Journal: Biomaterials

Article Title: Role of sustained antigen release from nanoparticle vaccines in shaping the T cell memory phenotype

doi: 10.1016/j.biomaterials.2012.03.041

Figure Lengend Snippet: Humoral and cellular responses after vaccination with liposomes and PLGA nanoparticles. Mice (N = 4) were subcutaneously administered a single dose of 100 μg of OVA encapsulated in liposomes or PLGA nanoparticles. Mice were bled approximately every 2 weeks and analyzed for antigen-specific (A) total IgG, (B) IgG1, and (C) IgG2b titers by ELISA. At 11 weeks, splenocytes were pulsed ex vivo with OVA at 25 and 50 μg/mL and analyzed for (D) IFN-γ in the supernatant after 72 h by ELISA. Data shown are from a single experiment that was performed twice with the same results. * Indicate P value less than 0.05.

Article Snippet: Supernatant was collected and analyzed for IFN-γ secretion by ELISA (BD Pharmingen).

Techniques: Enzyme-linked Immunosorbent Assay, Ex Vivo

Journal: Biomaterials

Article Title: Role of sustained antigen release from nanoparticle vaccines in shaping the T cell memory phenotype

doi: 10.1016/j.biomaterials.2012.03.041

Figure Lengend Snippet: Post-vaccination recall of Listeria monocytogenes (LM-OVA). (A) Study timeline. Mice received a single subcutaneous administration of OVA adsorbed to aluminum hydroxide or encapsulated in liposomes or PLGA nanoparticles. At 13 weeks, mice were divided into two groups(N = 3) and received intravenous (I.V) tail vein injections with either 1 × 105 (group I) or 5 × 104 (group II) CFU of LM-OVA. At 3 days post-challenge, splenocytes from Group I mice were assayed for bacterial titers. At 7 days post-challenge, splenocytes from group II mice were stained for effector T cell surface markers and intracellular IFN-γ and analyzed by flow cytometry. (B) Humoral response at time of challenge. Serum anti-OVA IgG was measured by ELISA. * Indicate a P value less than 0.001. C) LM-OVA titers in the spleen. Three days post-LM-OVA challenge, splenocytes from Group I mice were permeabilized and plated on agar overnight. Colony forming units (CFU) were calculated and normalized by mass of the organ. Study was repeated twice with same results. *Indicates a P value less than 0.05.

Article Snippet: Supernatant was collected and analyzed for IFN-γ secretion by ELISA (BD Pharmingen).

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Biomaterials

Article Title: Role of sustained antigen release from nanoparticle vaccines in shaping the T cell memory phenotype

doi: 10.1016/j.biomaterials.2012.03.041

Figure Lengend Snippet: Effector-like T cell analysis. Splenocytes from group II mice (N = 3) were pulsed with SIINFEKL and (A) IFN-γ expressing CD8+ T cell population was enumerated. (B) Activated antigen-specific CD8+ CD44 + T cells were also determined using a SIINFEKL tetramer. This population in C) was analyzed for surface markers indicative of an effector-like phenotype (KLRG1hi, CD127lo, CD27lo, CD62Llo). Shaded = PBS, Black = PLGA, Red = Liposome, Blue = Alum. Study was repeated twice with same results. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Supernatant was collected and analyzed for IFN-γ secretion by ELISA (BD Pharmingen).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Development of a TLR7/8 agonist adjuvant formulation to overcome early life hyporesponsiveness to DTaP vaccination

doi: 10.1038/s41598-022-20346-w

Figure Lengend Snippet: Imidazoquinoline UM-3001 demonstrated age-specific potency, effectiveness and IFNγ production in human newborn cord blood. ( A – B ) Human neonatal cord blood cultured in vitro for 6 h with buffer control (RPMI) or with increasing concentrations of various UM adjuvants. ( C – D ) Newborn versus adult blood cultured in vitro for 6 h with UM-3001. Supernatants were collected for ELISA. Results represent mean ± SEM, A-B; n = 7, C-D; n = 7. Cell supernatants were analyzed for cytokine expression by multiplex assay. Data are shown as fold change for newborn cord (blue line) over adult stimulated whole blood (black line) for 1 μM ( E ) and 10 μM ( F ) of UM-3001. (n = 6 adults, n = 8 newborns). For comparisons between overall groups (e.g., newborn vs. adult), two-way ANOVA followed by Tukey’s test for multiple comparisons was applied, and statistical significance denoted as * p < 0.033.

Article Snippet: Supernatants derived from human leukocyte stimulations were assayed by ELISA for TNF and IFNγ (BD Biosciences; San Jose, CA, USA).

Techniques: Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Multiplex Assay

Journal: Scientific Reports

Article Title: Development of a TLR7/8 agonist adjuvant formulation to overcome early life hyporesponsiveness to DTaP vaccination

doi: 10.1038/s41598-022-20346-w

Figure Lengend Snippet: UM-3003 enhances DTaP specific IgG2c production and Th1 polarization in adult mice. Adult C57BL/6 mice were immunized twice, 14 days apart ( A ) with DTaP (1/100th of the human dose) ± UM-3003 or alum at 10 µg per mouse in different formulations (aqueous or pre-adsorbed to alum). Serum was harvested 14 days following boost ( B – I ). Anti-FHA ( B - E ) and anti-PRN ( F – I ) serum Ab IgG ( B , F ), IgG1 ( C , G ) and IgG2c ( D , H ) titers were measured by ELISA. PBS control groups were represented as dotted line. ( E , I ) IgG2c/IgG1 ratios for FHA and PRN respectively. ( J , K ) Murine CD4 + T cell responses after FHA and PRN stimulation. Four weeks following booster, splenocytes from DTaP and adjuvanted vaccinates were isolated, stimulated with 2 μg/ml of either FHA or PRN along with CD28 (1 μg/ml) and CD49d (1 μg/ml) for 12 h followed by 6 h of BFA stimulation to block the extracellular cytokine secretion. After stimulation, cells were harvested, stained (intracellular cytokine staining) and analyzed by flow cytometry. Plots were gated on CD44 + CD4 + lymphocytes and analyzed for all combinations of simultaneous IFNγ, IL-4/5 and IL-17A productivity. Statistical analysis was performed by nonparametric Kruskal–Wallis test corrected for multiple comparisons; * p < 0.033, ** p < 0.002, *** p < 0.001 (n = 8–15 per group). Study is inclusive of three independent repeats.

Article Snippet: Supernatants derived from human leukocyte stimulations were assayed by ELISA for TNF and IFNγ (BD Biosciences; San Jose, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Blocking Assay, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: Development of a TLR7/8 agonist adjuvant formulation to overcome early life hyporesponsiveness to DTaP vaccination

doi: 10.1038/s41598-022-20346-w

Figure Lengend Snippet: UM-3003 enhances DTaP specific IgG2c production and Th1/Th17 polarization in neonatal mice. ( A ) 7 days old C57BL/6 were vaccinated (prime/ boost) with DTaP (1/100th of the human dose) ± UM-3003 or UM-3003 at 10 µg per mouse in alum absorbed formulations. Serum was harvested 14 days following boost (DOL 28). Anti-FHA ( B – E ) and anti-PRN ( F – I ) serum total IgG ( B , F ) titers, IgG1 ( C , G ) and IgG2c ( D , H ) were measured by ELISA. PBS control groups were represented as dotted line. ( E , I ) IgG2c/IgG1 ratios for FHA and PRN respectively. ( J , K ) Splenic CD4 + T cell responses after FHA and PRN stimulation. Four weeks following booster, splenocytes from vaccinates were isolated and stimulated with 2 μg/ml of either FHA or PRN along with CD28 (1 μg/ml) and CD49d (1 μg/ml) for 12 h followed by 6 h of BFA stimulation. After stimulation, cells were harvested and stained (intracellular cytokine staining) and analyzed by flow cytometry. Plots were gated on CD44 + CD4 + lymphocytes and analyzed for all combinations of simultaneous IFNγ, IL-4/5 and IL-17A productivity. ( L ) Fold change of FHA specific IFNγ production in splenic CD4 + T cell relative to DTaP are shown. Statistical comparison was performed either using one-way ANOVA (C, E) or nonparametric Kruskal–Wallis test corrected for multiple comparisons; * p < 0.033, ** p < 0.002, *** p < 0.001 (n = 9–13 per group), with comparison to DTaP alone. Study is inclusive of two independent repeats.

Article Snippet: Supernatants derived from human leukocyte stimulations were assayed by ELISA for TNF and IFNγ (BD Biosciences; San Jose, CA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Staining, Flow Cytometry