Journal:

Article Title: Gram-Positive Bacteria Are Potent Inducers of Monocytic Interleukin-12 (IL-12) while Gram-Negative Bacteria Preferentially Stimulate IL-10 Production

doi:

Figure Lengend Snippet: IL-12 p70 and IL-10 responses by monocytes from nine blood donors stimulated with each of seven gram-positive and seven gram-negative species separately. Concentrations ranged from 5 × 105 to 5 × 107 bacteria/ml. Cytokines were measured in 24-h culture supernatants by ELISA. Each bar represents the mean response of all donors to all gram-positive or gram-negative bacterial strains at a particular concentration. The error bars represent standard errors for the variability between the bacterial species.

Article Snippet: Microtiter plates (Maxisorp; Nunc) were coated overnight at 4°C with monoclonal mouse immunoglobulin G1 (IgG1) antihuman IL-12 antibody (clone B-P24; Diaclone, Besançon, France [unknown concentration]) diluted 1/200 in PBS.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal:

Article Title: Gram-Positive Bacteria Are Potent Inducers of Monocytic Interleukin-12 (IL-12) while Gram-Negative Bacteria Preferentially Stimulate IL-10 Production

doi:

Figure Lengend Snippet: IL-12 p70 responses in supernatants after addition of anti-IL-10 antibodies or control IgG1 antibodies to the monocyte cultures before stimulation with 5 × 106 bacteria/ml. IL-12 was measured after 24 h. Bars represent the mean responses of three donors, and the error bars represent standard errors of the means.

Article Snippet: Microtiter plates (Maxisorp; Nunc) were coated overnight at 4°C with monoclonal mouse immunoglobulin G1 (IgG1) antihuman IL-12 antibody (clone B-P24; Diaclone, Besançon, France [unknown concentration]) diluted 1/200 in PBS.

Techniques:

Journal: bioRxiv

Article Title: Influenza virus-like particle-based hybrid vaccine containing RBD induces immunity against influenza and SARS-CoV-2 viruses

doi: 10.1101/2022.02.01.478657

Figure Lengend Snippet: ( A ) Colloidal blue (lane 1), and Western blot (lanes 2-7) of the immunoaffinity column purified fusion protein from CHO-S cells probed with anti-RBD antibody (lanes 3 & 4), or anti-GM-CSF mAb (lanes 6 & 7). Lane 2 is control RBD probed with anti-RBD Ab, and lane 5 is control GM-CSF probed with anti-GM-CSF antibody. ( B ) ELISA for GPI-RBD-GM-CSF binding to antibodies in the human COVID-19 patients’ sera, and ( C) ELISA of purified GPI-RBD-GM-CSF binding to ACE-2. ( D ) FACS analysis of VLPs incorporated with GPI-IL-12 and GPI-RBD-GM-CSF fusion protein by protein transfer, and ( E ) BMDC proliferation by GPI-GM-CSF, GPI-RBD-GM-CSF and hybrid vaccine.

Article Snippet: Purified anti-mouse GM-CSF (clone MP1-22E9) and anti-mouse IL-12 (clone C17.8) were from BioXcell and used for affinity chromatography purification of GPI-RBD-GM-CSF fusion protein and GPI-IL-12, respectively.

Techniques: Western Blot, Purification, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: bioRxiv

Article Title: Influenza virus-like particle-based hybrid vaccine containing RBD induces immunity against influenza and SARS-CoV-2 viruses

doi: 10.1101/2022.02.01.478657

Figure Lengend Snippet: ELISA plates were coated with GPI-RBD-GM-CSF and serum samples from various groups of mice (n=5) immunized with GPI-RBD-GM-CSF, VLP vaccine containing the GPI-RBD-GM-CSF or hybrid vaccine (VLP vaccine containing the GPI-RBD-GM-CSF + GPI-IL-12) were diluted and added to the wells after blocking the plates. (A) Total IgG levels 6 weeks after booster dose, and (B) IgG isotype in the sera 10 weeks after the booster dose. Anti-mouse IgG (A) or isotype specific anti-mouse IgG-HRP conjugate (B) was used to detect the bound antibody.

Article Snippet: Purified anti-mouse GM-CSF (clone MP1-22E9) and anti-mouse IL-12 (clone C17.8) were from BioXcell and used for affinity chromatography purification of GPI-RBD-GM-CSF fusion protein and GPI-IL-12, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal: bioRxiv

Article Title: Influenza virus-like particle-based hybrid vaccine containing RBD induces immunity against influenza and SARS-CoV-2 viruses

doi: 10.1101/2022.02.01.478657

Figure Lengend Snippet: ELISA plates were coated with inactivated influenza A/PR8 H1N1. Serum samples from mice vaccinated with VLP, VLP with GPI-GM-CSF and GPI-IL-12 or hybrid vaccine were serially diluted (5-fold) and added to the wells after blocking the plates. ( A ) total IgG, ( B ) IgG1 or ( C ) IgG2a antibody response. Isotype specific anti-mouse Ig-HRP conjugate was used to detect the isotype of the antibody bound. * p<0.05

Article Snippet: Purified anti-mouse GM-CSF (clone MP1-22E9) and anti-mouse IL-12 (clone C17.8) were from BioXcell and used for affinity chromatography purification of GPI-RBD-GM-CSF fusion protein and GPI-IL-12, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal: bioRxiv

Article Title: Influenza virus-like particle-based hybrid vaccine containing RBD induces immunity against influenza and SARS-CoV-2 viruses

doi: 10.1101/2022.02.01.478657

Figure Lengend Snippet: BALB/c mice (n=10; 2-3 months old) administered with hybrid vaccine, hybrid vaccine without IL-12 (VLP RG) or VLP in 50 ul volume via intramuscular route. Control mice received either PBS or influenza VLP. Booster dose given on d33, and blood was collected 2 weeks later (week 7). Mice were challenged with influenza A/PR8 H1N1 virus (n=5) or mouse adapted SARS-CoV2 (n=5) 16-18 weeks after the first dose. ( A ) Neutralizing antibodies titers in the serum of BALB/c mice (n=5-6 per group). Serum collected from BALB/c mice 2 weeks after booster dose was serially diluted from 1:4 to 1:1024 and PRNT was conducted against SARS-CoV-2 (Wuhan virus). ( B ) BALB/c mice were inoculated intranasally with mouse-adapted SARS-CoV-2 (10 5 plaque-forming units) 3 months after booster dose. Percentage of daily body weight change in the animals. ( C ) The RNA levels of SARS-CoV-2 were determined in the lungs by qRT-PCR (n=4-5 per group). Error bars represent SEM. The data are expressed as genome copies/μg of RNA. Each data point represents an individual mouse. Data are expressed as mean log 10 titer. * p<0.05; ** p <0.01 ***p<0.001. VLP RG: VLP incorporated with GPI-RBD-GM-CSF; hybrid vaccine: VLP incorporated with GPI-RBD-GM-CSF and GPI-IL-12.

Article Snippet: Purified anti-mouse GM-CSF (clone MP1-22E9) and anti-mouse IL-12 (clone C17.8) were from BioXcell and used for affinity chromatography purification of GPI-RBD-GM-CSF fusion protein and GPI-IL-12, respectively.

Techniques: Quantitative RT-PCR

Journal: bioRxiv

Article Title: Influenza virus-like particle-based hybrid vaccine containing RBD induces immunity against influenza and SARS-CoV-2 viruses

doi: 10.1101/2022.02.01.478657

Figure Lengend Snippet: BALB/c mice administered with hybrid vaccine (10 μg/dose) or hybrid vaccine without GPI-IL-12 (VLP RG) as described in . Booster dose given after 33 days of the first dose. ( A ) HAI titer in the blood 2 weeks after the booster dose (day 48). Lung viral titer ( B ) and survival ( C ) of mice challenged with influenza A/PR8 H1N1 virus 3 months after the booster dose. Inoculation dose was 10 times LD50. VLP RG: VLP incorporated with GPI-RBD-GM-CSF; Hybrid vaccine: VLP incorporated with GPI-RBD-GM-CSF and GPI-IL-12. Statistical significance was calculated by one-way ANOVA and Dunnett’s post-multiple comparison tests. Error bars indicate the mean ± standard errors of the mean (SEM). ***; p < 0.001, ns; not significant

Article Snippet: Purified anti-mouse GM-CSF (clone MP1-22E9) and anti-mouse IL-12 (clone C17.8) were from BioXcell and used for affinity chromatography purification of GPI-RBD-GM-CSF fusion protein and GPI-IL-12, respectively.

Techniques:

Journal: bioRxiv

Article Title: Strain-Specific Human Natural Killer Cell Recognition of Influenza A Virus

doi: 10.1101/148528

Figure Lengend Snippet: (A) Cytokine concentrations assessed by Luminex®, values displayed represent the mean fluorescence intensity in pH1N1-infected/H3N2 infected conditions (MOI=3). Cytokines elevated by 2.5-fold over the level in mock-infected monocytes are plotted. (B) Impact of cytokine receptor blocking on the NK cell IFN-γ response evaluated by pre-incubating NK cells for 1 hr with blocking antibodies specific to IFNAR2, IL-12R, IL-15R, IL-18R, and IFNGR1 followed by co-culture with infected monocytes. 24 HPI, intracellular cytokine staining was used to assess NK cell IFN-γ + frequency compared to treatment with an isotype control antibody (H3N2: n =2-6, pH1N1: n =9). (C) NK cells were incubated for 1 hr with antibodies specific to IFNAR2 and CD226 or CD54 followed by co-culture with autologous infected monocytes. At 24 HPI, intracellular cytokine staining was used to assess IFN-γ production compared to treatment with an isotype control antibody ( n =6). * P < 0.05, ** P < 0.005, Wilcoxon signed-rank test. (D) Model of strain-specific NK cell recognition of influenza A infection. pH1N1-infection of monocytes virus does not downregulate CD54 and CD112 to the same extent as H3N2-infected monocytes. CD54 expression is retained on Flu-NP + cells while CD112 expression is preferentially retained on exposed, uninfected monocytes. pH1N1 infection of monocytes elicits enhanced IFN-α secretion and blockade of IFNAR2 dampens NK cell anti-pH1N1 IFN-γ production.

Article Snippet: IL-12R β 2 Ab (R&D Systems AF1959-SP) was used at a final concentration of 83.33 μg/mL.

Techniques: Luminex, Fluorescence, Infection, Blocking Assay, Co-Culture Assay, Staining, Control, Incubation, Virus, Expressing