Journal: Biomaterials

Article Title: Enabling Sublingual Peptide Immunization with Molecular Self-assemblies

doi: 10.1016/j.biomaterials.2020.119903

Figure Lengend Snippet: Q11OVA with N-terminal mPEG blocks of 350, 1000, and 2000 average molecular weight formed nanofibers, as evidenced by negative stained TEM: (a) mPEG350-Q11OVA (b) mPEG1000-Q11OVA (c) mPEG2000Q11OVA. (d) Isothermal titration calorimetry indicated that increasing PEG length diminished interactions with mucin. * p < 0.05, R2 = 0.94 by linear regression of PEG size vs. percent change in binding stoichiometry. (e) Increasing PEG length improved OVA-specific titers raised against OVAQ11-PEG nanofibers. Mice were immunized sublingually with 7 μL of 5 mM peptide with 2 μg of cholera toxin per mouse and boosted with the same dose at weeks 1, 3, and 6. IgG against the pOVA epitope was measured at week 8 by ELISA. A, B, C: Groups in (e) that do not share a letter are statistically different (p < 0.05) by 2-way ANOVA with Tukey’s multiple comparisons test, n=6 (0 PEG) or n=8/group from two independent experiments. Complete titer data is shown in Fig. S11. (f) Proline-Alanine-Serine modification (PASylation) had a similar effect as PEGylation, enabling sublingual immunization against PAS20-Q11OVA nanofibers in a fully peptidic formulation. Mice were immunized with 8 μL of 5 mM peptide and 10 μg CTB at weeks 0, 1, 3, and 5. Data from OVAQ11-PEG + CTB immunizations are re-presented from Figure 4f as a comparison. Arrows indicate timepoints of immunizations. (g) Sublingual immunization was achieved against the ESAT651–70 epitope from M. tuberculosis using cyclic-di-AMP adjuvant. CBA/J mice (n=5) were immunized at weeks 0, 1, 3, 7, and 9 with 8 μL of 5 mM mPEG2000-Q11ESAT651–70 with 10 μg of cyclic-di-AMP adjuvant per mouse; IgG against the ESAT651–70 and pOVA epitopes were measured by ELISA.

Article Snippet: Adjuvanted formulations included 2 μg of cholera toxin (List Biological Laboratories, 101B), 10 μg of CTB (List Biological Laboratories, 103B), or 10 μg of cyclic-di-AMP (Invivogen, vac-nacda) as described in figure captions.

Techniques: Molecular Weight, Staining, Isothermal Titration Calorimetry, Binding Assay, Enzyme-linked Immunosorbent Assay, Modification, Formulation, Comparison, Adjuvant

Journal: The Journal of Biological Chemistry

Article Title: Identification, Characterization, and Structure Analysis of the Cyclic di-AMP-binding P II -like Signal Transduction Protein DarA *

doi: 10.1074/jbc.M114.619619

Figure Lengend Snippet: Identification of the c-di-AMP receptor DarA in B. subtilis. A, a pulldown assay was performed with B. subtilis crude extract to identify c-di-AMP-binding proteins. After incubation of Strep-Tactin-covered beads with 2′-AHC-biotinylated c-di-AMP and a B. subtilis crude extract, the samples were washed to eliminate nonspecifically bound proteins. Elution was performed with 5 mm biotin. Samples were analyzed by SDS-PAGE with subsequent silver staining. As the negative control, the crude extract was incubated with Strep-Tactin-covered beads alone (−). B, DarA was overexpressed in E. coli and subsequently incubated with 2′-AHC-biotinylated c-di-AMP. After incubation, the sample was purified using a Strep-Tactin column, and bound protein was eluted with 2.5 mm d-desthiobiotin. As a negative control, the overexpressed protein was purified without addition of 2′-AHC-biotinylated c-di-AMP (−). The last wash fraction (W7) and elution fractions (E1–E3) were analyzed by SDS-PAGE with subsequent silver staining.

Article Snippet: To isolate c-di-AMP-binding proteins from B. subtilis , biotinylated c-di-AMP (2′-[biotin]-AHC-c-di-AMP; BIOLOG, Bremen, Germany) was coupled to Strep-Tactin-covered MagStrep “type 2HC” beads (IBA, Göttingen, Germany).

Techniques: Binding Assay, Incubation, SDS Page, Silver Staining, Negative Control, Purification