S-0.8 (y): S 0.8 µg boost sera of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant boost sera of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant boost sera of old aged mice, S1-4 (y): S1 4 µg boost immune sera of young adult mice, S1-4 + adj (y): S1 4 µg + adjuvant boost immune sera of young adult mice, S2-4 (y): S2 4 µg boost immune sera of young adult mice, S2-4 + adj (y): S2 4 µg + adjuvant boost immune sera of young adult mice, S-0.8 + adj (y, x3): S 0.8 µg + adjuvant 2nd boost immune sera of young adult mice, S-0.8 + adj (y, x3, 19W): S 0.8 µg + adjuvant immune sera collected at week 19 post 2nd boost of young adult mice, S-4 + adj (a, x3): S 4 µg + adjuvant 2nd boost sera of old aged mice. IV-0.8-10 + adj (y, x3): inactivated adjuvanted SARS-CoV-2 vaccination in young age mice (prime 0.8 µg of heat-inactivated and gamma-irradiated virus, 2 times boost with 10 µg inactivated adjuvanted SARS-CoV-2 of heat-inactivated and gamma-irradiated virus). Adj: adjuvants (MPL + QS-21, 1 µg + 10 µg). Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. ( D – F ) ACE2 receptor binding inhibition titers in pooled immune sera ( n = 6–8) with triplicate wells. Inhibition percentage (%) of hACE2 binding to RBD was measured after incubation with serially diluted immune sera in the plate precoated with hACE2 protein. Immune sera of groups are the same as in ( A – C ). Statistical significance was calculated using two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. **; p

    Journal: Vaccines

    Article Title: Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice

    doi: 10.3390/vaccines9040316

    Figure Lengend Snippet: Adjuvanted S and S1 immune sera exhibit high titers of SARS-CoV-2 pseudovirus neutralization and receptor binding inhibition activities. ( A – C ) Reduction percentage (%) in relative luminometer units (RLU) as a measure of luciferase activity for SARS-CoV-2 spike pseudotyped lentivirus infection in HEK293 cells expressing human ACE2 receptor. Data were obtained from pooled sera ( n = 6 to 8) with triplicate wells. S-0.8 (y): S 0.8 µg boost sera of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant boost sera of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant boost sera of old aged mice, S1-4 (y): S1 4 µg boost immune sera of young adult mice, S1-4 + adj (y): S1 4 µg + adjuvant boost immune sera of young adult mice, S2-4 (y): S2 4 µg boost immune sera of young adult mice, S2-4 + adj (y): S2 4 µg + adjuvant boost immune sera of young adult mice, S-0.8 + adj (y, x3): S 0.8 µg + adjuvant 2nd boost immune sera of young adult mice, S-0.8 + adj (y, x3, 19W): S 0.8 µg + adjuvant immune sera collected at week 19 post 2nd boost of young adult mice, S-4 + adj (a, x3): S 4 µg + adjuvant 2nd boost sera of old aged mice. IV-0.8-10 + adj (y, x3): inactivated adjuvanted SARS-CoV-2 vaccination in young age mice (prime 0.8 µg of heat-inactivated and gamma-irradiated virus, 2 times boost with 10 µg inactivated adjuvanted SARS-CoV-2 of heat-inactivated and gamma-irradiated virus). Adj: adjuvants (MPL + QS-21, 1 µg + 10 µg). Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. ( D – F ) ACE2 receptor binding inhibition titers in pooled immune sera ( n = 6–8) with triplicate wells. Inhibition percentage (%) of hACE2 binding to RBD was measured after incubation with serially diluted immune sera in the plate precoated with hACE2 protein. Immune sera of groups are the same as in ( A – C ). Statistical significance was calculated using two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. **; p

    Article Snippet: Recombinant Proteins and ReagentsSARS-CoV-2 different recombinant S and receptor proteins were obtained from Sino Biologicals (Wayne, PA, USA): Full-length S (S1–S2) ectodomain amino acid (aa) residues 16-1213 (40589-V08B1, 134.36 kDa, expressed in baculovirus-insect cells), S1 subunit (aa 16-685) with RBD domain (40591-V08H, 76.5 kDa, expressed in HEK293 cells); S2 subunit (aa 686-1213) with fusion domain (40589-V08B1, 59.36 kDa, expressed in baculovirus-insect cells); human angiotensin-converting enzyme 2 (hACE2) protein (aa 1-740) fused to Fc tag (10108-H02H, expressed in HEK293 cells).

    Techniques: Neutralization, Binding Assay, Inhibition, Luciferase, Activity Assay, Infection, Expressing, Mouse Assay, Irradiation, Incubation

    B cell and T cell immune responses to SARS-CoV-2 S vaccination in young adult and old aged mice. To determine cellular immunity, spleen cells were prepared from immunized young adult ( n = 6) and old aged mice ( n = 8). ( A ) Antibody-secreting cells (ASCs) specific for full-length S protein were determined on the ELISpot plate precoated with full-length S protein. ( B ) IFN-γ-secreting cells were analyzed by in vitro stimulation with pooled S peptides or full-length S protein using ELISpot assay. ( C , D ). IFN-γ + CD4 and IFN-γ + CD8 T cells were determined by flow cytometry after in vitro stimulation with pooled S peptides and intracellular cytokine antibi staining. S-0.8 (y): S 0.8 µg vaccination of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant vaccination of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant vaccination of old aged mice. Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. Statistical significance was calculated using one-way ANOVA and a Dunnett’s multiple-comparison test. Error bars indicate the mean ± SEM. *; p

    Journal: Vaccines

    Article Title: Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice

    doi: 10.3390/vaccines9040316

    Figure Lengend Snippet: B cell and T cell immune responses to SARS-CoV-2 S vaccination in young adult and old aged mice. To determine cellular immunity, spleen cells were prepared from immunized young adult ( n = 6) and old aged mice ( n = 8). ( A ) Antibody-secreting cells (ASCs) specific for full-length S protein were determined on the ELISpot plate precoated with full-length S protein. ( B ) IFN-γ-secreting cells were analyzed by in vitro stimulation with pooled S peptides or full-length S protein using ELISpot assay. ( C , D ). IFN-γ + CD4 and IFN-γ + CD8 T cells were determined by flow cytometry after in vitro stimulation with pooled S peptides and intracellular cytokine antibi staining. S-0.8 (y): S 0.8 µg vaccination of young adult mice, S-0.8 + adj (y): S 0.8 µg + adjuvant vaccination of young adult mice, S-0.8 + adj (a): S 0.8 µg + adjuvant vaccination of old aged mice. Mock: sera from mice with adjuvant (MPL + QS-21, 1 + 10 µg) only. Statistical significance was calculated using one-way ANOVA and a Dunnett’s multiple-comparison test. Error bars indicate the mean ± SEM. *; p

    Article Snippet: Recombinant Proteins and ReagentsSARS-CoV-2 different recombinant S and receptor proteins were obtained from Sino Biologicals (Wayne, PA, USA): Full-length S (S1–S2) ectodomain amino acid (aa) residues 16-1213 (40589-V08B1, 134.36 kDa, expressed in baculovirus-insect cells), S1 subunit (aa 16-685) with RBD domain (40591-V08H, 76.5 kDa, expressed in HEK293 cells); S2 subunit (aa 686-1213) with fusion domain (40589-V08B1, 59.36 kDa, expressed in baculovirus-insect cells); human angiotensin-converting enzyme 2 (hACE2) protein (aa 1-740) fused to Fc tag (10108-H02H, expressed in HEK293 cells).

    Techniques: Mouse Assay, Enzyme-linked Immunospot, In Vitro, Flow Cytometry, Staining

    SARS-CoV-2 full-length spike (S) ectodomain and subunit proteins and receptor binding activities. ( A ) Full-length S (S1–S2) ectodomain contains aa residues 16-1213, S1 subunit aa 16-685 (green), and S2 subunit aa 686-1213. NTD: N-terminal domain (blue), RBD: receptor binding domain. ( B , C ) The receptor binding properties were determined using serially diluted soluble hACE2-Fc (0.5–2 µg/mL) on the 96-well plates precoated with 0.8 µg ( B ) or 2 µg ( C ) of S (S1–S2) and S1 subunit proteins. Due to different molecular masses of S and S1 proteins despite the same concentration, molarity in nanomoles (nM) is indicated for each protein coated.

    Journal: Vaccines

    Article Title: Immunogenicity and Neutralizing Activity Comparison of SARS-CoV-2 Spike Full-Length and Subunit Domain Proteins in Young Adult and Old-Aged Mice

    doi: 10.3390/vaccines9040316

    Figure Lengend Snippet: SARS-CoV-2 full-length spike (S) ectodomain and subunit proteins and receptor binding activities. ( A ) Full-length S (S1–S2) ectodomain contains aa residues 16-1213, S1 subunit aa 16-685 (green), and S2 subunit aa 686-1213. NTD: N-terminal domain (blue), RBD: receptor binding domain. ( B , C ) The receptor binding properties were determined using serially diluted soluble hACE2-Fc (0.5–2 µg/mL) on the 96-well plates precoated with 0.8 µg ( B ) or 2 µg ( C ) of S (S1–S2) and S1 subunit proteins. Due to different molecular masses of S and S1 proteins despite the same concentration, molarity in nanomoles (nM) is indicated for each protein coated.

    Article Snippet: Recombinant Proteins and ReagentsSARS-CoV-2 different recombinant S and receptor proteins were obtained from Sino Biologicals (Wayne, PA, USA): Full-length S (S1–S2) ectodomain amino acid (aa) residues 16-1213 (40589-V08B1, 134.36 kDa, expressed in baculovirus-insect cells), S1 subunit (aa 16-685) with RBD domain (40591-V08H, 76.5 kDa, expressed in HEK293 cells); S2 subunit (aa 686-1213) with fusion domain (40589-V08B1, 59.36 kDa, expressed in baculovirus-insect cells); human angiotensin-converting enzyme 2 (hACE2) protein (aa 1-740) fused to Fc tag (10108-H02H, expressed in HEK293 cells).

    Techniques: Binding Assay, Concentration Assay

    HKU1 antibodies are prevalent in healthy children and children with acute COVID-19 and MIS-C. SARS-CoV-2 (A) and HKU1 (B) spike IgG antibody titers and FRNT neutralization titers (C) in healthy pediatric controls compared to children hospitalized with acute COVID-19 and MIS-C. * P

    Journal: medRxiv

    Article Title: Original antigenic sin responses to heterologous Betacoronavirus spike proteins are observed in mice following intramuscular administration, but are not apparent in children following SARS-CoV-2 infection

    doi: 10.1101/2021.04.29.21256344

    Figure Lengend Snippet: HKU1 antibodies are prevalent in healthy children and children with acute COVID-19 and MIS-C. SARS-CoV-2 (A) and HKU1 (B) spike IgG antibody titers and FRNT neutralization titers (C) in healthy pediatric controls compared to children hospitalized with acute COVID-19 and MIS-C. * P

    Article Snippet: Three weeks later the same mice were immunized with 10 µg alum-adjuvanted SARS-CoV-2 S1+S2 ECD-His (SinoBiological, 40589-V08B1) IM, followed by a final boost 21 days later.

    Techniques: Neutralization

    Schematic of intramuscular spike protein administrations in groups of five BALB/c mice. Group 1 received prime and boost with SARS-CoV-2 spike, followed by prime and boost with HKU1 spike. Group 2 received a reciprocal administration regimen, with prime and boost with HKU1 spike, followed by prime and boost by HKU1 spike. D, days post-administration; S, spike; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. *These mice were immunized with nucleocapsid protein 21 and 42 days prior to utilization for this study.

    Journal: medRxiv

    Article Title: Original antigenic sin responses to heterologous Betacoronavirus spike proteins are observed in mice following intramuscular administration, but are not apparent in children following SARS-CoV-2 infection

    doi: 10.1101/2021.04.29.21256344

    Figure Lengend Snippet: Schematic of intramuscular spike protein administrations in groups of five BALB/c mice. Group 1 received prime and boost with SARS-CoV-2 spike, followed by prime and boost with HKU1 spike. Group 2 received a reciprocal administration regimen, with prime and boost with HKU1 spike, followed by prime and boost by HKU1 spike. D, days post-administration; S, spike; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. *These mice were immunized with nucleocapsid protein 21 and 42 days prior to utilization for this study.

    Article Snippet: Three weeks later the same mice were immunized with 10 µg alum-adjuvanted SARS-CoV-2 S1+S2 ECD-His (SinoBiological, 40589-V08B1) IM, followed by a final boost 21 days later.

    Techniques: Mouse Assay

    HKU1 spike IgG antibodies correlated positively with both SAR-CoV-2 spike IgG and SARS-CoV-2 neutralizing antibodies in children with acute COVID-19 and MIS-C. Linear regression analyses compared the log-transformed antibody titers of (A) SARS-CoV-2 spike IgG vs. HKU1 spike IgG; (B) HKU1 spike IgG vs. SARS-CoV-2 neutralization titers; and (C) SARS-CoV-2 spike IgG vs. SARS-CoV-2 neutralization titers among children with acute COVID-19 or MIS-C. Spearman’s correlation coefficients (r) and P-values are shown.

    Journal: medRxiv

    Article Title: Original antigenic sin responses to heterologous Betacoronavirus spike proteins are observed in mice following intramuscular administration, but are not apparent in children following SARS-CoV-2 infection

    doi: 10.1101/2021.04.29.21256344

    Figure Lengend Snippet: HKU1 spike IgG antibodies correlated positively with both SAR-CoV-2 spike IgG and SARS-CoV-2 neutralizing antibodies in children with acute COVID-19 and MIS-C. Linear regression analyses compared the log-transformed antibody titers of (A) SARS-CoV-2 spike IgG vs. HKU1 spike IgG; (B) HKU1 spike IgG vs. SARS-CoV-2 neutralization titers; and (C) SARS-CoV-2 spike IgG vs. SARS-CoV-2 neutralization titers among children with acute COVID-19 or MIS-C. Spearman’s correlation coefficients (r) and P-values are shown.

    Article Snippet: Three weeks later the same mice were immunized with 10 µg alum-adjuvanted SARS-CoV-2 S1+S2 ECD-His (SinoBiological, 40589-V08B1) IM, followed by a final boost 21 days later.

    Techniques: Transformation Assay, Neutralization

    Priming mice with HKU1 spike protein prior to boosting with SARS-CoV-2 spike protein completely impeded the development of SARS-CoV-2 neutralizing antibodies. SARS-CoV-2 (A,B) and HKU1 (C,D) full-length spike IgG binding and SARS-CoV-2 neutralizing (E, F) antibodies in mice are shown as log(end-point titer). Group 1 was primed with two doses of alum-adjuvanted SARS-CoV-2 spike and boosted with two doses of alum-adjuvanted HKU1 spike (A, C, E). Group 2 received the reciprocal regimen of HKU1 spike prime and SARS-CoV-2 spike boost (B, D, F). * P

    Journal: medRxiv

    Article Title: Original antigenic sin responses to heterologous Betacoronavirus spike proteins are observed in mice following intramuscular administration, but are not apparent in children following SARS-CoV-2 infection

    doi: 10.1101/2021.04.29.21256344

    Figure Lengend Snippet: Priming mice with HKU1 spike protein prior to boosting with SARS-CoV-2 spike protein completely impeded the development of SARS-CoV-2 neutralizing antibodies. SARS-CoV-2 (A,B) and HKU1 (C,D) full-length spike IgG binding and SARS-CoV-2 neutralizing (E, F) antibodies in mice are shown as log(end-point titer). Group 1 was primed with two doses of alum-adjuvanted SARS-CoV-2 spike and boosted with two doses of alum-adjuvanted HKU1 spike (A, C, E). Group 2 received the reciprocal regimen of HKU1 spike prime and SARS-CoV-2 spike boost (B, D, F). * P

    Article Snippet: Three weeks later the same mice were immunized with 10 µg alum-adjuvanted SARS-CoV-2 S1+S2 ECD-His (SinoBiological, 40589-V08B1) IM, followed by a final boost 21 days later.

    Techniques: Mouse Assay, Binding Assay

    Fragments of spike protein generated due to cleavage in S1/S2 domains. From the docking software, fragments created from cleavage by two or more cathepsins at two sites in the S1/S2 region of spike protein are shown for (a), G700 – G769, 7 kDa fragment (b), T768‐V860, a 10 kDa fragment and (c) G700 – V860, a 17 kDa fragment. Fragments are shown from the protomer and from the trimer for each cleavage site pair

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Multiple sites on SARS‐CoV‐2 spike protein are susceptible to proteolysis by cathepsins B, K, L, S, and V, et al. Multiple sites on SARS‐CoV‐2 spike protein are susceptible to proteolysis by cathepsins B, K, L, S, and V

    doi: 10.1002/pro.4073

    Figure Lengend Snippet: Fragments of spike protein generated due to cleavage in S1/S2 domains. From the docking software, fragments created from cleavage by two or more cathepsins at two sites in the S1/S2 region of spike protein are shown for (a), G700 – G769, 7 kDa fragment (b), T768‐V860, a 10 kDa fragment and (c) G700 – V860, a 17 kDa fragment. Fragments are shown from the protomer and from the trimer for each cleavage site pair

    Article Snippet: A 5 p mol of recombinant human cathepsins B, K, L, S, and V (Enzo) was incubated with 5 ng/μl of SARS‐CoV‐2 (2019‐nCoV) Spike S1 + S2 ECD‐His Recombinant Protein (Sino Biological #40589‐V08B1) in phosphate buffer (pH 6.0), 2 mM DTT, 1 mM EDTA for at 37°C for defined time periods.

    Techniques: Generated, Software

    Identification of putative cleavage sites in the S1/S2 region of spike protein based on PACMANS scoring. For each protease, spike protein cleavage sites in the S1 and S2 regions were ranked using PACMANS, and scores are shown with a heat map. Heat scores of ranks are colored from green to red, with green indicating a higher rank (closer to 1) and red indicating lower rank (greater numbers) that are less likely to be susceptible to cleavage by the specific protease. Boxed sequences indicate published cleavage sites of spike protein that promote conformational change for entry to cell. Stars indicate locations where multiple proteases have higher scores of cleavage probability: yellow for furin, blue for the cathepsins K, L, S, and V near S1/S2 site, and pink for cathepsins K, L, S, and V cleaving outside the S2' site

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Multiple sites on SARS‐CoV‐2 spike protein are susceptible to proteolysis by cathepsins B, K, L, S, and V, et al. Multiple sites on SARS‐CoV‐2 spike protein are susceptible to proteolysis by cathepsins B, K, L, S, and V

    doi: 10.1002/pro.4073

    Figure Lengend Snippet: Identification of putative cleavage sites in the S1/S2 region of spike protein based on PACMANS scoring. For each protease, spike protein cleavage sites in the S1 and S2 regions were ranked using PACMANS, and scores are shown with a heat map. Heat scores of ranks are colored from green to red, with green indicating a higher rank (closer to 1) and red indicating lower rank (greater numbers) that are less likely to be susceptible to cleavage by the specific protease. Boxed sequences indicate published cleavage sites of spike protein that promote conformational change for entry to cell. Stars indicate locations where multiple proteases have higher scores of cleavage probability: yellow for furin, blue for the cathepsins K, L, S, and V near S1/S2 site, and pink for cathepsins K, L, S, and V cleaving outside the S2' site

    Article Snippet: A 5 p mol of recombinant human cathepsins B, K, L, S, and V (Enzo) was incubated with 5 ng/μl of SARS‐CoV‐2 (2019‐nCoV) Spike S1 + S2 ECD‐His Recombinant Protein (Sino Biological #40589‐V08B1) in phosphate buffer (pH 6.0), 2 mM DTT, 1 mM EDTA for at 37°C for defined time periods.

    Techniques:

    Putative sites susceptible to cleavage by four cathepsins. From PACMANS analysis, there were two amino acid sequences susceptible to cleavage by four cathepsins as indicated by high normalized scores and relatively high rank orders. From the three‐dimensional model of spike protein, the domains are color coded, (a) and susceptible sequences are highlighted in red. (b) Cleavage after H49 is in the N‐terminus domain (blue) of spike protein, and (c) cleavage after G700 was even more highly ranked and cleaves in the S1/S2 domain

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Multiple sites on SARS‐CoV‐2 spike protein are susceptible to proteolysis by cathepsins B, K, L, S, and V, et al. Multiple sites on SARS‐CoV‐2 spike protein are susceptible to proteolysis by cathepsins B, K, L, S, and V

    doi: 10.1002/pro.4073

    Figure Lengend Snippet: Putative sites susceptible to cleavage by four cathepsins. From PACMANS analysis, there were two amino acid sequences susceptible to cleavage by four cathepsins as indicated by high normalized scores and relatively high rank orders. From the three‐dimensional model of spike protein, the domains are color coded, (a) and susceptible sequences are highlighted in red. (b) Cleavage after H49 is in the N‐terminus domain (blue) of spike protein, and (c) cleavage after G700 was even more highly ranked and cleaves in the S1/S2 domain

    Article Snippet: A 5 p mol of recombinant human cathepsins B, K, L, S, and V (Enzo) was incubated with 5 ng/μl of SARS‐CoV‐2 (2019‐nCoV) Spike S1 + S2 ECD‐His Recombinant Protein (Sino Biological #40589‐V08B1) in phosphate buffer (pH 6.0), 2 mM DTT, 1 mM EDTA for at 37°C for defined time periods.

    Techniques:

    Optimization of the SARS-CoV-2-S antigen concentration. The sera from four COVID-19 patients and negative controls (NCs) at 1:100 dilution were tested against decreasing concentrations of the SARS-CoV-2 spike protein (4 μg/ml to 0.5 μg/ml) for IgG and IgM reactivity.

    Journal: Journal of Taibah University Medical Sciences

    Article Title: A serological assay to detect human SARS-CoV-2 antibodies

    doi: 10.1016/j.jtumed.2020.11.011

    Figure Lengend Snippet: Optimization of the SARS-CoV-2-S antigen concentration. The sera from four COVID-19 patients and negative controls (NCs) at 1:100 dilution were tested against decreasing concentrations of the SARS-CoV-2 spike protein (4 μg/ml to 0.5 μg/ml) for IgG and IgM reactivity.

    Article Snippet: SARS-CoV-2 recombinant proteinsRecombinant SARS-CoV-2-S full length (cat no. 40589-V08B1), SARS-CoV-2-S1 (cat no. 40591-V08H), and SARS-CoV-2-S2 (cat no. 40590-V08B) proteins were all purchased from Sino Biological, USA.

    Techniques: Concentration Assay