sars cov 2 2019 ncov spike s1 ntd his avi recombinant protein  (Sino Biological)


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    Name:
    SARS CoV 2 2019 nCoV Spike S1 NTD His AVI Recombinant Protein
    Description:
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike S1 NTD YP 009724390 1 Met1 Ser305 was expressed with a c terminal polyhistidine tagged AVI tag at the C terminus
    Catalog Number:
    40591-V49H
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    coronavirus spike Protein 2019-nCoV, cov spike Protein 2019-nCoV, ncov RBD Protein 2019-nCoV, ncov s1 Protein 2019-nCoV, ncov s2 Protein 2019-nCoV, ncov spike Protein 2019-nCoV, NCP-CoV RBD Protein 2019-nCoV, NCP-CoV s1 Protein 2019-nCoV, NCP-CoV s2 Protein 2019-nCoV, NCP-CoV Spike Protein 2019-nCoV, novel coronavirus RBD Protein 2019-nCoV, novel coronavirus s1 Protein 2019-nCoV, novel coronavirus s2 Protein 2019-nCoV, novel coronavirus spike Protein 2019-nCoV, RBD Protein 2019-nCoV, S1 Protein 2019-nCoV, S2 Protein 2019-nCoV, Spike RBD Protein 2019-nCoV
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological sars cov 2 2019 ncov spike s1 ntd his avi recombinant protein
    <t>SARS-CoV-2</t> vaccine-elicited functional antibody responses in infant rhesus macaques. (A): The ACE2 blocking assay was performed at 1:10 plasma dilution and data are reported as %ACE2 blocking. (B-C) : Neutralization capacity was measured using a pseudovirus assay (B) and whole virus assay (C) ; results are expressed as reciprocal 80% inhibitory dilution (ID 80 ). Different symbols represent individual animals ( Table S1 ).
    A DNA sequence encoding the SARS CoV 2 2019 nCoV Spike S1 NTD YP 009724390 1 Met1 Ser305 was expressed with a c terminal polyhistidine tagged AVI tag at the C terminus
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    sars cov 2 2019 ncov spike s1 ntd his avi recombinant protein - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques"

    Article Title: SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques

    Journal: bioRxiv

    doi: 10.1101/2021.04.05.438479

    SARS-CoV-2 vaccine-elicited functional antibody responses in infant rhesus macaques. (A): The ACE2 blocking assay was performed at 1:10 plasma dilution and data are reported as %ACE2 blocking. (B-C) : Neutralization capacity was measured using a pseudovirus assay (B) and whole virus assay (C) ; results are expressed as reciprocal 80% inhibitory dilution (ID 80 ). Different symbols represent individual animals ( Table S1 ).
    Figure Legend Snippet: SARS-CoV-2 vaccine-elicited functional antibody responses in infant rhesus macaques. (A): The ACE2 blocking assay was performed at 1:10 plasma dilution and data are reported as %ACE2 blocking. (B-C) : Neutralization capacity was measured using a pseudovirus assay (B) and whole virus assay (C) ; results are expressed as reciprocal 80% inhibitory dilution (ID 80 ). Different symbols represent individual animals ( Table S1 ).

    Techniques Used: Functional Assay, Blocking Assay, Neutralization

    Spike-specific T cell responses in LN following SARS-CoV-2 vaccination. Intracellular cytokine staining as in Figure 5 in lymph node biopsy samples at week 6 to measure Spike-specific T cell responses. Panels A and B show mRNA-LNP and Protein+3M-052+SE CD4 + T-cell responses, respectively. Panels C and D represent CD8 + T cell cytokine responses from mRNA-LNP and Protein+3M-052+SE animals, respectively. Symbols and the legend mirror those from Figure 6 (see also Table S1 ). Dotted lines represent 2 standard deviations from SARS-CoV-2 naïve LN samples.
    Figure Legend Snippet: Spike-specific T cell responses in LN following SARS-CoV-2 vaccination. Intracellular cytokine staining as in Figure 5 in lymph node biopsy samples at week 6 to measure Spike-specific T cell responses. Panels A and B show mRNA-LNP and Protein+3M-052+SE CD4 + T-cell responses, respectively. Panels C and D represent CD8 + T cell cytokine responses from mRNA-LNP and Protein+3M-052+SE animals, respectively. Symbols and the legend mirror those from Figure 6 (see also Table S1 ). Dotted lines represent 2 standard deviations from SARS-CoV-2 naïve LN samples.

    Techniques Used: Staining

    Spike-specific CD8 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining was performed as described in Figure 5 at weeks 0, 6, 4, 8, and 14 to assess CD8 + T-cell responses. Panels A, B, C and D show responses detected in PBMC from the mRNA-LNP group at weeks 4, 6, 8, and 14, respectively. Panels E, F, G and H show responses in Protein+3M-052+SE vaccinees at weeks 4, 6, 8 and 14, respectively. The legend and symbols used mirror those from Figure 6 (see also Table S1 ). Dotted lines represent the cut-off for cytokine-positive responses, determined as 2 standard deviations above median values of SARS-CoV-2 naïve animals.
    Figure Legend Snippet: Spike-specific CD8 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining was performed as described in Figure 5 at weeks 0, 6, 4, 8, and 14 to assess CD8 + T-cell responses. Panels A, B, C and D show responses detected in PBMC from the mRNA-LNP group at weeks 4, 6, 8, and 14, respectively. Panels E, F, G and H show responses in Protein+3M-052+SE vaccinees at weeks 4, 6, 8 and 14, respectively. The legend and symbols used mirror those from Figure 6 (see also Table S1 ). Dotted lines represent the cut-off for cytokine-positive responses, determined as 2 standard deviations above median values of SARS-CoV-2 naïve animals.

    Techniques Used: Staining

    Weight gain of SARS-CoV-2 immunized infant rhesus macaques. Longitudinal weight data for all female (top) and male (bottom) animals from this study with their respective symbol shapes and colors ( Table S1 ) overlaid with historical weight data from age and sex-matched infant rhesus monkeys housed outdoors at the CNPRC, Davis California (open black circles-female; filled gray circles-male).
    Figure Legend Snippet: Weight gain of SARS-CoV-2 immunized infant rhesus macaques. Longitudinal weight data for all female (top) and male (bottom) animals from this study with their respective symbol shapes and colors ( Table S1 ) overlaid with historical weight data from age and sex-matched infant rhesus monkeys housed outdoors at the CNPRC, Davis California (open black circles-female; filled gray circles-male).

    Techniques Used:

    Polyfunctional CD4 + T cells in SARS-CoV-2 immunized rhesus macaques at week 14. Panels A and B show the frequencies of CD4 + T cells that co-produced IL-17 and IFN-γ in response to stimulation with spike protein overlapping peptides in animals of the mRNA-LNP or Protein-3M-052-SE vaccine group, respectively. Individual symbols represent individual animals in each group ( Table S1 ).
    Figure Legend Snippet: Polyfunctional CD4 + T cells in SARS-CoV-2 immunized rhesus macaques at week 14. Panels A and B show the frequencies of CD4 + T cells that co-produced IL-17 and IFN-γ in response to stimulation with spike protein overlapping peptides in animals of the mRNA-LNP or Protein-3M-052-SE vaccine group, respectively. Individual symbols represent individual animals in each group ( Table S1 ).

    Techniques Used: Produced

    Characterization of Spike-specific B cell responses two weeks post boost. CD20 + CD27 + memory B cells that co-stained with fluorochrome-conjugated SARS-CoV-2 spike protein in mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccinees in blood ( A - B ) or LN ( C - D ). Frequencies are expressed as percent of total memory B cells. The gating strategy is provided in Supplementary Figure S8. ( E–F) portray antibody secreting cell (ASC) as measured by B cell ELISpot in PBMC from mRNA-LNP or Protein+3M-052-SE vaccinees, while (G–H) contain mRNA-LNP and Protein+3M-052-SE ASC responses, respectively, in LN at W6. Different symbols represent individual animals ( Table S1 ). Solid lines represent median values.
    Figure Legend Snippet: Characterization of Spike-specific B cell responses two weeks post boost. CD20 + CD27 + memory B cells that co-stained with fluorochrome-conjugated SARS-CoV-2 spike protein in mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccinees in blood ( A - B ) or LN ( C - D ). Frequencies are expressed as percent of total memory B cells. The gating strategy is provided in Supplementary Figure S8. ( E–F) portray antibody secreting cell (ASC) as measured by B cell ELISpot in PBMC from mRNA-LNP or Protein+3M-052-SE vaccinees, while (G–H) contain mRNA-LNP and Protein+3M-052-SE ASC responses, respectively, in LN at W6. Different symbols represent individual animals ( Table S1 ). Solid lines represent median values.

    Techniques Used: Staining, Enzyme-linked Immunospot

    Study Design: evaluation of immunogenicity of two SARS-CoV-2 vaccines in infant rhesus macaques. Infant rhesus macaques (median age of 2.2 months at study initiation) were immunized at 0 and 4 weeks with either 30 µg mRNA encoding S-2P (Vaccine Research Center, NIH) in lipid nanoparticles (mRNA-LNP) or 15 µg S-2P protein formulated with 3M-052 adjuvant, a TLR7/8 agonist, as a stable emulsion (3M-052-SE). Each group consisted of 8 animals. Blood and saliva samples were collected at weeks 0, 4, 6, 8, 14, 18 and 22, and lymph node biopsies were obtained at week 6.
    Figure Legend Snippet: Study Design: evaluation of immunogenicity of two SARS-CoV-2 vaccines in infant rhesus macaques. Infant rhesus macaques (median age of 2.2 months at study initiation) were immunized at 0 and 4 weeks with either 30 µg mRNA encoding S-2P (Vaccine Research Center, NIH) in lipid nanoparticles (mRNA-LNP) or 15 µg S-2P protein formulated with 3M-052 adjuvant, a TLR7/8 agonist, as a stable emulsion (3M-052-SE). Each group consisted of 8 animals. Blood and saliva samples were collected at weeks 0, 4, 6, 8, 14, 18 and 22, and lymph node biopsies were obtained at week 6.

    Techniques Used:

    Spike-specific CD4 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining for IL-2, IL-17, IFN-γ, and TNF-α was performed on PBMC at weeks 0, 6, 4, 8, and 14 to assess T-cell responses to a peptide pool encompassing the entire SARS-CoV-2 spike protein. (A–D) display responses detected in Protein+3M-052+SE vaccinees (blue). (E-H) portray cytokine responses from mRNA-LNP recipients (red). The dashed lines represent week 0 values plus 2 standard deviations and define the cutoff for positive cytokine responses. Different symbols represent individual animals ( Table S1 ).
    Figure Legend Snippet: Spike-specific CD4 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining for IL-2, IL-17, IFN-γ, and TNF-α was performed on PBMC at weeks 0, 6, 4, 8, and 14 to assess T-cell responses to a peptide pool encompassing the entire SARS-CoV-2 spike protein. (A–D) display responses detected in Protein+3M-052+SE vaccinees (blue). (E-H) portray cytokine responses from mRNA-LNP recipients (red). The dashed lines represent week 0 values plus 2 standard deviations and define the cutoff for positive cytokine responses. Different symbols represent individual animals ( Table S1 ).

    Techniques Used: Staining

    S-specific B cell gating strategy. Panel A shows a representative plot from RM7; Panel B shows a naive, age-matched control macaque without SARS-CoV-2 S protein conjugates; Panel C shows the same donor macaque with the SARS-CoV-2 S protein conjugates included.
    Figure Legend Snippet: S-specific B cell gating strategy. Panel A shows a representative plot from RM7; Panel B shows a naive, age-matched control macaque without SARS-CoV-2 S protein conjugates; Panel C shows the same donor macaque with the SARS-CoV-2 S protein conjugates included.

    Techniques Used:

    Th1 and Th2 cytokines in plasma of infant rhesus macaques prior to and following SARS-CoV-2 vaccination. Plasma levels of IFN-γ ( panel A ) IL-2 ( panel B ), IL-4 ( panel C ) and IL-13 ( panel D ) were measured by multiplex Luminex assay. Different symbols represent individual animals in the mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccine groups, respectively ( Table S1 ). Horizontal lines represent median values.
    Figure Legend Snippet: Th1 and Th2 cytokines in plasma of infant rhesus macaques prior to and following SARS-CoV-2 vaccination. Plasma levels of IFN-γ ( panel A ) IL-2 ( panel B ), IL-4 ( panel C ) and IL-13 ( panel D ) were measured by multiplex Luminex assay. Different symbols represent individual animals in the mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccine groups, respectively ( Table S1 ). Horizontal lines represent median values.

    Techniques Used: Multiplex Assay, Luminex

    SARS-CoV-2 vaccine-elicited binding antibody responses in infant rhesus macaques. Plasma and saliva were collected before vaccination (W0), at W4 -just prior to the boost-, two weeks post boost (W6), at W8, W14, W18 and W22 from infant RM vaccinated with 30 µg mRNA encoding S-2P spike protein in lipid nanoparticles (n=8; red) or with 15 µg prefusion SARS-CoV-2 S-2P spike protein formulated with 3M-052 adjuvant (n=8; blue). (A): S-2P protein-specific antibody responses were measured by enzyme-linked immunosorbent assay (ELISA). Serial dilutions of plasma starting at 1:40 were assayed for IgG binding to SARS-CoV-2 spike. Data are reported as log 10 area under the curve (AUC) values. (B): Salivary RBD-specific IgG was measured by binding antigen multiplex assay (BAMA) using serial dilutions of saliva. (C): Antibody epitope specificity measured by BAMA. Plasma was diluted 1:10,000 to measure binding to different domains of the spike protein, including the full-length S protein, S1, RBD, NTD, and S2. Binding antibody responses are reported as log 10 transformed mean fluorescence intensity (MFI) after subtraction of background values. Red or blue lines and symbols represent the mRNA or protein vaccine groups, respectively, with different symbols representing individual animals ( Table S1 ).
    Figure Legend Snippet: SARS-CoV-2 vaccine-elicited binding antibody responses in infant rhesus macaques. Plasma and saliva were collected before vaccination (W0), at W4 -just prior to the boost-, two weeks post boost (W6), at W8, W14, W18 and W22 from infant RM vaccinated with 30 µg mRNA encoding S-2P spike protein in lipid nanoparticles (n=8; red) or with 15 µg prefusion SARS-CoV-2 S-2P spike protein formulated with 3M-052 adjuvant (n=8; blue). (A): S-2P protein-specific antibody responses were measured by enzyme-linked immunosorbent assay (ELISA). Serial dilutions of plasma starting at 1:40 were assayed for IgG binding to SARS-CoV-2 spike. Data are reported as log 10 area under the curve (AUC) values. (B): Salivary RBD-specific IgG was measured by binding antigen multiplex assay (BAMA) using serial dilutions of saliva. (C): Antibody epitope specificity measured by BAMA. Plasma was diluted 1:10,000 to measure binding to different domains of the spike protein, including the full-length S protein, S1, RBD, NTD, and S2. Binding antibody responses are reported as log 10 transformed mean fluorescence intensity (MFI) after subtraction of background values. Red or blue lines and symbols represent the mRNA or protein vaccine groups, respectively, with different symbols representing individual animals ( Table S1 ).

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Transformation Assay, Fluorescence

    Vaccine-elicited SARS-CoV-2 neutralization responses. Panel A shows the ID 50 neutralization titers obtained using the pseudovirus neutralization assay. Panel B depicts the ID 50 neutralization titers in infant and adult rhesus macaques vaccinated with the mRNA-LNP vaccine ( 16 ). Infant RM were vaccinated with 30 µg of mRNA-LNP at week 0 and at week 4. Adult RM followed the same vaccine schedule but received 10 µg or 100 µg of the mRNA-LNP vaccine. Neutralization was assessed with a pseudovirus assay at week 8 (4 weeks after the second vaccination). Panel C shows the ID 50 neutralization titers obtained using the whole virus neutralization assay. Panel D illustrates the correlation of ID 50 (left graph) and ID 80 (right graph) neutralizing titers from both vaccine groups obtained in the pseudovirus or whole virus neutralization assay at week 6. Correlation was assessed using Spearman rank test.
    Figure Legend Snippet: Vaccine-elicited SARS-CoV-2 neutralization responses. Panel A shows the ID 50 neutralization titers obtained using the pseudovirus neutralization assay. Panel B depicts the ID 50 neutralization titers in infant and adult rhesus macaques vaccinated with the mRNA-LNP vaccine ( 16 ). Infant RM were vaccinated with 30 µg of mRNA-LNP at week 0 and at week 4. Adult RM followed the same vaccine schedule but received 10 µg or 100 µg of the mRNA-LNP vaccine. Neutralization was assessed with a pseudovirus assay at week 8 (4 weeks after the second vaccination). Panel C shows the ID 50 neutralization titers obtained using the whole virus neutralization assay. Panel D illustrates the correlation of ID 50 (left graph) and ID 80 (right graph) neutralizing titers from both vaccine groups obtained in the pseudovirus or whole virus neutralization assay at week 6. Correlation was assessed using Spearman rank test.

    Techniques Used: Neutralization

    2) Product Images from "A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model"

    Article Title: A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model

    Journal: bioRxiv

    doi: 10.1101/2021.04.16.440101

    Characterization of recombinant human TRES antibodies (A) Flow cytometric analysis of HEK-293T cells expressing the SARS-CoV-2-S protein and stained with recombinant humanized IgG1 TRES (TREShu) antibodies and a fluorochrome-labeled secondary antibody against human IgG-Fc. A non-S binding human antibody served as a negative control. (B, C) HEK-293T cells expressing the SARS-CoV-2 spike protein were incubated with recombinant TRES antibodies with a human Fcγ1 region and serially diluted TRES hybridoma antibodies with a murine Fcγ. Bound recombinant human TRES224hu (B) or TRES618hu (C) were detected with a mouse Alexa647-labeled antibody directed against the human Fcγ region. The mean percentages of binding and SEM of one experiment performed in triplicates are shown. (D) The SARS-CoV-2 neutralizing activity of the human recombinant TRES antibodies was analyzed as described in Fig. 4B . Shown are means and SEM of triplicates of one representative experiment out of three. Also given are the mean and standard deviation of IC50s, given in ng/ml, of the three independent experiments, calculated as described in Fig. 4B .
    Figure Legend Snippet: Characterization of recombinant human TRES antibodies (A) Flow cytometric analysis of HEK-293T cells expressing the SARS-CoV-2-S protein and stained with recombinant humanized IgG1 TRES (TREShu) antibodies and a fluorochrome-labeled secondary antibody against human IgG-Fc. A non-S binding human antibody served as a negative control. (B, C) HEK-293T cells expressing the SARS-CoV-2 spike protein were incubated with recombinant TRES antibodies with a human Fcγ1 region and serially diluted TRES hybridoma antibodies with a murine Fcγ. Bound recombinant human TRES224hu (B) or TRES618hu (C) were detected with a mouse Alexa647-labeled antibody directed against the human Fcγ region. The mean percentages of binding and SEM of one experiment performed in triplicates are shown. (D) The SARS-CoV-2 neutralizing activity of the human recombinant TRES antibodies was analyzed as described in Fig. 4B . Shown are means and SEM of triplicates of one representative experiment out of three. Also given are the mean and standard deviation of IC50s, given in ng/ml, of the three independent experiments, calculated as described in Fig. 4B .

    Techniques Used: Recombinant, Expressing, Staining, Labeling, Binding Assay, Negative Control, Incubation, Activity Assay, Standard Deviation

    Efficacy of TRES6 and TRES328 in a post-exposure prophylactic model. Reduction of viral load in hACE2-transgenic mice treated with TRES6, TRES328 or TRES480 isotype control antibody. Mice were inoculated intranasally with 300 FFU of SARS-CoV-2 on day 0. One day later, mice were treated intravenously with 5.25 mg/kg TRES6, TRES328 or TRES480 control antibody. Viral loads were determined on day 4 (A) or day 10 (B) after virus inoculation by RT-qPCR in the indicated organ samples. Data points represent the viral copy number of individual animals with the geometric means of each group depicted as lines, circles (•) indicate the survival of 4 or 10 days post-infection, and triangles indicate euthanized mice according to humane endpoints at day 6 (▲) or day 8 (▼). Calculated reduction of viral RNA is shown in comparison to the TRES480 control group. (C) Infectious virus load in BAL samples from antibody and isotype treated mice. Infectious virus was measured by focus-forming assay. (D) Body weight and (E) clinical score of antibody treated and isotype treated mice. Animals reaching humane endpoints were euthanized and are marked by a cross (†). (F) Survival curve of antibody treated and isotype control treated animals. Percent survival as the fraction of animals surviving humane endpoints (Kaplan-Meier analysis). Statistical analysis of the presented data was performed by Kruskal-Wallistest (one way ANOVA) and Dunn’s Pairwise Multiple Comparison Procedures as post hoc test in comparison to the TRES-480 control (ns: non-significant, *: p
    Figure Legend Snippet: Efficacy of TRES6 and TRES328 in a post-exposure prophylactic model. Reduction of viral load in hACE2-transgenic mice treated with TRES6, TRES328 or TRES480 isotype control antibody. Mice were inoculated intranasally with 300 FFU of SARS-CoV-2 on day 0. One day later, mice were treated intravenously with 5.25 mg/kg TRES6, TRES328 or TRES480 control antibody. Viral loads were determined on day 4 (A) or day 10 (B) after virus inoculation by RT-qPCR in the indicated organ samples. Data points represent the viral copy number of individual animals with the geometric means of each group depicted as lines, circles (•) indicate the survival of 4 or 10 days post-infection, and triangles indicate euthanized mice according to humane endpoints at day 6 (▲) or day 8 (▼). Calculated reduction of viral RNA is shown in comparison to the TRES480 control group. (C) Infectious virus load in BAL samples from antibody and isotype treated mice. Infectious virus was measured by focus-forming assay. (D) Body weight and (E) clinical score of antibody treated and isotype treated mice. Animals reaching humane endpoints were euthanized and are marked by a cross (†). (F) Survival curve of antibody treated and isotype control treated animals. Percent survival as the fraction of animals surviving humane endpoints (Kaplan-Meier analysis). Statistical analysis of the presented data was performed by Kruskal-Wallistest (one way ANOVA) and Dunn’s Pairwise Multiple Comparison Procedures as post hoc test in comparison to the TRES-480 control (ns: non-significant, *: p

    Techniques Used: Transgenic Assay, Mouse Assay, Quantitative RT-PCR, Infection, Focus Forming Assay

    Immunization of TRIANNI mice for induction of SARS-CoV-2 neutralizing antibodies. TRIANNI mice harboring the entire human Ig variable region repertoire (A) were primed by intramuscular electroporation with expression plasmids for wild type SARS-CoV-2-S (M1, M2) or a hybrid SARS-CoV-2-S containing the intracytoplasmic domain of VSV-G (M3, M4) (B) Mice were boosted with the expression plasmids used for priming (M1, M3), soluble trimeric S protein (M2), or exosomes carrying the hybrid SARS-CoV-2-S protein (M4). (C) A flow cytometric assay assessed the binding of sera at a 1:200 dilution to the SARS-CoV-2-S protein with HEK-293T cells transiently expressing the S protein. Numbers indicate the relative mean fluorescence intensities of sera drawn two weeks after the booster immunizations. (D) Scheme of hACE2-Fc competition assay. HEK-293T cells expressing the SARS-CoV-2-S protein were incubated with hACE2-Fc fusion protein in the presence or absence of sera from immunized mice before staining with an AF647-labelled anti-human Fc antibody. (E) Competitive inhibition of hACE2-Fc binding to trimeric S protein by sera (1:200) from control mice and mice at the indicated time points after the first immunization. The mean percentage of binding as compared to control binding is shown (two experiments each performed in triplicates). (F) For the neutralization assay, Vero-E6 cells were infected with the SARS-CoV-2 isolate MUC-IMB-1 in the presence or absence of week 5 sera. SARS-CoV-2 infection was quantitated after 20 to 24 hours by staining with purified IgG from a convalescent COVID-19 patient and a fluorescence-labeled anti-human IgG using an ELISPOT reader. The mean and SEM of triplicates of one experiment are shown.
    Figure Legend Snippet: Immunization of TRIANNI mice for induction of SARS-CoV-2 neutralizing antibodies. TRIANNI mice harboring the entire human Ig variable region repertoire (A) were primed by intramuscular electroporation with expression plasmids for wild type SARS-CoV-2-S (M1, M2) or a hybrid SARS-CoV-2-S containing the intracytoplasmic domain of VSV-G (M3, M4) (B) Mice were boosted with the expression plasmids used for priming (M1, M3), soluble trimeric S protein (M2), or exosomes carrying the hybrid SARS-CoV-2-S protein (M4). (C) A flow cytometric assay assessed the binding of sera at a 1:200 dilution to the SARS-CoV-2-S protein with HEK-293T cells transiently expressing the S protein. Numbers indicate the relative mean fluorescence intensities of sera drawn two weeks after the booster immunizations. (D) Scheme of hACE2-Fc competition assay. HEK-293T cells expressing the SARS-CoV-2-S protein were incubated with hACE2-Fc fusion protein in the presence or absence of sera from immunized mice before staining with an AF647-labelled anti-human Fc antibody. (E) Competitive inhibition of hACE2-Fc binding to trimeric S protein by sera (1:200) from control mice and mice at the indicated time points after the first immunization. The mean percentage of binding as compared to control binding is shown (two experiments each performed in triplicates). (F) For the neutralization assay, Vero-E6 cells were infected with the SARS-CoV-2 isolate MUC-IMB-1 in the presence or absence of week 5 sera. SARS-CoV-2 infection was quantitated after 20 to 24 hours by staining with purified IgG from a convalescent COVID-19 patient and a fluorescence-labeled anti-human IgG using an ELISPOT reader. The mean and SEM of triplicates of one experiment are shown.

    Techniques Used: Mouse Assay, Electroporation, Expressing, Flow Cytometry, Binding Assay, Fluorescence, Competitive Binding Assay, Incubation, Staining, Inhibition, Neutralization, Infection, Purification, Labeling, Enzyme-linked Immunospot

    Characterization of monoclonal TRES antibodies. (A) ELISA-based hACE2-competition assay with TRES antibodies. Plates were coated with RBD and incubated with serial dilutions of TRES antibodies and soluble hACE2 (400 ng/ml). Bound hACE2 was quantitated with HRP-coupled antibodies against the hFcγ1-Tag of hACE2. Mean and standard deviation of quadruplicates of one representative experiment out of two are shown in the graphs. The mean EC50s of all experiments are also shown. (B) Neutralization assay. Vero-E6 cells were incubated with the CoV-2 ER-1 isolate with increasing concentrations of the respective TRES antibodies. SARS-CoV-2 infection after 20 to 24 hours was quantitated as described in Fig. 1 F . Graphs show the mean and SEM of triplicates of one representative experiment of at least three experiments. The mean IC50 and standard deviation, in ng/ml, of all experiments, is also given. IC50s were calculated with inhibitor vs. variable slope fitting curve with GraphPad Prism 7.02.
    Figure Legend Snippet: Characterization of monoclonal TRES antibodies. (A) ELISA-based hACE2-competition assay with TRES antibodies. Plates were coated with RBD and incubated with serial dilutions of TRES antibodies and soluble hACE2 (400 ng/ml). Bound hACE2 was quantitated with HRP-coupled antibodies against the hFcγ1-Tag of hACE2. Mean and standard deviation of quadruplicates of one representative experiment out of two are shown in the graphs. The mean EC50s of all experiments are also shown. (B) Neutralization assay. Vero-E6 cells were incubated with the CoV-2 ER-1 isolate with increasing concentrations of the respective TRES antibodies. SARS-CoV-2 infection after 20 to 24 hours was quantitated as described in Fig. 1 F . Graphs show the mean and SEM of triplicates of one representative experiment of at least three experiments. The mean IC50 and standard deviation, in ng/ml, of all experiments, is also given. IC50s were calculated with inhibitor vs. variable slope fitting curve with GraphPad Prism 7.02.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Competitive Binding Assay, Incubation, Standard Deviation, Neutralization, Infection

    Breadth of neutralization by TRES antibodies and emergence of escape mutants. A) Neutralizing activity of hybridoma (TRES) or recombinant human TRES (TREShu) antibodies towards B.1.1.7 or B.1.351 determined as described in Fig. 4B . Shown are IC50s calculated from two to three experiments each performed in triplicates. (B) Emergence of escape mutants in cell culture during 5 passages on Vero-E6 cells in the presence of TRES6 or TRES328 in the absence or presence of increasing antibody concentrations. IC50s were determined in two independent experiments performed in triplicates as described previously in Fig. 4B . (C) Mutations in the S-Protein (not scaled) of the SARS-CoV-2 TRES6 (pink) and TRES328 (blue) escape mutants and the P5 variant passaged without antibody (white) as determined by whole-genome sequencing. The percentage describes the frequency of the mutations in the virus population. Exp (Experiment), NTD (N-terminal domain), RBD (receptorbinding domain), FP (fusion peptide), HR (heptat repeat), TM (transmembrane domain), CT (cytoplasmic tail).
    Figure Legend Snippet: Breadth of neutralization by TRES antibodies and emergence of escape mutants. A) Neutralizing activity of hybridoma (TRES) or recombinant human TRES (TREShu) antibodies towards B.1.1.7 or B.1.351 determined as described in Fig. 4B . Shown are IC50s calculated from two to three experiments each performed in triplicates. (B) Emergence of escape mutants in cell culture during 5 passages on Vero-E6 cells in the presence of TRES6 or TRES328 in the absence or presence of increasing antibody concentrations. IC50s were determined in two independent experiments performed in triplicates as described previously in Fig. 4B . (C) Mutations in the S-Protein (not scaled) of the SARS-CoV-2 TRES6 (pink) and TRES328 (blue) escape mutants and the P5 variant passaged without antibody (white) as determined by whole-genome sequencing. The percentage describes the frequency of the mutations in the virus population. Exp (Experiment), NTD (N-terminal domain), RBD (receptorbinding domain), FP (fusion peptide), HR (heptat repeat), TM (transmembrane domain), CT (cytoplasmic tail).

    Techniques Used: Neutralization, Activity Assay, Recombinant, Cell Culture, Variant Assay, Sequencing

    Binding pattern of hybridoma supernatants and purified antibodies. ELISA plates were coated with recombinant RBD ( A ), S2 ( B ), S1 ( C ), NTD ( D ), or SARS-CoV-2 spike protein stabilized in a prefusion state affinity-purified from medium of transfected HEK-293F cells ( E ) and incubated with TRES hybridoma culture medium. Bound antibodies were detected with HRP-labeled anti-mouse IgG antibodies and the RLUs measured. Mean and standard deviations of triplicates of one experiment are shown.
    Figure Legend Snippet: Binding pattern of hybridoma supernatants and purified antibodies. ELISA plates were coated with recombinant RBD ( A ), S2 ( B ), S1 ( C ), NTD ( D ), or SARS-CoV-2 spike protein stabilized in a prefusion state affinity-purified from medium of transfected HEK-293F cells ( E ) and incubated with TRES hybridoma culture medium. Bound antibodies were detected with HRP-labeled anti-mouse IgG antibodies and the RLUs measured. Mean and standard deviations of triplicates of one experiment are shown.

    Techniques Used: Binding Assay, Purification, Enzyme-linked Immunosorbent Assay, Recombinant, Affinity Purification, Transfection, Incubation, Labeling

    Screening of hybridoma supernatants. (A) The binding of antibodies from undiluted TRES hybridoma supernatants to HEK-293T cells expressing the SARS-CoV-2 spike protein was detected with a fluorescence-conjugated murine pan IgG antibody. Numbers indicate the relative mean fluorescence intensity. (B) Detection of hACE2-competing TRES antibodies as described in Fig. 1 D . Numbers depict the relative mean fluorescence intensity. (C) Vero-E6 cells were infected with the SARS-CoV-2 MUC-IMB-1 isolate in the presence or absence of undiluted TRES hybridoma supernatants. SARS-CoV-2 infection was quantified after 20 to 24 hours by staining as described in Fig. 1F . The mean and standard deviation of triplicates of one experiment are shown.
    Figure Legend Snippet: Screening of hybridoma supernatants. (A) The binding of antibodies from undiluted TRES hybridoma supernatants to HEK-293T cells expressing the SARS-CoV-2 spike protein was detected with a fluorescence-conjugated murine pan IgG antibody. Numbers indicate the relative mean fluorescence intensity. (B) Detection of hACE2-competing TRES antibodies as described in Fig. 1 D . Numbers depict the relative mean fluorescence intensity. (C) Vero-E6 cells were infected with the SARS-CoV-2 MUC-IMB-1 isolate in the presence or absence of undiluted TRES hybridoma supernatants. SARS-CoV-2 infection was quantified after 20 to 24 hours by staining as described in Fig. 1F . The mean and standard deviation of triplicates of one experiment are shown.

    Techniques Used: Binding Assay, Expressing, Fluorescence, Infection, Staining, Standard Deviation

    Related Articles

    Binding Assay:

    Article Title: A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model
    Article Snippet: EC50 values were calculated by plotting hACE2 activity against antibody concentrations and applying a 4-parameter curve fit using GraphPad Prism 7.02 (San Diego, USA). .. Detection of SARS-CoV-2 spike binding antibodies by ELISA96-well microtiter plates were coated over night at 4°C with 100ng per well of recombinant NTD (Sino Biological; Beijing, China #40591-V49H), SARS-CoV-2 Spike S2 or S1 Glycoprotein (Virion\Serion, Würzburg, Germany), RBD or a S protein stabilized in a prefusion state (both affinity purified from the supernatant of HEK-293F cells, as described previously [ , ]). .. The plates were washed and blocked with 5% skimmed milk for 1 hour at room temperature and then incubated with purified TRES antibodies or hybridoma supernatants for 1 hour.

    Recombinant:

    Article Title: A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model
    Article Snippet: EC50 values were calculated by plotting hACE2 activity against antibody concentrations and applying a 4-parameter curve fit using GraphPad Prism 7.02 (San Diego, USA). .. Detection of SARS-CoV-2 spike binding antibodies by ELISA96-well microtiter plates were coated over night at 4°C with 100ng per well of recombinant NTD (Sino Biological; Beijing, China #40591-V49H), SARS-CoV-2 Spike S2 or S1 Glycoprotein (Virion\Serion, Würzburg, Germany), RBD or a S protein stabilized in a prefusion state (both affinity purified from the supernatant of HEK-293F cells, as described previously [ , ]). .. The plates were washed and blocked with 5% skimmed milk for 1 hour at room temperature and then incubated with purified TRES antibodies or hybridoma supernatants for 1 hour.

    Affinity Purification:

    Article Title: A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model
    Article Snippet: EC50 values were calculated by plotting hACE2 activity against antibody concentrations and applying a 4-parameter curve fit using GraphPad Prism 7.02 (San Diego, USA). .. Detection of SARS-CoV-2 spike binding antibodies by ELISA96-well microtiter plates were coated over night at 4°C with 100ng per well of recombinant NTD (Sino Biological; Beijing, China #40591-V49H), SARS-CoV-2 Spike S2 or S1 Glycoprotein (Virion\Serion, Würzburg, Germany), RBD or a S protein stabilized in a prefusion state (both affinity purified from the supernatant of HEK-293F cells, as described previously [ , ]). .. The plates were washed and blocked with 5% skimmed milk for 1 hour at room temperature and then incubated with purified TRES antibodies or hybridoma supernatants for 1 hour.

    Produced:

    Article Title: SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques
    Article Snippet: Concentrations of antibody measured in BAMA were normalized relative to the total IgG and IgA measured by ELISA as describedusing plates coated with goat anti-monkey IgG or IgA and the secondary antibodies above. .. Mapping plasma S-specific IgGSARS-CoV-2 antigens, including whole spike (produced by PPF), S1 (Sinobiological cat# 40591-V08H), S2 (Sinobiological cat# 40590-V08B), RBD (Sinobiological cat# 40592-V08H) and NTD (Sinobiological cat# 40591-V49H) were conjugated to Magplex beads (Bio-Rad, Hercules, CA). .. The conjugated beads were incubated on filter plates (Millipore, Stafford, VA) for 30 min before plasma samples were added.

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    Sino Biological sars cov 2 2019 ncov spike s1 ntd his avi recombinant protein
    <t>SARS-CoV-2</t> vaccine-elicited functional antibody responses in infant rhesus macaques. (A): The ACE2 blocking assay was performed at 1:10 plasma dilution and data are reported as %ACE2 blocking. (B-C) : Neutralization capacity was measured using a pseudovirus assay (B) and whole virus assay (C) ; results are expressed as reciprocal 80% inhibitory dilution (ID 80 ). Different symbols represent individual animals ( Table S1 ).
    Sars Cov 2 2019 Ncov Spike S1 Ntd His Avi Recombinant Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological biotinylated sars cov 2 2019 ncov spike s1 ntd his avi recombinant protein
    <t>SARS-CoV-2</t> vaccine-elicited functional antibody responses in infant rhesus macaques. (A): The ACE2 blocking assay was performed at 1:10 plasma dilution and data are reported as %ACE2 blocking. (B-C) : Neutralization capacity was measured using a pseudovirus assay (B) and whole virus assay (C) ; results are expressed as reciprocal 80% inhibitory dilution (ID 80 ). Different symbols represent individual animals ( Table S1 ).
    Biotinylated Sars Cov 2 2019 Ncov Spike S1 Ntd His Avi Recombinant Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological sars cov 2 2019 ncov spike s1 ntd his avi recombinant protein biotinylated
    <t>SARS-CoV-2</t> vaccine-elicited functional antibody responses in infant rhesus macaques. (A): The ACE2 blocking assay was performed at 1:10 plasma dilution and data are reported as %ACE2 blocking. (B-C) : Neutralization capacity was measured using a pseudovirus assay (B) and whole virus assay (C) ; results are expressed as reciprocal 80% inhibitory dilution (ID 80 ). Different symbols represent individual animals ( Table S1 ).
    Sars Cov 2 2019 Ncov Spike S1 Ntd His Avi Recombinant Protein Biotinylated, supplied by Sino Biological, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SARS-CoV-2 vaccine-elicited functional antibody responses in infant rhesus macaques. (A): The ACE2 blocking assay was performed at 1:10 plasma dilution and data are reported as %ACE2 blocking. (B-C) : Neutralization capacity was measured using a pseudovirus assay (B) and whole virus assay (C) ; results are expressed as reciprocal 80% inhibitory dilution (ID 80 ). Different symbols represent individual animals ( Table S1 ).

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques

    doi: 10.1101/2021.04.05.438479

    Figure Lengend Snippet: SARS-CoV-2 vaccine-elicited functional antibody responses in infant rhesus macaques. (A): The ACE2 blocking assay was performed at 1:10 plasma dilution and data are reported as %ACE2 blocking. (B-C) : Neutralization capacity was measured using a pseudovirus assay (B) and whole virus assay (C) ; results are expressed as reciprocal 80% inhibitory dilution (ID 80 ). Different symbols represent individual animals ( Table S1 ).

    Article Snippet: Mapping plasma S-specific IgGSARS-CoV-2 antigens, including whole spike (produced by PPF), S1 (Sinobiological cat# 40591-V08H), S2 (Sinobiological cat# 40590-V08B), RBD (Sinobiological cat# 40592-V08H) and NTD (Sinobiological cat# 40591-V49H) were conjugated to Magplex beads (Bio-Rad, Hercules, CA).

    Techniques: Functional Assay, Blocking Assay, Neutralization

    Spike-specific T cell responses in LN following SARS-CoV-2 vaccination. Intracellular cytokine staining as in Figure 5 in lymph node biopsy samples at week 6 to measure Spike-specific T cell responses. Panels A and B show mRNA-LNP and Protein+3M-052+SE CD4 + T-cell responses, respectively. Panels C and D represent CD8 + T cell cytokine responses from mRNA-LNP and Protein+3M-052+SE animals, respectively. Symbols and the legend mirror those from Figure 6 (see also Table S1 ). Dotted lines represent 2 standard deviations from SARS-CoV-2 naïve LN samples.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques

    doi: 10.1101/2021.04.05.438479

    Figure Lengend Snippet: Spike-specific T cell responses in LN following SARS-CoV-2 vaccination. Intracellular cytokine staining as in Figure 5 in lymph node biopsy samples at week 6 to measure Spike-specific T cell responses. Panels A and B show mRNA-LNP and Protein+3M-052+SE CD4 + T-cell responses, respectively. Panels C and D represent CD8 + T cell cytokine responses from mRNA-LNP and Protein+3M-052+SE animals, respectively. Symbols and the legend mirror those from Figure 6 (see also Table S1 ). Dotted lines represent 2 standard deviations from SARS-CoV-2 naïve LN samples.

    Article Snippet: Mapping plasma S-specific IgGSARS-CoV-2 antigens, including whole spike (produced by PPF), S1 (Sinobiological cat# 40591-V08H), S2 (Sinobiological cat# 40590-V08B), RBD (Sinobiological cat# 40592-V08H) and NTD (Sinobiological cat# 40591-V49H) were conjugated to Magplex beads (Bio-Rad, Hercules, CA).

    Techniques: Staining

    Spike-specific CD8 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining was performed as described in Figure 5 at weeks 0, 6, 4, 8, and 14 to assess CD8 + T-cell responses. Panels A, B, C and D show responses detected in PBMC from the mRNA-LNP group at weeks 4, 6, 8, and 14, respectively. Panels E, F, G and H show responses in Protein+3M-052+SE vaccinees at weeks 4, 6, 8 and 14, respectively. The legend and symbols used mirror those from Figure 6 (see also Table S1 ). Dotted lines represent the cut-off for cytokine-positive responses, determined as 2 standard deviations above median values of SARS-CoV-2 naïve animals.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques

    doi: 10.1101/2021.04.05.438479

    Figure Lengend Snippet: Spike-specific CD8 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining was performed as described in Figure 5 at weeks 0, 6, 4, 8, and 14 to assess CD8 + T-cell responses. Panels A, B, C and D show responses detected in PBMC from the mRNA-LNP group at weeks 4, 6, 8, and 14, respectively. Panels E, F, G and H show responses in Protein+3M-052+SE vaccinees at weeks 4, 6, 8 and 14, respectively. The legend and symbols used mirror those from Figure 6 (see also Table S1 ). Dotted lines represent the cut-off for cytokine-positive responses, determined as 2 standard deviations above median values of SARS-CoV-2 naïve animals.

    Article Snippet: Mapping plasma S-specific IgGSARS-CoV-2 antigens, including whole spike (produced by PPF), S1 (Sinobiological cat# 40591-V08H), S2 (Sinobiological cat# 40590-V08B), RBD (Sinobiological cat# 40592-V08H) and NTD (Sinobiological cat# 40591-V49H) were conjugated to Magplex beads (Bio-Rad, Hercules, CA).

    Techniques: Staining

    Weight gain of SARS-CoV-2 immunized infant rhesus macaques. Longitudinal weight data for all female (top) and male (bottom) animals from this study with their respective symbol shapes and colors ( Table S1 ) overlaid with historical weight data from age and sex-matched infant rhesus monkeys housed outdoors at the CNPRC, Davis California (open black circles-female; filled gray circles-male).

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques

    doi: 10.1101/2021.04.05.438479

    Figure Lengend Snippet: Weight gain of SARS-CoV-2 immunized infant rhesus macaques. Longitudinal weight data for all female (top) and male (bottom) animals from this study with their respective symbol shapes and colors ( Table S1 ) overlaid with historical weight data from age and sex-matched infant rhesus monkeys housed outdoors at the CNPRC, Davis California (open black circles-female; filled gray circles-male).

    Article Snippet: Mapping plasma S-specific IgGSARS-CoV-2 antigens, including whole spike (produced by PPF), S1 (Sinobiological cat# 40591-V08H), S2 (Sinobiological cat# 40590-V08B), RBD (Sinobiological cat# 40592-V08H) and NTD (Sinobiological cat# 40591-V49H) were conjugated to Magplex beads (Bio-Rad, Hercules, CA).

    Techniques:

    Polyfunctional CD4 + T cells in SARS-CoV-2 immunized rhesus macaques at week 14. Panels A and B show the frequencies of CD4 + T cells that co-produced IL-17 and IFN-γ in response to stimulation with spike protein overlapping peptides in animals of the mRNA-LNP or Protein-3M-052-SE vaccine group, respectively. Individual symbols represent individual animals in each group ( Table S1 ).

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques

    doi: 10.1101/2021.04.05.438479

    Figure Lengend Snippet: Polyfunctional CD4 + T cells in SARS-CoV-2 immunized rhesus macaques at week 14. Panels A and B show the frequencies of CD4 + T cells that co-produced IL-17 and IFN-γ in response to stimulation with spike protein overlapping peptides in animals of the mRNA-LNP or Protein-3M-052-SE vaccine group, respectively. Individual symbols represent individual animals in each group ( Table S1 ).

    Article Snippet: Mapping plasma S-specific IgGSARS-CoV-2 antigens, including whole spike (produced by PPF), S1 (Sinobiological cat# 40591-V08H), S2 (Sinobiological cat# 40590-V08B), RBD (Sinobiological cat# 40592-V08H) and NTD (Sinobiological cat# 40591-V49H) were conjugated to Magplex beads (Bio-Rad, Hercules, CA).

    Techniques: Produced

    Characterization of Spike-specific B cell responses two weeks post boost. CD20 + CD27 + memory B cells that co-stained with fluorochrome-conjugated SARS-CoV-2 spike protein in mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccinees in blood ( A - B ) or LN ( C - D ). Frequencies are expressed as percent of total memory B cells. The gating strategy is provided in Supplementary Figure S8. ( E–F) portray antibody secreting cell (ASC) as measured by B cell ELISpot in PBMC from mRNA-LNP or Protein+3M-052-SE vaccinees, while (G–H) contain mRNA-LNP and Protein+3M-052-SE ASC responses, respectively, in LN at W6. Different symbols represent individual animals ( Table S1 ). Solid lines represent median values.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques

    doi: 10.1101/2021.04.05.438479

    Figure Lengend Snippet: Characterization of Spike-specific B cell responses two weeks post boost. CD20 + CD27 + memory B cells that co-stained with fluorochrome-conjugated SARS-CoV-2 spike protein in mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccinees in blood ( A - B ) or LN ( C - D ). Frequencies are expressed as percent of total memory B cells. The gating strategy is provided in Supplementary Figure S8. ( E–F) portray antibody secreting cell (ASC) as measured by B cell ELISpot in PBMC from mRNA-LNP or Protein+3M-052-SE vaccinees, while (G–H) contain mRNA-LNP and Protein+3M-052-SE ASC responses, respectively, in LN at W6. Different symbols represent individual animals ( Table S1 ). Solid lines represent median values.

    Article Snippet: Mapping plasma S-specific IgGSARS-CoV-2 antigens, including whole spike (produced by PPF), S1 (Sinobiological cat# 40591-V08H), S2 (Sinobiological cat# 40590-V08B), RBD (Sinobiological cat# 40592-V08H) and NTD (Sinobiological cat# 40591-V49H) were conjugated to Magplex beads (Bio-Rad, Hercules, CA).

    Techniques: Staining, Enzyme-linked Immunospot

    Spike-specific CD4 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining for IL-2, IL-17, IFN-γ, and TNF-α was performed on PBMC at weeks 0, 6, 4, 8, and 14 to assess T-cell responses to a peptide pool encompassing the entire SARS-CoV-2 spike protein. (A–D) display responses detected in Protein+3M-052+SE vaccinees (blue). (E-H) portray cytokine responses from mRNA-LNP recipients (red). The dashed lines represent week 0 values plus 2 standard deviations and define the cutoff for positive cytokine responses. Different symbols represent individual animals ( Table S1 ).

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques

    doi: 10.1101/2021.04.05.438479

    Figure Lengend Snippet: Spike-specific CD4 + T cell responses in SARS-CoV-2 immunized infant macaques. Intracellular cytokine staining for IL-2, IL-17, IFN-γ, and TNF-α was performed on PBMC at weeks 0, 6, 4, 8, and 14 to assess T-cell responses to a peptide pool encompassing the entire SARS-CoV-2 spike protein. (A–D) display responses detected in Protein+3M-052+SE vaccinees (blue). (E-H) portray cytokine responses from mRNA-LNP recipients (red). The dashed lines represent week 0 values plus 2 standard deviations and define the cutoff for positive cytokine responses. Different symbols represent individual animals ( Table S1 ).

    Article Snippet: Mapping plasma S-specific IgGSARS-CoV-2 antigens, including whole spike (produced by PPF), S1 (Sinobiological cat# 40591-V08H), S2 (Sinobiological cat# 40590-V08B), RBD (Sinobiological cat# 40592-V08H) and NTD (Sinobiological cat# 40591-V49H) were conjugated to Magplex beads (Bio-Rad, Hercules, CA).

    Techniques: Staining

    S-specific B cell gating strategy. Panel A shows a representative plot from RM7; Panel B shows a naive, age-matched control macaque without SARS-CoV-2 S protein conjugates; Panel C shows the same donor macaque with the SARS-CoV-2 S protein conjugates included.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques

    doi: 10.1101/2021.04.05.438479

    Figure Lengend Snippet: S-specific B cell gating strategy. Panel A shows a representative plot from RM7; Panel B shows a naive, age-matched control macaque without SARS-CoV-2 S protein conjugates; Panel C shows the same donor macaque with the SARS-CoV-2 S protein conjugates included.

    Article Snippet: Mapping plasma S-specific IgGSARS-CoV-2 antigens, including whole spike (produced by PPF), S1 (Sinobiological cat# 40591-V08H), S2 (Sinobiological cat# 40590-V08B), RBD (Sinobiological cat# 40592-V08H) and NTD (Sinobiological cat# 40591-V49H) were conjugated to Magplex beads (Bio-Rad, Hercules, CA).

    Techniques:

    Th1 and Th2 cytokines in plasma of infant rhesus macaques prior to and following SARS-CoV-2 vaccination. Plasma levels of IFN-γ ( panel A ) IL-2 ( panel B ), IL-4 ( panel C ) and IL-13 ( panel D ) were measured by multiplex Luminex assay. Different symbols represent individual animals in the mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccine groups, respectively ( Table S1 ). Horizontal lines represent median values.

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques

    doi: 10.1101/2021.04.05.438479

    Figure Lengend Snippet: Th1 and Th2 cytokines in plasma of infant rhesus macaques prior to and following SARS-CoV-2 vaccination. Plasma levels of IFN-γ ( panel A ) IL-2 ( panel B ), IL-4 ( panel C ) and IL-13 ( panel D ) were measured by multiplex Luminex assay. Different symbols represent individual animals in the mRNA-LNP (red) or Protein+3M-052-SE (blue) vaccine groups, respectively ( Table S1 ). Horizontal lines represent median values.

    Article Snippet: Mapping plasma S-specific IgGSARS-CoV-2 antigens, including whole spike (produced by PPF), S1 (Sinobiological cat# 40591-V08H), S2 (Sinobiological cat# 40590-V08B), RBD (Sinobiological cat# 40592-V08H) and NTD (Sinobiological cat# 40591-V49H) were conjugated to Magplex beads (Bio-Rad, Hercules, CA).

    Techniques: Multiplex Assay, Luminex

    SARS-CoV-2 vaccine-elicited binding antibody responses in infant rhesus macaques. Plasma and saliva were collected before vaccination (W0), at W4 -just prior to the boost-, two weeks post boost (W6), at W8, W14, W18 and W22 from infant RM vaccinated with 30 µg mRNA encoding S-2P spike protein in lipid nanoparticles (n=8; red) or with 15 µg prefusion SARS-CoV-2 S-2P spike protein formulated with 3M-052 adjuvant (n=8; blue). (A): S-2P protein-specific antibody responses were measured by enzyme-linked immunosorbent assay (ELISA). Serial dilutions of plasma starting at 1:40 were assayed for IgG binding to SARS-CoV-2 spike. Data are reported as log 10 area under the curve (AUC) values. (B): Salivary RBD-specific IgG was measured by binding antigen multiplex assay (BAMA) using serial dilutions of saliva. (C): Antibody epitope specificity measured by BAMA. Plasma was diluted 1:10,000 to measure binding to different domains of the spike protein, including the full-length S protein, S1, RBD, NTD, and S2. Binding antibody responses are reported as log 10 transformed mean fluorescence intensity (MFI) after subtraction of background values. Red or blue lines and symbols represent the mRNA or protein vaccine groups, respectively, with different symbols representing individual animals ( Table S1 ).

    Journal: bioRxiv

    Article Title: SARS-CoV-2 Vaccines Elicit Durable Immune Responses in Infant Rhesus Macaques

    doi: 10.1101/2021.04.05.438479

    Figure Lengend Snippet: SARS-CoV-2 vaccine-elicited binding antibody responses in infant rhesus macaques. Plasma and saliva were collected before vaccination (W0), at W4 -just prior to the boost-, two weeks post boost (W6), at W8, W14, W18 and W22 from infant RM vaccinated with 30 µg mRNA encoding S-2P spike protein in lipid nanoparticles (n=8; red) or with 15 µg prefusion SARS-CoV-2 S-2P spike protein formulated with 3M-052 adjuvant (n=8; blue). (A): S-2P protein-specific antibody responses were measured by enzyme-linked immunosorbent assay (ELISA). Serial dilutions of plasma starting at 1:40 were assayed for IgG binding to SARS-CoV-2 spike. Data are reported as log 10 area under the curve (AUC) values. (B): Salivary RBD-specific IgG was measured by binding antigen multiplex assay (BAMA) using serial dilutions of saliva. (C): Antibody epitope specificity measured by BAMA. Plasma was diluted 1:10,000 to measure binding to different domains of the spike protein, including the full-length S protein, S1, RBD, NTD, and S2. Binding antibody responses are reported as log 10 transformed mean fluorescence intensity (MFI) after subtraction of background values. Red or blue lines and symbols represent the mRNA or protein vaccine groups, respectively, with different symbols representing individual animals ( Table S1 ).

    Article Snippet: Mapping plasma S-specific IgGSARS-CoV-2 antigens, including whole spike (produced by PPF), S1 (Sinobiological cat# 40591-V08H), S2 (Sinobiological cat# 40590-V08B), RBD (Sinobiological cat# 40592-V08H) and NTD (Sinobiological cat# 40591-V49H) were conjugated to Magplex beads (Bio-Rad, Hercules, CA).

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Transformation Assay, Fluorescence

    Characterization of recombinant human TRES antibodies (A) Flow cytometric analysis of HEK-293T cells expressing the SARS-CoV-2-S protein and stained with recombinant humanized IgG1 TRES (TREShu) antibodies and a fluorochrome-labeled secondary antibody against human IgG-Fc. A non-S binding human antibody served as a negative control. (B, C) HEK-293T cells expressing the SARS-CoV-2 spike protein were incubated with recombinant TRES antibodies with a human Fcγ1 region and serially diluted TRES hybridoma antibodies with a murine Fcγ. Bound recombinant human TRES224hu (B) or TRES618hu (C) were detected with a mouse Alexa647-labeled antibody directed against the human Fcγ region. The mean percentages of binding and SEM of one experiment performed in triplicates are shown. (D) The SARS-CoV-2 neutralizing activity of the human recombinant TRES antibodies was analyzed as described in Fig. 4B . Shown are means and SEM of triplicates of one representative experiment out of three. Also given are the mean and standard deviation of IC50s, given in ng/ml, of the three independent experiments, calculated as described in Fig. 4B .

    Journal: bioRxiv

    Article Title: A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model

    doi: 10.1101/2021.04.16.440101

    Figure Lengend Snippet: Characterization of recombinant human TRES antibodies (A) Flow cytometric analysis of HEK-293T cells expressing the SARS-CoV-2-S protein and stained with recombinant humanized IgG1 TRES (TREShu) antibodies and a fluorochrome-labeled secondary antibody against human IgG-Fc. A non-S binding human antibody served as a negative control. (B, C) HEK-293T cells expressing the SARS-CoV-2 spike protein were incubated with recombinant TRES antibodies with a human Fcγ1 region and serially diluted TRES hybridoma antibodies with a murine Fcγ. Bound recombinant human TRES224hu (B) or TRES618hu (C) were detected with a mouse Alexa647-labeled antibody directed against the human Fcγ region. The mean percentages of binding and SEM of one experiment performed in triplicates are shown. (D) The SARS-CoV-2 neutralizing activity of the human recombinant TRES antibodies was analyzed as described in Fig. 4B . Shown are means and SEM of triplicates of one representative experiment out of three. Also given are the mean and standard deviation of IC50s, given in ng/ml, of the three independent experiments, calculated as described in Fig. 4B .

    Article Snippet: Detection of SARS-CoV-2 spike binding antibodies by ELISA96-well microtiter plates were coated over night at 4°C with 100ng per well of recombinant NTD (Sino Biological; Beijing, China #40591-V49H), SARS-CoV-2 Spike S2 or S1 Glycoprotein (Virion\Serion, Würzburg, Germany), RBD or a S protein stabilized in a prefusion state (both affinity purified from the supernatant of HEK-293F cells, as described previously [ , ]).

    Techniques: Recombinant, Expressing, Staining, Labeling, Binding Assay, Negative Control, Incubation, Activity Assay, Standard Deviation

    Efficacy of TRES6 and TRES328 in a post-exposure prophylactic model. Reduction of viral load in hACE2-transgenic mice treated with TRES6, TRES328 or TRES480 isotype control antibody. Mice were inoculated intranasally with 300 FFU of SARS-CoV-2 on day 0. One day later, mice were treated intravenously with 5.25 mg/kg TRES6, TRES328 or TRES480 control antibody. Viral loads were determined on day 4 (A) or day 10 (B) after virus inoculation by RT-qPCR in the indicated organ samples. Data points represent the viral copy number of individual animals with the geometric means of each group depicted as lines, circles (•) indicate the survival of 4 or 10 days post-infection, and triangles indicate euthanized mice according to humane endpoints at day 6 (▲) or day 8 (▼). Calculated reduction of viral RNA is shown in comparison to the TRES480 control group. (C) Infectious virus load in BAL samples from antibody and isotype treated mice. Infectious virus was measured by focus-forming assay. (D) Body weight and (E) clinical score of antibody treated and isotype treated mice. Animals reaching humane endpoints were euthanized and are marked by a cross (†). (F) Survival curve of antibody treated and isotype control treated animals. Percent survival as the fraction of animals surviving humane endpoints (Kaplan-Meier analysis). Statistical analysis of the presented data was performed by Kruskal-Wallistest (one way ANOVA) and Dunn’s Pairwise Multiple Comparison Procedures as post hoc test in comparison to the TRES-480 control (ns: non-significant, *: p

    Journal: bioRxiv

    Article Title: A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model

    doi: 10.1101/2021.04.16.440101

    Figure Lengend Snippet: Efficacy of TRES6 and TRES328 in a post-exposure prophylactic model. Reduction of viral load in hACE2-transgenic mice treated with TRES6, TRES328 or TRES480 isotype control antibody. Mice were inoculated intranasally with 300 FFU of SARS-CoV-2 on day 0. One day later, mice were treated intravenously with 5.25 mg/kg TRES6, TRES328 or TRES480 control antibody. Viral loads were determined on day 4 (A) or day 10 (B) after virus inoculation by RT-qPCR in the indicated organ samples. Data points represent the viral copy number of individual animals with the geometric means of each group depicted as lines, circles (•) indicate the survival of 4 or 10 days post-infection, and triangles indicate euthanized mice according to humane endpoints at day 6 (▲) or day 8 (▼). Calculated reduction of viral RNA is shown in comparison to the TRES480 control group. (C) Infectious virus load in BAL samples from antibody and isotype treated mice. Infectious virus was measured by focus-forming assay. (D) Body weight and (E) clinical score of antibody treated and isotype treated mice. Animals reaching humane endpoints were euthanized and are marked by a cross (†). (F) Survival curve of antibody treated and isotype control treated animals. Percent survival as the fraction of animals surviving humane endpoints (Kaplan-Meier analysis). Statistical analysis of the presented data was performed by Kruskal-Wallistest (one way ANOVA) and Dunn’s Pairwise Multiple Comparison Procedures as post hoc test in comparison to the TRES-480 control (ns: non-significant, *: p

    Article Snippet: Detection of SARS-CoV-2 spike binding antibodies by ELISA96-well microtiter plates were coated over night at 4°C with 100ng per well of recombinant NTD (Sino Biological; Beijing, China #40591-V49H), SARS-CoV-2 Spike S2 or S1 Glycoprotein (Virion\Serion, Würzburg, Germany), RBD or a S protein stabilized in a prefusion state (both affinity purified from the supernatant of HEK-293F cells, as described previously [ , ]).

    Techniques: Transgenic Assay, Mouse Assay, Quantitative RT-PCR, Infection, Focus Forming Assay

    Immunization of TRIANNI mice for induction of SARS-CoV-2 neutralizing antibodies. TRIANNI mice harboring the entire human Ig variable region repertoire (A) were primed by intramuscular electroporation with expression plasmids for wild type SARS-CoV-2-S (M1, M2) or a hybrid SARS-CoV-2-S containing the intracytoplasmic domain of VSV-G (M3, M4) (B) Mice were boosted with the expression plasmids used for priming (M1, M3), soluble trimeric S protein (M2), or exosomes carrying the hybrid SARS-CoV-2-S protein (M4). (C) A flow cytometric assay assessed the binding of sera at a 1:200 dilution to the SARS-CoV-2-S protein with HEK-293T cells transiently expressing the S protein. Numbers indicate the relative mean fluorescence intensities of sera drawn two weeks after the booster immunizations. (D) Scheme of hACE2-Fc competition assay. HEK-293T cells expressing the SARS-CoV-2-S protein were incubated with hACE2-Fc fusion protein in the presence or absence of sera from immunized mice before staining with an AF647-labelled anti-human Fc antibody. (E) Competitive inhibition of hACE2-Fc binding to trimeric S protein by sera (1:200) from control mice and mice at the indicated time points after the first immunization. The mean percentage of binding as compared to control binding is shown (two experiments each performed in triplicates). (F) For the neutralization assay, Vero-E6 cells were infected with the SARS-CoV-2 isolate MUC-IMB-1 in the presence or absence of week 5 sera. SARS-CoV-2 infection was quantitated after 20 to 24 hours by staining with purified IgG from a convalescent COVID-19 patient and a fluorescence-labeled anti-human IgG using an ELISPOT reader. The mean and SEM of triplicates of one experiment are shown.

    Journal: bioRxiv

    Article Title: A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model

    doi: 10.1101/2021.04.16.440101

    Figure Lengend Snippet: Immunization of TRIANNI mice for induction of SARS-CoV-2 neutralizing antibodies. TRIANNI mice harboring the entire human Ig variable region repertoire (A) were primed by intramuscular electroporation with expression plasmids for wild type SARS-CoV-2-S (M1, M2) or a hybrid SARS-CoV-2-S containing the intracytoplasmic domain of VSV-G (M3, M4) (B) Mice were boosted with the expression plasmids used for priming (M1, M3), soluble trimeric S protein (M2), or exosomes carrying the hybrid SARS-CoV-2-S protein (M4). (C) A flow cytometric assay assessed the binding of sera at a 1:200 dilution to the SARS-CoV-2-S protein with HEK-293T cells transiently expressing the S protein. Numbers indicate the relative mean fluorescence intensities of sera drawn two weeks after the booster immunizations. (D) Scheme of hACE2-Fc competition assay. HEK-293T cells expressing the SARS-CoV-2-S protein were incubated with hACE2-Fc fusion protein in the presence or absence of sera from immunized mice before staining with an AF647-labelled anti-human Fc antibody. (E) Competitive inhibition of hACE2-Fc binding to trimeric S protein by sera (1:200) from control mice and mice at the indicated time points after the first immunization. The mean percentage of binding as compared to control binding is shown (two experiments each performed in triplicates). (F) For the neutralization assay, Vero-E6 cells were infected with the SARS-CoV-2 isolate MUC-IMB-1 in the presence or absence of week 5 sera. SARS-CoV-2 infection was quantitated after 20 to 24 hours by staining with purified IgG from a convalescent COVID-19 patient and a fluorescence-labeled anti-human IgG using an ELISPOT reader. The mean and SEM of triplicates of one experiment are shown.

    Article Snippet: Detection of SARS-CoV-2 spike binding antibodies by ELISA96-well microtiter plates were coated over night at 4°C with 100ng per well of recombinant NTD (Sino Biological; Beijing, China #40591-V49H), SARS-CoV-2 Spike S2 or S1 Glycoprotein (Virion\Serion, Würzburg, Germany), RBD or a S protein stabilized in a prefusion state (both affinity purified from the supernatant of HEK-293F cells, as described previously [ , ]).

    Techniques: Mouse Assay, Electroporation, Expressing, Flow Cytometry, Binding Assay, Fluorescence, Competitive Binding Assay, Incubation, Staining, Inhibition, Neutralization, Infection, Purification, Labeling, Enzyme-linked Immunospot

    Characterization of monoclonal TRES antibodies. (A) ELISA-based hACE2-competition assay with TRES antibodies. Plates were coated with RBD and incubated with serial dilutions of TRES antibodies and soluble hACE2 (400 ng/ml). Bound hACE2 was quantitated with HRP-coupled antibodies against the hFcγ1-Tag of hACE2. Mean and standard deviation of quadruplicates of one representative experiment out of two are shown in the graphs. The mean EC50s of all experiments are also shown. (B) Neutralization assay. Vero-E6 cells were incubated with the CoV-2 ER-1 isolate with increasing concentrations of the respective TRES antibodies. SARS-CoV-2 infection after 20 to 24 hours was quantitated as described in Fig. 1 F . Graphs show the mean and SEM of triplicates of one representative experiment of at least three experiments. The mean IC50 and standard deviation, in ng/ml, of all experiments, is also given. IC50s were calculated with inhibitor vs. variable slope fitting curve with GraphPad Prism 7.02.

    Journal: bioRxiv

    Article Title: A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model

    doi: 10.1101/2021.04.16.440101

    Figure Lengend Snippet: Characterization of monoclonal TRES antibodies. (A) ELISA-based hACE2-competition assay with TRES antibodies. Plates were coated with RBD and incubated with serial dilutions of TRES antibodies and soluble hACE2 (400 ng/ml). Bound hACE2 was quantitated with HRP-coupled antibodies against the hFcγ1-Tag of hACE2. Mean and standard deviation of quadruplicates of one representative experiment out of two are shown in the graphs. The mean EC50s of all experiments are also shown. (B) Neutralization assay. Vero-E6 cells were incubated with the CoV-2 ER-1 isolate with increasing concentrations of the respective TRES antibodies. SARS-CoV-2 infection after 20 to 24 hours was quantitated as described in Fig. 1 F . Graphs show the mean and SEM of triplicates of one representative experiment of at least three experiments. The mean IC50 and standard deviation, in ng/ml, of all experiments, is also given. IC50s were calculated with inhibitor vs. variable slope fitting curve with GraphPad Prism 7.02.

    Article Snippet: Detection of SARS-CoV-2 spike binding antibodies by ELISA96-well microtiter plates were coated over night at 4°C with 100ng per well of recombinant NTD (Sino Biological; Beijing, China #40591-V49H), SARS-CoV-2 Spike S2 or S1 Glycoprotein (Virion\Serion, Würzburg, Germany), RBD or a S protein stabilized in a prefusion state (both affinity purified from the supernatant of HEK-293F cells, as described previously [ , ]).

    Techniques: Enzyme-linked Immunosorbent Assay, Competitive Binding Assay, Incubation, Standard Deviation, Neutralization, Infection

    Breadth of neutralization by TRES antibodies and emergence of escape mutants. A) Neutralizing activity of hybridoma (TRES) or recombinant human TRES (TREShu) antibodies towards B.1.1.7 or B.1.351 determined as described in Fig. 4B . Shown are IC50s calculated from two to three experiments each performed in triplicates. (B) Emergence of escape mutants in cell culture during 5 passages on Vero-E6 cells in the presence of TRES6 or TRES328 in the absence or presence of increasing antibody concentrations. IC50s were determined in two independent experiments performed in triplicates as described previously in Fig. 4B . (C) Mutations in the S-Protein (not scaled) of the SARS-CoV-2 TRES6 (pink) and TRES328 (blue) escape mutants and the P5 variant passaged without antibody (white) as determined by whole-genome sequencing. The percentage describes the frequency of the mutations in the virus population. Exp (Experiment), NTD (N-terminal domain), RBD (receptorbinding domain), FP (fusion peptide), HR (heptat repeat), TM (transmembrane domain), CT (cytoplasmic tail).

    Journal: bioRxiv

    Article Title: A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model

    doi: 10.1101/2021.04.16.440101

    Figure Lengend Snippet: Breadth of neutralization by TRES antibodies and emergence of escape mutants. A) Neutralizing activity of hybridoma (TRES) or recombinant human TRES (TREShu) antibodies towards B.1.1.7 or B.1.351 determined as described in Fig. 4B . Shown are IC50s calculated from two to three experiments each performed in triplicates. (B) Emergence of escape mutants in cell culture during 5 passages on Vero-E6 cells in the presence of TRES6 or TRES328 in the absence or presence of increasing antibody concentrations. IC50s were determined in two independent experiments performed in triplicates as described previously in Fig. 4B . (C) Mutations in the S-Protein (not scaled) of the SARS-CoV-2 TRES6 (pink) and TRES328 (blue) escape mutants and the P5 variant passaged without antibody (white) as determined by whole-genome sequencing. The percentage describes the frequency of the mutations in the virus population. Exp (Experiment), NTD (N-terminal domain), RBD (receptorbinding domain), FP (fusion peptide), HR (heptat repeat), TM (transmembrane domain), CT (cytoplasmic tail).

    Article Snippet: Detection of SARS-CoV-2 spike binding antibodies by ELISA96-well microtiter plates were coated over night at 4°C with 100ng per well of recombinant NTD (Sino Biological; Beijing, China #40591-V49H), SARS-CoV-2 Spike S2 or S1 Glycoprotein (Virion\Serion, Würzburg, Germany), RBD or a S protein stabilized in a prefusion state (both affinity purified from the supernatant of HEK-293F cells, as described previously [ , ]).

    Techniques: Neutralization, Activity Assay, Recombinant, Cell Culture, Variant Assay, Sequencing

    Binding pattern of hybridoma supernatants and purified antibodies. ELISA plates were coated with recombinant RBD ( A ), S2 ( B ), S1 ( C ), NTD ( D ), or SARS-CoV-2 spike protein stabilized in a prefusion state affinity-purified from medium of transfected HEK-293F cells ( E ) and incubated with TRES hybridoma culture medium. Bound antibodies were detected with HRP-labeled anti-mouse IgG antibodies and the RLUs measured. Mean and standard deviations of triplicates of one experiment are shown.

    Journal: bioRxiv

    Article Title: A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model

    doi: 10.1101/2021.04.16.440101

    Figure Lengend Snippet: Binding pattern of hybridoma supernatants and purified antibodies. ELISA plates were coated with recombinant RBD ( A ), S2 ( B ), S1 ( C ), NTD ( D ), or SARS-CoV-2 spike protein stabilized in a prefusion state affinity-purified from medium of transfected HEK-293F cells ( E ) and incubated with TRES hybridoma culture medium. Bound antibodies were detected with HRP-labeled anti-mouse IgG antibodies and the RLUs measured. Mean and standard deviations of triplicates of one experiment are shown.

    Article Snippet: Detection of SARS-CoV-2 spike binding antibodies by ELISA96-well microtiter plates were coated over night at 4°C with 100ng per well of recombinant NTD (Sino Biological; Beijing, China #40591-V49H), SARS-CoV-2 Spike S2 or S1 Glycoprotein (Virion\Serion, Würzburg, Germany), RBD or a S protein stabilized in a prefusion state (both affinity purified from the supernatant of HEK-293F cells, as described previously [ , ]).

    Techniques: Binding Assay, Purification, Enzyme-linked Immunosorbent Assay, Recombinant, Affinity Purification, Transfection, Incubation, Labeling

    Screening of hybridoma supernatants. (A) The binding of antibodies from undiluted TRES hybridoma supernatants to HEK-293T cells expressing the SARS-CoV-2 spike protein was detected with a fluorescence-conjugated murine pan IgG antibody. Numbers indicate the relative mean fluorescence intensity. (B) Detection of hACE2-competing TRES antibodies as described in Fig. 1 D . Numbers depict the relative mean fluorescence intensity. (C) Vero-E6 cells were infected with the SARS-CoV-2 MUC-IMB-1 isolate in the presence or absence of undiluted TRES hybridoma supernatants. SARS-CoV-2 infection was quantified after 20 to 24 hours by staining as described in Fig. 1F . The mean and standard deviation of triplicates of one experiment are shown.

    Journal: bioRxiv

    Article Title: A pair of non-competing neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model

    doi: 10.1101/2021.04.16.440101

    Figure Lengend Snippet: Screening of hybridoma supernatants. (A) The binding of antibodies from undiluted TRES hybridoma supernatants to HEK-293T cells expressing the SARS-CoV-2 spike protein was detected with a fluorescence-conjugated murine pan IgG antibody. Numbers indicate the relative mean fluorescence intensity. (B) Detection of hACE2-competing TRES antibodies as described in Fig. 1 D . Numbers depict the relative mean fluorescence intensity. (C) Vero-E6 cells were infected with the SARS-CoV-2 MUC-IMB-1 isolate in the presence or absence of undiluted TRES hybridoma supernatants. SARS-CoV-2 infection was quantified after 20 to 24 hours by staining as described in Fig. 1F . The mean and standard deviation of triplicates of one experiment are shown.

    Article Snippet: Detection of SARS-CoV-2 spike binding antibodies by ELISA96-well microtiter plates were coated over night at 4°C with 100ng per well of recombinant NTD (Sino Biological; Beijing, China #40591-V49H), SARS-CoV-2 Spike S2 or S1 Glycoprotein (Virion\Serion, Würzburg, Germany), RBD or a S protein stabilized in a prefusion state (both affinity purified from the supernatant of HEK-293F cells, as described previously [ , ]).

    Techniques: Binding Assay, Expressing, Fluorescence, Infection, Staining, Standard Deviation