Journal: Vaccines

Article Title: Adjuvantation of an Influenza Hemagglutinin Antigen with TLR4 and NOD2 Agonists Encapsulated in Poly(D,L-Lactide-Co-Glycolide) Nanoparticles Enhances Immunogenicity and Protection against Lethal Influenza Virus Infection in Mice

doi: 10.3390/vaccines8030519

Figure Lengend Snippet: PLGA NPs containing both MPLA and MDP induce stronger antigen-specific CD4+ T-cell response in mice than NPs containing individual PRR agonists. C57BL/6 mice ( n = 5/group) were i.m. immunized three times with PLGA NPs: PLGA-HA, PLGA-HA-MPLA, PLGA-HA-MDP, and PLGA-HA-MPLA + MDP with a two-week interval. Non-immunized control mice received PBS. Splenocytes were harvested from mice 14 days after the last immunization. Splenocytes were stained with CFSE, treated with whole hemagglutinin (1 μg/mL). 72 h after cell were harvested for cellular proliferation analysis, cell culture was collected for IFN-γ detection. ( A ) Gating strategy for assessment of proliferating T cells in culture. Single lymphocytes were gated using the forward and side scatter characteristics. Live (DAPI negative) CD3 + T cells were then obtained from the single cells gate. CFSE dim cells were gated in each CD3 + CD4+ as well as CD3 + CD8+ populations. ( B ) % of proliferating CD4 + T-cells (referred to fraction diluted) in response to hemagglutinin restimulation. ( C ) IFN-γ levels in supernatants from HA-stimulated spleen cells, determined by a bio-plex assay 72 h after restimulation. Boxes show interquartile range, whiskers show range, and horizontal lines represent median values. Dots show individual data points. Significant differences ( p

Article Snippet: PLGA NPs were diluted to 0.1 μg/mL concentration in particle-free 0.02 μm filtered PBS (Whatman, Maidstone, UK).

Techniques: Mouse Assay, Staining, Cell Culture, Plex Assay

Journal: Vaccines

Article Title: Adjuvantation of an Influenza Hemagglutinin Antigen with TLR4 and NOD2 Agonists Encapsulated in Poly(D,L-Lactide-Co-Glycolide) Nanoparticles Enhances Immunogenicity and Protection against Lethal Influenza Virus Infection in Mice

doi: 10.3390/vaccines8030519

Figure Lengend Snippet: PLGA NPs containing both MPLA and MDP are more effectively phagocytosed by bone marrow-derived dendritic cells (BMDCs) compared to NPs containing individual PRR agonists. ( A ) A time course of phagocytosis quantification by BMDCs of PLGA NPs containing pHRodo-stained hemagglutinin (HA). Immature BMDCs isolated from naïve C57BL/6 mice were seeded in a 96-well plate at 2 × 10 5 cells per well in complete RPMI medium. The next day, PLGA-HA, PLGA-HA-MPLA, PLGA-HA-MDP, and PLGA-HA-MDP-MPLA formulations produced with pHRodo-labeled hemagglutinin were added in a 100 μg/mL final concentration. The medium was removed at the indicated time points, BMDCs were washed with PBS, and the integral fluorescence of each well was determined using a Biotek microplate reader. Each bar represents the mean fold increase ± SD (whiskers) in relative fluorescence units (RFU) over intact (untreated) cells. The mean value of untreated cells (= 1088 ± 10 RFU) was taken equal to 1 (100%). Experiment was performed in triplicate and repeated twice. ( B ) Representative fluorescence microscopy images of BMDCs treated with pHRodo-stained PLGA NPs for 120 min. BMD Cs were stained with anti-CD11c antibodies (green). The cell nucleus was visualized with DAPI (blue). Phagocytosed PLGA NPs were specifically visualized by pHRodo (red) fluorescence. Scale bar represents 20 μm. ( C ) Representative gating strategy used to assess the phagocytic activity of BMDCs. BMDCs were initially gated in an forward versus side scatter (FSC vs. SSC) dot plot and then identified as double positive (MHCII+ CD11c+) population. Phagocytic activity of BMDCs was determined by ( D ) % of pHRodo-positive BMDCs as well as ( E ) fold increase in mean fluorescence intensity (MFI) of total MHCII+ CD11c+ cells relative to untreated cells 120 min after the addition of PLGA NPs. Mean value of untreated cells (= 749.5 ± 9.2 RFU) was taken equal to 1 (100%). Each bar represents the mean ± SD from three independent experiments, each performed in triplicate. Significant differences between PLGA NPs and intact cells are indicated by hashes: ## for p

Article Snippet: PLGA NPs were diluted to 0.1 μg/mL concentration in particle-free 0.02 μm filtered PBS (Whatman, Maidstone, UK).

Techniques: Derivative Assay, Staining, Isolation, Mouse Assay, Produced, Labeling, Concentration Assay, Fluorescence, Microscopy, Activity Assay

Journal: Vaccines

Article Title: Adjuvantation of an Influenza Hemagglutinin Antigen with TLR4 and NOD2 Agonists Encapsulated in Poly(D,L-Lactide-Co-Glycolide) Nanoparticles Enhances Immunogenicity and Protection against Lethal Influenza Virus Infection in Mice

doi: 10.3390/vaccines8030519

Figure Lengend Snippet: PLGA NPs containing both MPLA and MDP led to a stronger protection in mice than NPs containing individual PRR agonists. Kaplan–Meier survival curves of mice i.m. immunized three times with ( A ) 1 dose (1 mg/mouse, n = 8 per group) or ( B ) 2 doses (2 mg/mouse, n = 10 per group) of PLGA NPs: PLGA-HA, PLGA-HA-MPLA, PLGA-HA-MDP, and PLGA-HA-MDP-MPLA. Non-immunized control mice received PBS. Two weeks after last immunization, mice were infected using a lethal dose (50LD50) of the A/California/07/2009 (H1N1) strain virus. Mortality was monitored daily for 14 days post-challenge. # indicates a significant difference ( p

Article Snippet: PLGA NPs were diluted to 0.1 μg/mL concentration in particle-free 0.02 μm filtered PBS (Whatman, Maidstone, UK).

Techniques: Mouse Assay, Infection

Journal: Vaccines

Article Title: Adjuvantation of an Influenza Hemagglutinin Antigen with TLR4 and NOD2 Agonists Encapsulated in Poly(D,L-Lactide-Co-Glycolide) Nanoparticles Enhances Immunogenicity and Protection against Lethal Influenza Virus Infection in Mice

doi: 10.3390/vaccines8030519

Figure Lengend Snippet: PLGA NPs containing both MPLA and MDP lead to a stronger humoral adaptive response in mice than NPs containing individual PRR agonists. BALB/c mice ( n = 5/group) were i.m. immunized three times with PLGA NPs: PLGA-HA, PLGA-HA-MPLA, PLGA-HA-MDP, and PLGA-HA-MDP-MPLA. Non-immunized control mice received PBS. Blood was collected from mice 14 days after the last immunization for serum preparation. The geometric mean for 5 mice/group with 95%CI is shown. ( A ) The reciprocal titers of hemagglutinin-specific total IgG antibodies and IgG1, IgG2a, IgG2b, and IgG2c subtypes were detected in serum by ELISA. The geometric mean for 5 mice/group with 95% CI is shown. ( B ) Ratios of IgG2a/IgG1 isotype HA-specific antibodies. ( C ) The reciprocal titers of hemagglutination inhibition (HI) antibodies against the H1N1 virus in the sera of mice immunized with PLGA-HA, PLGA-HA-MPLA, PLGA-HA-MDP, and PLGA-HA-MDP-MPLA. Significant differences between PLGA NPs and intact cells are indicated by hashes: # for p

Article Snippet: PLGA NPs were diluted to 0.1 μg/mL concentration in particle-free 0.02 μm filtered PBS (Whatman, Maidstone, UK).

Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, HI Assay

Journal: Vaccines

Article Title: Adjuvantation of an Influenza Hemagglutinin Antigen with TLR4 and NOD2 Agonists Encapsulated in Poly(D,L-Lactide-Co-Glycolide) Nanoparticles Enhances Immunogenicity and Protection against Lethal Influenza Virus Infection in Mice

doi: 10.3390/vaccines8030519

Figure Lengend Snippet: Phagocytosis rate and cell response after the addition of equal amounts of PLGA NPs and H1N1 influenza viral particles to RAW-Blue cells. ( A ) Phagocytosis quantification of PLGA NPs and H1N1 influenza viral particles. RAW-Blue cells were seeded in 96-well plate at 2 × 10 5 cells per well in complete RPMI medium. The next day, normalized to 10 10 particles/well PLGA (roughly equal to 100µg/ml) and virus particles were added to the cells. The medium was removed at the indicated time points, the cells were washed with PBS, and the integral fluorescence (RFU) of each well was determined using a Biotek reader. Mean values (indicated above the bars) of intact cells were taken equal to 1 (100%). Data represent the mean ( n = 3) fold increase over intact cells ± SD. ( B ) NF-κB/AP-1-dependent SEAP reporter gene expression in cell-free culture supernatants from RAW-Blue cells treated for 18 h with PLGA and with virus particles normalized by quantity (10 10 particles/well). Mean values (indicated above the bars) of intact cells were taken equal to 1 (100%). Data represent the mean ( n = 3) fold increase over intact cells ± SD. ( C ) Cytokine levels were measured in cell-free culture supernatants from the same wells using bead-based immunoassay. Bars represent mean fold increase ± SD (whiskers) over intact group. Values (mean concentrations ± SD are indicated above the bars) of intact groups were taken equal to 1 (100%). Significant differences between treated and intact cells are indicated by hashes: # for p

Article Snippet: PLGA NPs were diluted to 0.1 μg/mL concentration in particle-free 0.02 μm filtered PBS (Whatman, Maidstone, UK).

Techniques: Fluorescence, Expressing, Bead-based Assay

Journal: Vaccines

Article Title: Adjuvantation of an Influenza Hemagglutinin Antigen with TLR4 and NOD2 Agonists Encapsulated in Poly(D,L-Lactide-Co-Glycolide) Nanoparticles Enhances Immunogenicity and Protection against Lethal Influenza Virus Infection in Mice

doi: 10.3390/vaccines8030519

Figure Lengend Snippet: PLGA NPs containing both MPLA and MDP induce stronger NF-κB transcriptional response in BALB/c-Tg (Rela-luc) 31Xen transgenic mice than NPs with individual PRR agonists. ( A ) Representative bioluminescent pseudocolored images of NF-κB-luciferase activity in live NF-κB-Luc transgenic mice 3 h after i.m. injection of PBS, PLGA-HA, PLGA-HA-MPLA, PLGA-HA-MDP, or PLGA-HA-MPLA + MDP (all in 1 mg/mouse in 100 μL). Prior to imaging, mice received an i.p. injection of luciferin followed by anesthesia. Non-immunized control mice received PBS. The intensity of bioluminescence (i.e., NF- κB activity) is shown in color according to the scale on the right. Two additional mice in each group gave similar results. ( B ) NF-κB-dependent bioluminescence in tissue homogenates prepared from NF-κB-Luc transgenic mice at the same time point after the injection of PLGA NPs. Control mice were injected with PBS. Values of control group (mean fluorescence intensities ± SD are indicated above the bars) were taken equal to 1 (100%). Results are expressed as the fold increase in relative luminescence units over PBS-treated control animals. Each bar represents the mean of three mice per group ± SD (error bars). Significant differences between PLGA NPs and PBS treated mice are indicated by hashes: ## for p

Article Snippet: PLGA NPs were diluted to 0.1 μg/mL concentration in particle-free 0.02 μm filtered PBS (Whatman, Maidstone, UK).

Techniques: Transgenic Assay, Mouse Assay, Luciferase, Activity Assay, Injection, Imaging, Fluorescence

Journal: Vaccines

Article Title: Adjuvantation of an Influenza Hemagglutinin Antigen with TLR4 and NOD2 Agonists Encapsulated in Poly(D,L-Lactide-Co-Glycolide) Nanoparticles Enhances Immunogenicity and Protection against Lethal Influenza Virus Infection in Mice

doi: 10.3390/vaccines8030519

Figure Lengend Snippet: PLGA NPs containing both MPLA and MDP induce a stronger local cytokine response in mice than NPs containing individual PRR agonists. BALB/c mice were intramuscularly injected with 1 mg/dose of PLGA-HA, PLGA-HA-MPLA, PLGA-HA-MDP, or PLGA-HA-MPLA + MDP. Non-immunized control mice received PBS. Three hours later, inguinal lymph nodes, the liver, the spleen, and the thigh muscle (corresponding to the injection site) were isolated. Cytokine levels were measured in tissue homogenates using bead-based immunoassay. Control mice were injected with PBS. Results are expressed as the mean fold change in cytokine concentration relative to the mean concentration in PBS-treated control animals. Values of control group (mean concentrations ± SD are indicated above the bars) were taken equal to 1 (100%). Each point represents the mean of three mice per group ± SD (error bars). Significant differences between PLGA NPs and intact cells are indicated by hashes: # for p

Article Snippet: PLGA NPs were diluted to 0.1 μg/mL concentration in particle-free 0.02 μm filtered PBS (Whatman, Maidstone, UK).

Techniques: Mouse Assay, Injection, Isolation, Bead-based Assay, Concentration Assay

Journal: bioRxiv

Article Title: A replication-competent vesicular stomatitis virus for studies of SARS-CoV-2 spike-mediated cell entry and its inhibition

doi: 10.1101/2020.05.20.105247

Figure Lengend Snippet: Inhibition of syncytium formation by sera from convalescent donors. Vero cells were infected with pre-titrated amounts of rVSV-SARS-CoV-2. At 2 h post-infection, cells were washed with PBS to remove residual virus and then exposed to the indicated dilutions of each convalescent serum. Cells were fixed, their nuclei counterstained, and they were imaged for syncytium formation by eGFP expression at 16 h post-infection. Representative images from one of two independent experiments are shown.

Article Snippet: S-mediated antibody depletion assayHigh-protein binding 96-well ELISA plates (Corning) coated with PBS alone or with 2 µg/ml of SARS-CoV-2 S protein in PBS overnight at 4°C were blocked for 1 hour with 3% nonfat dry milk (Biorad) in PBS.

Techniques: Inhibition, Infection, Expressing

Journal: BMC Infectious Diseases

Article Title: SPS-neutralization in tissue samples for efficacy testing of antimicrobial peptides

doi: 10.1186/s12879-019-4700-1

Figure Lengend Snippet: SPS-neutralization in efficacy tests of SAAP-148 against gram-positive and gram-negative bacteria. Ninety μL of 10 7 CFU/mL MRSA (LUH14616 and Mu50), E. faecalis (ATCC 29212) , P. aeruginosa (PAO1; ATCC BAA47), E. coli (ATCC 35218) and A. baumannii were exposed to 10 μL of 1% (wt/v) SAAP-148 in PBS or PBS for 30 min. Subsequently, samples were homogenized in 500 μL of PBS with or without 0.05% (wt/v) SPS. The means and SD of six independent experiments performed in duplicate are shown. Results are expressed as the number of surviving bacteria in log10 CFU/mL. * indicates significant difference as compared to the samples without SPS (* p

Article Snippet: SPS-neutralization of antimicrobial activity Ten μL of PBS or one of the antimicrobial agents: 1% (wt/v) SAAP-148 in PBS, 1% (wt/v) pexiganan in PBS, 1% (wt/wt) SSD, 0.5% (v/v) chlorhexidine in 70% alcohol, 2% (wt/wt) Bactroban or 2% (wt/wt) Fucidin were added to polypropylene vials containing 400 μL of PBS, 0.05, 0.1, 0.5% or 1% (wt/v; final concentrations) SPS (Fig. ) in PBS (Merck, KGaA, Darmstadt, Germany).

Techniques: Neutralization

Journal: BMC Infectious Diseases

Article Title: SPS-neutralization in tissue samples for efficacy testing of antimicrobial peptides

doi: 10.1186/s12879-019-4700-1

Figure Lengend Snippet: Effect of SPS on the antimicrobial activity of various antimicrobial agents. Mixtures of 10 μL of SAAP-148 (1% wt/v), pexiganan (1% wt/v), chlorhexidine (0.5% v/v in 70% alcohol) or PBS and 400 μL of PBS, 0.05, 0.1, 0.5% or 1% (wt/v; final concentrations) SPS in PBS were prepared. Ninety μL of LUH14616 with a final concentration of 10 5 CFU/mL were added to these mixtures to determine the antimicrobial activity after 30 min incubation at 37 °C and 5% CO 2 . The means and standard deviations (SD) of three independent experiments performed in duplicate are shown. Results are expressed as the number of surviving bacteria in log10 CFU/mL. * indicates significant difference as compared to the samples without SPS (* p

Article Snippet: SPS-neutralization of antimicrobial activity Ten μL of PBS or one of the antimicrobial agents: 1% (wt/v) SAAP-148 in PBS, 1% (wt/v) pexiganan in PBS, 1% (wt/wt) SSD, 0.5% (v/v) chlorhexidine in 70% alcohol, 2% (wt/wt) Bactroban or 2% (wt/wt) Fucidin were added to polypropylene vials containing 400 μL of PBS, 0.05, 0.1, 0.5% or 1% (wt/v; final concentrations) SPS (Fig. ) in PBS (Merck, KGaA, Darmstadt, Germany).

Techniques: Activity Assay, Concentration Assay, Incubation

Journal: BMC Infectious Diseases

Article Title: SPS-neutralization in tissue samples for efficacy testing of antimicrobial peptides

doi: 10.1186/s12879-019-4700-1

Figure Lengend Snippet: SPS-neutralization of residual activity of various antimicrobial agents. Excision wound models were inoculated with 10 5 CFU/mL LUH14616 for 1 h and exposed to 20 μL of SAAP-148 (1% wt/v), pexiganan (1% wt/v), chlorhexidine (0.5% v/v in 70% alcohol) or PBS for 1 h. Subsequently, the models were homogenized in 1 mL of PBS with or without 0.05% (wt/v) SPS. The means and SD of at least eight independent experiments performed in triplicate are shown. Results are expressed as the number of surviving bacteria in log10 CFU/mL. * indicates significant difference as compared to the samples without SPS (* p

Article Snippet: SPS-neutralization of antimicrobial activity Ten μL of PBS or one of the antimicrobial agents: 1% (wt/v) SAAP-148 in PBS, 1% (wt/v) pexiganan in PBS, 1% (wt/wt) SSD, 0.5% (v/v) chlorhexidine in 70% alcohol, 2% (wt/wt) Bactroban or 2% (wt/wt) Fucidin were added to polypropylene vials containing 400 μL of PBS, 0.05, 0.1, 0.5% or 1% (wt/v; final concentrations) SPS (Fig. ) in PBS (Merck, KGaA, Darmstadt, Germany).

Techniques: Neutralization, Activity Assay