Maternal and Fetal Priming by DNA Immunization Figure 4. Effect of maternal DNA immunization on TGF-b1 levels in milk and on the generation of CD4+CD25+ FoxP3+ T cells in offspring. Female mice were primed and boosted with 50 mg of Lamp/gag (LG), gag (G), or Lamp (L) plasmid DNA and mated one day after the boost. (A) Breast-milk samples collected 7 days after delivery; (B) percentages of CD4+CD25+Foxp3+ T cells in offspring splenocytes at 7 d-o; (C) CD4+CD25+FoxP3+ T cells in individual offspring at 7 d-o, non-immunized (NI), or immunized with L, G or LG. The results of 8–12 animals per group are expressed as the mean 6 SEM. Schematic diagram of the immunization protocol is shown. #p#0.05 and ##p,0.01 when compared with the non-immunized (NI) group; **p,0.01 when compared with the gag-immunized group. doi:10.1371/journal.pone.0031608.g004 in 6-month-old offspring from immunized or non-immunized mother. Figure 6A shows that maternal immunization had no negative effect on IFN-c producing cells in the LAMP/gag– immunized offspring for up to 6 months when compared to the control group (LG offspring – NI mothers). Similarly, as expected, maternal immunization with gag DNA did not interfere with the long-lasting IFN-c response in offspring. However, an anti-Gag IgG Ab response was detected only in 6-month-old offspring immunized with LAMP/gag DNA following an immunization boost probably due to the high CD4 T cell augmented response given by the chimeric vaccine (Figure 6B). After the 6-month LG DNA boost, the immunized offspring from immune mother showed similar levels of Anti-Gag IgG levels than the immunized offspring from non-immune mother (Figure 6B). These results indicated that although maternal immunization with chimeric LAMP/gag DNA affected the early response (10-days after the second boost), it did not impair the late T- and B-cell response (6 months after vaccination). Intravenous injection of LAMP/gag DNA vaccine during pregnancy primes the fetal immune system We also investigated whether it was possible to prime the fetus immune system with the chimeric DNA Lamp/Gag or conventional (Gag) DNA vaccine by immunizing pregnant mice. We designed a second protocol aiming at (i) decreasing the maternal Ab levels, favoring the priming of the immune system of both mother and offspring, (ii) priming the offspring during the fetal period and (iii) avoiding the presence of high maternal Ab levels transferred to the offspring during their priming with the DNA vaccine. As it is well known that intravenous (IV) delivery is not an immunogenic route, and considering the role of the chimeric LAMP/gag DNA on antigen presentation-cell and CD4+ T cell activation, we hypothesized that injecting the DNA vaccines intravenously, seven to five days before delivery, could prime the offspring. We thus injected a 100 mg single dose of the DNA vaccine during the last 7 to 5 days of pregnancy and assessed the effects in the neonate. The results showed that one intravenous injection with 100 mg of LAMP/gag or gag DNA during pregnancy was able to prime mothers as determined by the induction of IFN-c SFCs that were generated against the immunodominant MHC class I and II epitopes and the p24 protein (Fig. 7A). We then compared the non-immunized offspring and offspring that were immunized with 5 mg DNA vaccine at 7–d-o. Non-immunized offspring of mothers that received a LAMP/gag DNA injection during pregnancy produced IFN-c SFCs against p24 (average 69.4 SFCs/106MSCs), and this number of IFN-c SFCs was significantly higher than that of non-immunized offspring of G-immunized mothers (2.54 SFC/ 106 MSCs). These results strongly suggest that there was in utero priming following IV injection of the chimeric LAMP/gag DNA vaccine (Figure 7B). In fact, when the 7-d-o offspring from LAMP/ gag-immunized mothers were then immunized with LAMP/gag, the amount of IFN-c produced in response to the immunodominant Figure 5. Transfer of IgG from immune mice decreases CTL numbers and anti-Gag IgG Abs in offspring immunized with LAMP/gag. Offspring from non-immunized mothers were immunized at 7- and 25-d-o with 5 mg of LAMP/gag (LG) and treated with IgG from non-immune (IgG- NI) or LG (IgG-LG)-immunized mice at 7-, 13-, and 25-d-o or without IgG treatment (none). (A) Offspring serum samples from 35-d-o mice (n = 6 per group) were evaluated for anti-Gag IgG Ab levels by ELISA using HIV-1 lysates. (B) In vivo T-cell cytotoxicity was evaluated ten days after LG immunization. Mice were IV injected with target cells from NI mice stained with low and high concentrations of CFSE. The CFSE-high target cells were pulsed with a class I immunodominant peptide (AMQMLKETI), and after 18 h, spleen cells were evaluated. (C) IFN-c SFCs from 35-d-o offspring (n = 3 per group) were evaluated after stimulation with Gag MHC class I and II epitopes or recombinant p55 of HIV-1. Bars represent the mean 6 SEM. Schematic diagram of the immunization protocol is shown. **p#0.01 compared with offspring treated with IgG from NI mice; #p#0.05 compared with offspring without IgG treatment. doi:10.1371/journal.pone.0031608.g005 PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e31608