Figure 2: Humoral and cellular responses to carrier proteins.

Mice were immunized i.m. with 1 μg Pfs25 conjugated to TT (Pfs25-TT), EPA (Pfs25-EPA), mouse serum albumin (Pfs25-MSA), or self-conjugated (Pfs25-Pfs25) formulated in Alhydrogel (AH) or GLA-LSQ on day 0 and day 28. (a,b) Anti-Pfs25 IgG ELISA titers were collected at various time points after immunization with conjugate vaccines in Alhydrogel (a) or GLA-LSQ (b). (c) Anti-EPA IgG ELISA titers collected at various time points after immunization. (d) Anti-TT IgG ELISA titers collected at various time points after immunization. Shown is the geometric mean with 95% confidence interval from 5–10 mice per group. Inguinal LNs (e,f) and spleens (g,h) were harvested at the indicated time points and processed for flow cytometry. Lymphocytes were left untreated (Ctrl), restimulated with EPA protein (10 μg/ml) or with TT protein (20 μg/ml) for 2 hours and then brefeldin A was added for 5 additional hours. Representative flow cytometry plots showing the frequency of TNF-α⁺ and/or IFN-γ⁺ within the activated CD44^hiFoxp3⁻CD4⁺ T cell population 13 (e) and 250 (g) days after immunization. Frequency of TNF-α producers within the activated CD44^hiFoxp3⁻CD4⁺ T cell population 13 (f) and 250 (h) days after immunization. Shown is the mean ± SEM, n = 5–10 mice per group. (*P < 0.05, **P < 0.01, ***P < 0.001; Mann-Whitney U test). All data presented here are representative of 3 independent experiments.