Journal: Biomaterials science

Article Title: Nanoparticle vaccines can be designed to induce pDC support of mDCs for increased antigen display

doi: 10.1039/d2bm01132h

Figure Lengend Snippet: A) Reaction scheme for the conjugation of CpG1826, CpG1018, or ssRNA adjuvants to the interior cysteines of E2 (D381C) nanoparticles. B) SDS-PAGE of nanoparticles encapsulating TLR agonist. Lanes: (1,6) molecular weight ladder; (2) E2; (3) CpG1826-E2; (4) CpG1018-E2; (5) ssRNA-E2. Construct bands at ~35 kDa confirm conjugation. C) Representative hydrodynamic diameter measurements of E2 encapsulating TLR agonists and E2 control. D) Representative TEM images of CpG1826-E2, CpG1018-E2, and ssRNA-E2. Scale bars = 50 nm. E) Reaction scheme for the conjugation of SIINFEKL to the exterior lysines of E2 nanoparticles. F) SDS-PAGE of complete E2 vaccine formulation or reaction intermediates. Lanes: (1,5) molecular weight ladder; (2) E2; (3) CpG-E2, (4) CpG-S-E2. G) Representative hydrodynamic diameter measurements of E2, CpG-E2, or CpG-S-E2. CpG: CpG1826. S: SIINFEKL.

Article Snippet: Adjuvants CpG1826 (5’-tccatgacgttcctgacgtt-3’), CpG1018 (5’-tgactgtgaacgttcgagatga-3’), and ssRNA (5’-gcccgucuguugugugacuc-3’) were custom-ordered from IDT or TriLink BioTechnologies, with or without 5’ benzaldehyde modification (for nanoparticle conjugation and controls, respectively).

Techniques: Conjugation Assay, SDS Page, Molecular Weight, Construct, Control, Formulation

Journal: Biomaterials science

Article Title: Nanoparticle vaccines can be designed to induce pDC support of mDCs for increased antigen display

doi: 10.1039/d2bm01132h

Figure Lengend Snippet: Myeloid DC (A-C) or plasmacytoid DC (D-F) MHC II+ MFI fold increase versus PBS control. For each condition or formulation, corresponding concentrations of E2 and adjuvant are displayed. White bars are PBS- and E2-only controls. Red bars indicate 100 ng/mL LPS (mDC positive control) or 200 ng/mL LPS (pDC positive control). Green, blue, and orange bars correspond to free (light bars) or encapsulated (dark bars) CpG1826, CpG1018, or ssRNA, respectively. A) MHC II expression of mDCs incubated with PBS, LPS, E2, and CpG1826 controls or CpG1826-E2. B) MHC II expression of mDCs incubated with CpG1018-E2 or control. C) MHC II expression of mDCs incubated with ssRNA-E2 or control. D) MHC II expression of pDCs incubated with CpG1826-E2 or control. E) MHC II expression of pDCs incubated with CpG1018-E2 or control. F) MHC II expression of pDCs incubated with ssRNA-E2 or control. Mean ± SEM. Statistics: 1-way ANOVA, Bonferroni’s test. ≥3 independent biological replicates, 2-3 technical replicates per independent biological replicate. Adj: adjuvant. *p ≤ 0.05, **p ≤ 0.01. Experimental procedure is outlined in Figure SI-1.

Article Snippet: Adjuvants CpG1826 (5’-tccatgacgttcctgacgtt-3’), CpG1018 (5’-tgactgtgaacgttcgagatga-3’), and ssRNA (5’-gcccgucuguugugugacuc-3’) were custom-ordered from IDT or TriLink BioTechnologies, with or without 5’ benzaldehyde modification (for nanoparticle conjugation and controls, respectively).

Techniques: Control, Formulation, Adjuvant, Positive Control, Expressing, Incubation

Journal: Biomaterials science

Article Title: Nanoparticle vaccines can be designed to induce pDC support of mDCs for increased antigen display

doi: 10.1039/d2bm01132h

Figure Lengend Snippet: A) Schematic for pDC supernatant transfer studies. For incubation steps, mDCs or pDCs were given 1 μg/mL CpG-S-E2 vaccine or controls (PBS negative control, 2 μg/mL SIINFEKL positive control, or 0.1 μg/mL CpG1826 with 0.1 μg/mL SIINFEKL). B) Myeloid DC SIINFEKL display with or without media transfer from pDCs that were incubated with PBS for 24 hr starting on Day 8. (C-E) Myeloid DC SIINFEKL display following supernatant transfer protocol for mDCs or pDCs incubated with C) PBS, D) 0.1 μg/mL CpG1826 with 0.1 μg/mL SIINFEKL, and E) 1 μg/mL CpG-S-E2 (containing 0.1 μg/mL CpG1826 with 0.1 μg/mL SIINFEKL) for 24 hr starting on Day 9 (mDCs) or Day 8 (pDCs). Shown is display fold increase in antigen display versus that of mDCs incubated with PBS. Mean ± SEM. Statistics: 1-way ANOVA, Bonferroni’s test. 3 independent biological replicates, 2-3 technical replicates per biological replicate. CpG: CpG1826. S: SIINFEKL. *p ≤ 0.05.

Article Snippet: Adjuvants CpG1826 (5’-tccatgacgttcctgacgtt-3’), CpG1018 (5’-tgactgtgaacgttcgagatga-3’), and ssRNA (5’-gcccgucuguugugugacuc-3’) were custom-ordered from IDT or TriLink BioTechnologies, with or without 5’ benzaldehyde modification (for nanoparticle conjugation and controls, respectively).

Techniques: Incubation, Negative Control, Positive Control

Journal: Biomaterials science

Article Title: Nanoparticle vaccines can be designed to induce pDC support of mDCs for increased antigen display

doi: 10.1039/d2bm01132h

Figure Lengend Snippet: Displayed are concentrations of cytokines (top graph of each set) and fold increase in cytokine concentration relative to PBS negative control (bottom graph of each set), secreted from pDCs after incubation with vaccine or control. For 24 hr or 48 hr incubation periods, pDC secretions of A) IFN-β, B) TNF-α, C) IL-6, D) IL-10, and E) IL-12p70 were determined by LegendPlex™ assay. Incubation conditions included PBS, 0.1 μg/mL CpG1826 with 0.1 μg/mL SIINFEKL (CpG + S), or CpG-S-E2 containing 0.1 μg/mL CpG1826 with 0.1 μg/mL SIINFEKL. Mean ± SEM. Statistics: 1-way ANOVA, Bonferroni’s test. 3 independent biological replicates, 2 technical replicates per biological replicate. CpG: CpG1826. S: SIINFEKL. *p ≤ 0.05.

Article Snippet: Adjuvants CpG1826 (5’-tccatgacgttcctgacgtt-3’), CpG1018 (5’-tgactgtgaacgttcgagatga-3’), and ssRNA (5’-gcccgucuguugugugacuc-3’) were custom-ordered from IDT or TriLink BioTechnologies, with or without 5’ benzaldehyde modification (for nanoparticle conjugation and controls, respectively).

Techniques: Concentration Assay, Negative Control, Incubation, Control