There is some evidence that maternal Ab generated after antigen vaccination negatively influence the vaccine response of the infant. However, there is strong evidence that DNA-vaccinated neonates, born from DNA-immunized mothers, can elicit effective immune responses against herpes simplex virus and malaria protozoa even in the presence of maternal Ab [10,11]. The use of DNA vaccines as a strategy to avoid the inhibitory effects of maternal Ab is based on the fact that the antigen encoded by the vaccine DNA is not exposed to maternal Ab; antigen presentation is carried out by the MHC class I and II molecules of cells that take up DNA [8]. Even if part of the DNA product is secreted and neutralized by maternal Abs, the remaining product will be processed and presented as an endogenous antigen. Our group has previously studied the LAMP/HIV-1-gag chimeric DNA vaccine in neonate models [12,13]. The lyso- some-associated membrane protein-1 (LAMP-1) is highly ex- pressed by all nucleated cells and is found in the MHC class II- enriched intracellular compartments (MIICs) that are associated with antigen presentation [14]. Intradermal delivery of the LAMP/ HIV-1-gag DNA vaccine has been shown to (i) elicit enhanced Ab production, (ii) amplify the anti- Gag CD4+, CD8+ T and B cells responses, and (iii) prolong immunological memory to Gag epitopes in adult mouse and monkey experimental models [14,15,16,12]. Recently, we have shown that mucosal and intradermal immunization of neonates with the LAMP/HIV-1- gag (LAMP/gag) construct correlates with strong anti-Gag cellular and humoral immune responses [12,13]. The rationale for using the LAMP/gag DNA strategy in maternal immunization was to verify whether subcutaneous immunization prior to conception could improve the newborn vaccine response by Ab transfer and allow the generation of immunological memory on offspring. Moreover, pregnant mice were also immunized by intravenous route to check whether this change in the maternal immunization protocol could prime the offspring in utero and prevent the inhibitory effects of maternal Ab. In fact, there have been reports showing that intravenous DNA injection of pregnant mice can prime the fetal immune system [17,18]. Furthermore, understanding the strong relationship between fetal and maternal immune systems is essential for Maternal and Fetal Priming by DNA Immunization developing strategies of maternal vaccination that avoid neonatal antigen tolerance. In the present work, we evaluated the effect of the LAMP/gag DNA vaccine which was administered to mice either before or during pregnancy on the neonatal immune response. The immunization before conception led to a robust transmission of maternal Ab to offspring, which lasted up to 2 months and did not interfere with the long-lasting cellular immune response after neonatal vaccination. Maternal vaccination via the intravenous route with the chimeric vaccine did not raise Ab and led to in utero priming of both maternal and fetal immune systems. Results LAMP/gag DNA immunization before conception induces anti-Gag IgG Abs transference through the transplacental and breastfeeding routes to offspring Transfer of maternal Ab is essential for protecting newborns against infectious agents; maternal vaccination can either prime the neonatal immune system or inhibit the neonatal vaccine response [9]. To evaluate the mechanisms triggered by maternal vaccination with LAMP/gag DNA, we first assessed the contribu- tion of IgG transfer to offspring by the transplacental and breastfeeding routes. Female BALB/c mice were immunized and boosted after 20 days by intradermal route with 50 mg LAMP/gag, gag, or LAMP plasmid DNA. The immunized mice were mated and maternal serum, amniotic fluid, and milk were collected. Fetal serum samples were collected by caesarean section, and serum samples from offspring were collected at different ages. We have previously demonstrated that immunization with LAMP/gag DNA preferentially induces production of the IgG1 subclass of Abs in adult and neonate mice [12]. Figure 1A shows that the LAMP/gag-immunized mothers showed high levels of anti- Gag IgG1 Abs which is also detected in the amniotic fluid (Figure 1B), neonatal serum (fetal serum, day 0, Figure 1D), and milk (Figure 1C). In fact, analysis of neonatal serum showed that there were significantly higher levels of anti-Gag IgG1 Ab in newborns from LAMP/gag–immunized mothers than in newborns from gag–immunized mothers (Figure 1D). Maternal anti-Gag Figure 1. Transfer of high levels of anti-Gag IgG1 Abs from Lamp/gag DNA-immunized mothers to offspring through transplacental and breastfeeding routes. Female BALB/c mice were primed and boosted with 50 mg of Lamp (L), gag (G) or Lamp/gag (LG) plasmid DNA and mated one day after the boost. (A) Maternal serum and (B) amniotic fluid were obtained by caesarean section from full-term pregnant mice. (C) Breast-milk samples (1:20) were collected on day 5 after delivery and (D) fetal serum from 0-, 30-, 60- and 90-d-o offspring were evaluated by ELISA using HIV-1 lysates. The results from 4–6 animals per group were expressed as the mean 6 SEM. Schematic diagram of the immunization protocol is shown. *p#0.05 and ***p,0.001 when compared to gag. doi:10.1371/journal.pone.0031608.g001 PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31608