Figure 5. Immunization with peptide nanofibers protected mice from LPS-induced inflammation.

Mice were challenged intraperitoneally with 10mg/kg LPS one week after final booster immunizations, and body temperature (a) and overall survival (b) were monitored. Humane endpoints of 32 °C (red line in a) or 20% body weight loss were employed. Group A, PADRE formulation 1: 1mM TNFQ11/0.05mM PADREQ11/0.95mM Q11; Group B, PADRE formulation 2: 0.2 mM TNFQ11/0.05 mM PADREQ11/1.75 mM Q11; Group C, VAC formulation: 1 mM TNFQ11/1.25 mM VACQ11/0.25 mM Q11; Group D, same VAC formulation as Group C, plus 10 µg CpG; Group E, negative control, missing any T-cell epitope; Group F, negative control, unvaccinated mice receiving LPS challenge; Group G, positive control: unvaccinated mice not receiving LPS challenge. Formulations containing TNFQ11 and either T-cell epitope offered significant protection, whereas unimmunized mice, vaccinations without T-cell epitopes, and CpG-containing vaccinations had poor survival. Data points in (a) represent individual mice. Box-and-whisker plots indicate medians and 25%/75% quartiles (horizontal line and box) and max/min values (whiskers). For Group D, quartile boxes are omitted where the number of surviving mice drops below 4. Statistical comparisons of survival between all groups in (b) were made using logrank test, *p<0.05, ns p>0.05. Individual p-values between all groups and descriptions of experimental replicates are provided in Table S2. All experiments shown in (a) were performed at Duke University, and (b) shows overall survival curves for the mice shown in (a) plus replicate experiments performed at the University of Chicago. ELISPOT analysis (c–d) of Th1 (IFNγ) and Th2 (IL-4)-secreting lymph node cells from unadjuvanted nanofiber immunizations (Group C), compared with nanofibers adjuvanted with CpG (Group D). Shown are total numbers of spots (c) and ratios of IFNγ/IL-4 spots (d). Mean ± SD is shown. *p<0.05 by ANOVA with Tukey’s multiple comparison (c) or two-tailed t-test (d).