Journal: EBioMedicine

Article Title: Adjuvant-enhanced CD4 T Cell Responses are Critical to Durable Vaccine Immunity

doi: 10.1016/j.ebiom.2015.11.041

Figure Lengend Snippet: Adjuvants impact the durability of protection conferred by eVLP vaccination. (a) C57BL/6 mice were vaccinated IM two times with 10 μg of eVLP and challenged four (short-term) weeks or twenty-two (long-term) weeks after the vaccine boost. Data in A are pooled from 8 individual studies with 6–10 animals/group. Fisher's exact test: survival in the short-term group was significantly higher than in the long-term group (p < 0.0001). (b) C57BL/6 mice were vaccinated IM two times with 10 μg of eVLP and challenged at the indicated days after the second vaccination. n = 9 or 10/group. Cochran-Armitage test: percentage surviving declined as time to challenge increased (p = 0.0046). There was a significant difference (p = 0.03) between survival on Day 77 and Day 175. (c) Serum samples collected from animals in (B) one week prior to challenge were subjected to an ELISA for the evaluation of anti-GP IgG, IgG1, and IgG2c antibody titers. Red symbols indicate titers of animals that succumbed to challenge while black indicate titers of survivors; red symbols with black outlines indicate that one of these two animals succumbed to challenge, but animal tags were indeterminate after challenge. Median and IQR shown. (d) C57BL/6 mice were vaccinated two times (IM) with VLP, with or without the indicated adjuvants. Animals were challenged twenty-two weeks after the vaccine boost. Data in D are pooled from at least 4 separate studies with a total of at least 35 animals per group. P-values comparing VLP alone to vaccination with VLP and adjuvant are shown, calculated using Fisher's exact tests with stepdown Bonferroni correction. V = VLP, VP = VLP + PolyICLC, VC = VLP + CpG, VM = VLP + MPLA, and VA = VLP + alhydrogel.

Article Snippet: Secondary antibodies included goat anti-mouse IgG-HRP (Southern Biotech 1030–05), IgG1-HRP (Southern Biotech 1070–05), IgG2c-HRP (Southern Biotech 1079–05), and IgG3-HRP (Southern Biotech 1100–05).

Techniques: Enzyme-linked Immunosorbent Assay, Adjuvant

Journal: EBioMedicine

Article Title: Adjuvant-enhanced CD4 T Cell Responses are Critical to Durable Vaccine Immunity

doi: 10.1016/j.ebiom.2015.11.041

Figure Lengend Snippet: Adjuvants have variable impact on IgG subclasses and antibody neutralization. (a) Serum was collected 14 days and 147 days after the vaccine boost (days 35 and 168, respectively) and evaluated for anti-GP IgG, IgG1, IgG2c, and IgG3 levels using an ELISA. Data shown are pooled from at least two separate experiments per group. (b) Pairwise comparison using DSCF multiple pairwise comparison was used and p values greater than 0.05 are shown as “ns”. (c) Summary of results shown in A, B and D. (d) Neutralizing antibody titers were evaluated using the PsVNA, with titers giving 80% neutralization shown; median and IQR shown. Samples within each group were selected randomly from three separate studies for evaluation in the assay. Pairwise comparison using post-hoc Tukey's studentized range test procedure was used to evaluate differences between groups at both days 35 and day 168, where “*” indicates 0.01 < p < 0.05, “**” indicates 0.001 < p < 0.01, “***” indicates 0.0001 < p < 0.001, and “****” indicates p < 0.0001.

Article Snippet: Secondary antibodies included goat anti-mouse IgG-HRP (Southern Biotech 1030–05), IgG1-HRP (Southern Biotech 1070–05), IgG2c-HRP (Southern Biotech 1079–05), and IgG3-HRP (Southern Biotech 1100–05).

Techniques: Neutralization, Enzyme-linked Immunosorbent Assay, Comparison

Journal: EBioMedicine

Article Title: Adjuvant-enhanced CD4 T Cell Responses are Critical to Durable Vaccine Immunity

doi: 10.1016/j.ebiom.2015.11.041

Figure Lengend Snippet: CD8 T cell deficiency does not impact short term or long term survival of vaccinated C57BL/6J mice. (a) Mice were treated IM twice with saline, VLP, VLP and polyICLC, or VLP and CpG. Four weeks after the second vaccination, mice were challenged. Closed symbols represent wild type C57BL/6J mice and open symbols indicate CD8-deficient mice. (b) Mice were vaccinated on the same schedule as in A, but challenge occurred 22 weeks after the second vaccination. (c) Two weeks after the second vaccination and 1 week prior to challenge, blood was collected from vaccinated animals and evaluated for anti-GP IgG antibody titers. (D) A subset of vaccinated animals was euthanized 4 days after the vaccine boost to evaluate CD4 + T cell responses. Median response of C57BL/6J mice and CD8-deficient mice is shown. N = 8–10 per treated group for A–C and D presents data pooled from two separate evaluations of 4–8 mice per group each. “*” indicates 0.005 < p < 0.05.

Article Snippet: Secondary antibodies included goat anti-mouse IgG-HRP (Southern Biotech 1030–05), IgG1-HRP (Southern Biotech 1070–05), IgG2c-HRP (Southern Biotech 1079–05), and IgG3-HRP (Southern Biotech 1100–05).

Techniques: Saline

Journal: Communications Medicine

Article Title: Application of machine learning algorithms to identify serological predictors of COVID-19 severity and outcomes

doi: 10.1038/s43856-024-00658-w

Figure Lengend Snippet: a – d Anti-nucleocapsid (N) and anti-spike (S) secretory IgA or ( e – h ) IgG responses were measured as median fluorescence intensity (MFI) in nasopharyngeal (NP) or oropharyngeal (OP) samples and compared among COVID-19 hospitalized patients classified as moderate (WHO score 3–4), severe (WHO score 5–7), or deceased (WHO score 8). Data are analyzed using Welch’s ANOVA and presented as means with standard deviations, indicated by error bars.

Article Snippet: Plates were incubated for 2 h at RT, washed, and 50 μL of anti-human HRP IgG (1:5000, #A18823, Invitrogen, Thermo Fisher Scientific), IgA (1:5000, #A18787, Invitrogen, Thermo Fisher Scientific), IgG1 (1:4000, #9054-05, Southern Biotech), IgG2 (1:4000, #9060-05, Southern Biotech), IgG3 (1:4000, #9210-05, Southern Biotech) or IgG4 (1:8000, #9200-05, Southern Biotech) secondary antibody was added.

Techniques: Fluorescence

Journal: Communications Medicine

Article Title: Application of machine learning algorithms to identify serological predictors of COVID-19 severity and outcomes

doi: 10.1038/s43856-024-00658-w

Figure Lengend Snippet: a – d IgG binding antibody responses against ancestral spike (S), spike receptor binding domain (S-RBD), and nucleocapsid (N) were quantified by ELISA and calculated as the binding antibody units (BAU) per ml if international standards were available or as the area under the curve (AUC) if standards were not available and titration curves only could be generated; ( e ) ACE2 binding inhibition antibodies were measured using MSD V-PLEX SARS-CoV-2 ACE2 kits; and ( f – h ) Fc effector antibody responses were quantified using complement fixation and antibody-dependent cellular cytotoxicity (ADCC) assays. All assays were run using ancestral SARS-CoV-2. Data were compared using linear regression analysis, controlling for age and biological sex, to look at differences between unvaccinated non-hospitalized and hospitalized patients at 1 MPE. Data are presented as means with standard deviations, indicated by error bars. The limit of detection (LOD) is indicated by the dashed lines. p -values for statistically significant differences (p < 0.05) are shown in the figures.

Article Snippet: Plates were incubated for 2 h at RT, washed, and 50 μL of anti-human HRP IgG (1:5000, #A18823, Invitrogen, Thermo Fisher Scientific), IgA (1:5000, #A18787, Invitrogen, Thermo Fisher Scientific), IgG1 (1:4000, #9054-05, Southern Biotech), IgG2 (1:4000, #9060-05, Southern Biotech), IgG3 (1:4000, #9210-05, Southern Biotech) or IgG4 (1:8000, #9200-05, Southern Biotech) secondary antibody was added.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Titration, Generated, Inhibition

Journal: Communications Medicine

Article Title: Application of machine learning algorithms to identify serological predictors of COVID-19 severity and outcomes

doi: 10.1038/s43856-024-00658-w

Figure Lengend Snippet: The binding ( a – c ) IgG and ( d ) IgA antibodies recognizing ancestral SARS-CoV-2 spike (S), spike receptor binding domain (S-RBD), or nucleocapsid (N) were quantified by ELISA, and measured as the binding antibody units (BAU) per mL if international standards were available or as the area under the curve (AUC) if standards were not available and titration curves could only be generated. e The percentage of ACE2 inhibition for the ancestral SARS-CoV-2 variant was calculated and arcsine transformed for analyses. f – h The Fc effector antibody responses were measured based on C1q complement fixation in response to either the spike or S-RBD or antibody dependent cellular cytotoxicity and reported as arbitrary units (AU). Antibody responses were compared among COVID-19 hospitalized patients classified as moderate (WHO score 3–4), severe (WHO score 5–7), or deceased (WHO score 8) using samples collected at enrollment vs. 1 MPE. Data are presented as means with standard deviations, indicated by error bars. p -values for statistically significant differences (p < 0.05) by linear mixed-effects regression to compare change over time or Welch’s ANOVA to compare across groups within a time point are indicated. Limit of detection (LOD) are indicated by the dashed lines.

Article Snippet: Plates were incubated for 2 h at RT, washed, and 50 μL of anti-human HRP IgG (1:5000, #A18823, Invitrogen, Thermo Fisher Scientific), IgA (1:5000, #A18787, Invitrogen, Thermo Fisher Scientific), IgG1 (1:4000, #9054-05, Southern Biotech), IgG2 (1:4000, #9060-05, Southern Biotech), IgG3 (1:4000, #9210-05, Southern Biotech) or IgG4 (1:8000, #9200-05, Southern Biotech) secondary antibody was added.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Titration, Generated, Inhibition, Variant Assay, Transformation Assay

Journal: Communications Medicine

Article Title: Application of machine learning algorithms to identify serological predictors of COVID-19 severity and outcomes

doi: 10.1038/s43856-024-00658-w

Figure Lengend Snippet: The binding of IgG1 ( a ), IgG2 ( b ), IgG3 ( c ), and IgG4 ( d ) to ancestral SARS-CoV-2 S antigen was measured as the area under the curve (AUC). Spearman correlation of IgG1 ( e ), IgG2 ( f ), IgG3 ( g ), and IgG4 ( h ) with % ACE2 inhibition at enrollment. Hospitalized COVID-19 patients were classified as moderate (WHO score 3–4), severe (WHO score 5–7), or deceased (WHO score 8). Data are presented as means with standard deviations, indicated by error bars. The limit of detection (LOD) is indicated by the dashed lines. p -values for statistically significant differences (p < 0.05) by linear mixed-effects regression to compare change over time or Welch’s ANOVA to compare across groups within a time point are indicated.

Article Snippet: Plates were incubated for 2 h at RT, washed, and 50 μL of anti-human HRP IgG (1:5000, #A18823, Invitrogen, Thermo Fisher Scientific), IgA (1:5000, #A18787, Invitrogen, Thermo Fisher Scientific), IgG1 (1:4000, #9054-05, Southern Biotech), IgG2 (1:4000, #9060-05, Southern Biotech), IgG3 (1:4000, #9210-05, Southern Biotech) or IgG4 (1:8000, #9200-05, Southern Biotech) secondary antibody was added.

Techniques: Binding Assay, Inhibition

Journal: Communications Medicine

Article Title: Application of machine learning algorithms to identify serological predictors of COVID-19 severity and outcomes

doi: 10.1038/s43856-024-00658-w

Figure Lengend Snippet: Linear mixed-effects regression models for ( a – d ) anti-spike (S), anti-spike receptor binding domain (S-RBD), or anti-nucleocapsid (N) IgG or IgA, measured as the binding antibody units (BAU) per ml if international standards were available or as the area under the curve (AUC) if standards were not available and only titration curves could be generated; ( e ) the percentage ACE2 inhibition against ancestral SARS-CoV-2 as a surrogate of virus neutralization, and ( f – h ) Fc effector antibody responses as measured by complement fixation against spike or S-RBD or antibody-dependent cellular cytotoxicity (ADCC) up until 100 DPE or death among hospitalized patients classified as moderate (WHO score 3–4; n = 41), severe (WHO score 5–7; n = 40), or deceased (WHO score 8; n = 24). P -values for statistically significant differences (p < 0.05) by linear mixed-effects regression contrasts are shown within the figures.

Article Snippet: Plates were incubated for 2 h at RT, washed, and 50 μL of anti-human HRP IgG (1:5000, #A18823, Invitrogen, Thermo Fisher Scientific), IgA (1:5000, #A18787, Invitrogen, Thermo Fisher Scientific), IgG1 (1:4000, #9054-05, Southern Biotech), IgG2 (1:4000, #9060-05, Southern Biotech), IgG3 (1:4000, #9210-05, Southern Biotech) or IgG4 (1:8000, #9200-05, Southern Biotech) secondary antibody was added.

Techniques: Binding Assay, Titration, Generated, Inhibition, Virus, Neutralization

Journal: Frontiers in Immunology

Article Title: Immunization With Fc-Based Recombinant Epstein–Barr Virus gp350 Elicits Potent Neutralizing Humoral Immune Response in a BALB/c Mice Model

doi: 10.3389/fimmu.2018.00932

Figure Lengend Snippet: Characterization of Fc-based recombinant gp350 proteins. (A) Diagrams for design of Fc-based recombinant gp350 proteins. The extracellular domains (D-I, D-II, and D-III), transmembrane domains, and cytoplasmic tail (TD/CT) are shown in gray. The 6-histidine tag is shown as a hatched box, the mouse IgG2a Fc domain is shown orange, and the glycine–serine (G 4 S) 3 linker is colored green. (B) Western blot of secreted recombinant gp350 constructs under reducing (+β-ME) and non-reducing conditions (−β-ME). gp350-ECD 123 -6His was detected by an anti-His tag antibody, and proteins fused with Fc domain were detected with an anti-mouse Fc antibody. For each fusion protein, the same volume (15 µl) of supernatants from identical passage 1 baculovirus stocks was loaded onto the gels under reducing and non-reducing conditions. Samples were resolved on 10% SDS-PAGE. (C) Detection of the indicated recombinant gp350 forms by conformation-dependent anti-gp350 mAb72A1 antibody in the supernatant of bacmid-transfected Sf9 cells using dot blot. Cell culture supernatants from mock-transfected cells were included as negative controls. (D) SDS-PAGE analysis of SEC-purified forms of recombinant gp350. Proteins from the peak fractions were resolved on 10% gel under reducing condition and stained with Coomassie brilliant blue.

Article Snippet: Then, the Ig isotypes were detected using HRP-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, and IgA (1/10,000, Promega).

Techniques: Recombinant, Western Blot, Construct, SDS Page, Transfection, Dot Blot, Cell Culture, Purification, Staining

Journal: Frontiers in Immunology

Article Title: Immunization With Fc-Based Recombinant Epstein–Barr Virus gp350 Elicits Potent Neutralizing Humoral Immune Response in a BALB/c Mice Model

doi: 10.3389/fimmu.2018.00932

Figure Lengend Snippet: Specific titers of anti-gp350 IgG isotypes and IgA in sera are markedly elevated post-immunization with Fc-based gp350 proteins. (A–D) Titers of gp350-specific IgG subtypes were determined in sera (5 weeks post-immunization) of mice immunized i.p. or i.n. with 20 µg of the indicated antigen. (E) Anti-gp350 IgA serum titers from mice immunized i.n. with 20 µg of the indicated antigens. Each symbol represents an individual serum. Horizontal bars represent mean values. Significance (* p ≤ 0.05, ** p < 0.01, and *** p < 0.001) between Fc fusion proteins versus monomer gp350 proteins is shown.

Article Snippet: Then, the Ig isotypes were detected using HRP-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, and IgA (1/10,000, Promega).

Techniques: