IgG1 Abs were detected in the serum of offspring from LAMP/gag- immunized mothers for up to one month after birth. Anti-Gag IgG2a and IgA Abs were also detected at low levels (Figure S1). Taken together, these results indicated that maternal immuniza- tion with LAMP/gag DNA before conception promoted a robust passive transfer of anti-Gag IgG Abs through the transplacental and breastfeeding routes both before and after birth. Effect of maternal DNA immunization on the vaccine response of offspring To investigate the effect of maternal immunization with LAMP/ gag DNA on the offspring immune response, adult female mice were immunized twice with 50 mg DNA at a 20–day interval according to the prime–boost protocol. Mice were mated one day after the boost. Seven–and 25–day old (d-o) offspring were immunized with 5 mg DNA vaccine and evaluated after 30 and 60 days for anti-Gag IgG Ab production. The frequency of IFN-c- secreting cells stimulated by Gag peptides was evaluated after 60 days. Females and offspring either non-immunized or immunized with the gag or LAMP DNA vaccine before mating were used as controls. Figure 2 shows that maternal immunization with LAMP/ gagDNA promoted the transfer of anti-Gag IgG1 Abs that lasted Maternal and Fetal Priming by DNA Immunization up to 30 days. LAMP/gag–immunized neonates from LAMP/gag– immunized mothers displayed a 70% reduction in anti-Gag IgG1 (Figure 2A) and anti-Gag IgG2a Ab levels (Figure S1) compared to immunized offspring from non-immune mothers; whereas non- immunized offspring from gag–immunized mothers had almost undetectable levels of maternal anti-Gag IgG1 Abs. When offspring of gag-immunized mothers were immunized, the detected Ab levels increased to 5% compared to gag-immune offspring from non-immune mother (Figure 2A). The control group of females and their respective offspring immunized with pITR-LAMP DNA did not show any anti-Gag response. Maternal LAMP/gag–immunization led to a decrease in the number of IFN-c spot–forming cells (SFCs) against Gag peptides in LAMP/gag–immunized offspring (Figure 3A). The number of IFN-c SFCs generated upon stimulation of several peptides, including those containing MHC class I–and II–restricted epitopes, decreased in LAMP/gag-immunized offspring from LAMP/gag-immunized mothers when compared to LAMP/gag- offspring from non-immunized mothers. The immunodominant Gag peptides recognized by MHC class I and II molecules of cells from adult BALB/c mice immunized with LAMP/gag and gag DNA constructs were peptide fragments from amino acids (aa)181 to aa 227 (MHC class I), aa 241 to aa 271, and aa 281 to aa 311 (MHC class II) as previously reported [15]. Interestingly, maternal Figure 2. Maternal immunization with the Lamp/gag DNA vaccine decreases anti-Gag IgG1 Ab production in immunized offspring. Female mice were immunized, and 20 days later, they were boosted with 50 mg Lamp (L), gag (G), or Lamp/gag (LG) plasmid DNA and mated one day after the boost. Offspring were either non-immunized or immunized with 5 mg of the same vaccine DNA at 7- and 25-d-o. Offspring serum samples were collected at (A) 30- and (B) 60-d-o (3–8 animals per group). Anti-Gag IgG1 was determined by ELISA using HIV-1 lysates. Bars represent the mean 6 SEM. Schematic diagram of the immunization protocols are shown. *p#0.05 and ***p,0.001 when compared to the respective control offspring from non-immunized (NI) mothers. doi:10.1371/journal.pone.0031608.g002 PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31608