Figure 4: DSPE-PEG conjugation protects peptide integrity; impact of linkage chemistries on priming.

A-D, EGP₂₀ peptide constructs were incubated overnight with 10% fresh mouse serum. Serum-treated or fresh constructs were incubated with pooled splenocytes from previously DSPE-PEG-EGP₂₀ immunized mice for 24 hours, and ICS was performed after 18 hours. A, Normalized IFNγ⁺ T-cell responses for EGP₂₀ peptide; B, DSPE-PEG-EGP₂₀ with N-terminal DSPE-PEG conjugation; C, DSPE-PEG-EGP₂₀ with C-terminal DSPE-PEG conjugation; and D, PEG-EGP₂₀ with N-terminal PEG conjugation. Representative of 2 independent experiments. E, C57BL6/J mice were primed on day 0 and boosted on day 14 with 25 µg c-di-GMP and 5 nmol EGP₂₀ peptide or peptide construct, and ICS was performed 7 days later. Mean T-cell responses quantified as % IFNγ⁺ of CD8⁺. n = 5/group; ANOVA with Tukey post-test. F, Mice were primed on day 0 and boosted on day 14 with 10µg DSPE-PEG-EGP₂₀ or indicated doses of EGP₂₀, and mean T-cell responses quantified as % IFNγ⁺ of CD8⁺ 6 days later. n = 5/group; ANOVA with Tukey post-test. *P<0.05, **P<0.01, ***P<0.001.