Maternal and Fetal Priming by DNA Immunization Figure 6. Maternal LAMP/gag immunization does not interfere with long-lasting IFN-c and B-cell responses in immunized offspring. Offspring from non-immune (NI) or immune (I) mothers were immunized at 7- and 25-d-o with 5 mg of either Lamp/gag (LG) or gag (G) DNA. Some neonates groups from I or NI mothers received a 3rd boost at 6-months-old. (A) IFN-c SFCs from 6-month-old offspring after stimulation with MHC H- 2d class I- and II-restricted pools of Gag peptides from HIV-1. The figure represents 4–5 assays per group (2–3 animals per group); *p#0.05 when compared with immune offspring from NI mothers. (B) Serum from offspring was collected when they were 6 months old (6 animals per group), and anti-Gag IgG was evaluated by ELISA using HIV-1 lysates. One group received a boost 10 days before analysis. Bars represent the mean 6SEM. *p#0.05 when compared with immune offspring from NI mothers. Schematic diagram of the immunization protocols are shown **p,0.01 when compared with gag-immunized offspring. doi:10.1371/journal.pone.0031608.g006 Figure 7. Intravenous DNA immunization during pregnancy primes the fetal immune system. Pregnant mice were IV immunized approximately 15–16 days into gestation with 100 mg of Lamp/gag (LG) or gag (G) vaccine DNA. Offspring were either immunized (IM) or not (NI) at 7- d-o with 5 mg of the DNA vaccine. (A) Mother: IFN-c SFCs from spleen cells of immunized mothers 35 days after delivery and (B) Offspring: IFN-c SFCs from spleen cells of 35-d-o offspring after stimulation with class I and class II Gag peptides and recombinant p24 of HIV-1. The figure represents 2–3 assays per group (2–3 animals per group). (C) Offspring: Serum samples from 35-d-o NI or IM offspring were evaluated by ELISA using HIV-1 lysates for anti-Gag IgG concentrations. Bars represent the mean 6 SEM. Schematic diagram of the immunization protocol is shown. *p#0.05, **p,0.01, and *** p,0.001 when compared with G-offspring from a G-mother; +++p,0.001 when compared with NI offspring from a G-immunized mother. doi:10.1371/journal.pone.0031608.g007 PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e31608