Maternal and Fetal Priming by DNA Immunization Figure 3. Maternal Lamp/gag immunization reduces the IFN-c response of immunized offspring. Offspring from mothers immunized with Lamp/gag (LG) or gag (G) were immunized with 5 mg of (A) LG or (B) G DNA, respectively. Spleen cells from 35-d-o offspring were cultured with 25 pooled HIV-1 Gag peptides. The figure represents 4–5 assays per group (2–3 animals per group). Bars show the mean 6 SEM. Schematic diagram of the immunization protocols are shown. *p#0.05 and **p,0.01 when compared with immunized offspring from non-immunized (NI) mothers. doi:10.1371/journal.pone.0031608.g003 gag immunization did not interfere with the cellular response (measured by the number of IFN-c SFCs) of gag-immunized offspring when compared to the control group (Figure 3B). The LAMP/gag immunization in turn generated broad epitope recognition in the neonatal period even after LAMP/gag-maternal immunization. In fact, when mothers were immunized with the chimeric vaccine and the offspring with the gag vaccine, the number of IFN-c SFCs in the offspring decreased (Figure 3B). This inhibitory effect mediated by LAMP/gag-maternal immunization was found to be Gag-specific, as there was no decrease in the IFN- c response to anti-CD3 stimulation. Furthermore, inhibitory effects after maternal LAMP/gag immunization were also con- firmed when offspring were immunized with 1 mg LAMP/gag DNA vaccine (Figure S2). Due to the inhibitory effect of maternal LAMP/gag immuni- zation on the offspring vaccine response, we also determined the level of TGF-b1 in milk. TGF-b is one of the important factors involved in the generation of T regulatory (T reg) cells, which contribute to the control of immune responses to vaccines. TGF-b1 levels were evaluated in milk samples that were taken from immune or non-immune mothers 7 days postpartum. Curiously, mothers immunized with either LAMP/ gag or gag DNA showed reduced TGF-b1 levels in their milk 7 days after delivery when compared to non-immunized mothers (Figure 4A). We next evaluated whether maternal immunization with LAMP/ gag DNA could interfere with the frequency of T reg cells in the offspring. We quantified the CD4+CD25+Foxp3+ T cells in spleen samples of 7-d-o offspring from immune or non-immune mothers (Figure 4B). Surprisingly, only maternal immunization with LAMP/ gag DNA augmented the percentage of CD4+CD25+Foxp3+ T cells in comparison to the control offspring. Therefore, maternal immunization with the chimeric LAMP/gag DNA down-modulated the immune response generated in immunized offspring, which could be related to the high levels of anti-Gag maternal Abs present in the offspring as well as to the generation of CD4+CD25+Foxp3+ T cells by the offspring. Effect of IgG treatment on LAMP/gag-immunized offspring We next evaluated whether maternal anti-Gag IgG Abs could be involved in the decreased humoral and cellular anti-Gag immune responses in LAMP/gag-immunized offspring. Purified IgG from either non-immune or LAMP/gag DNA-immunized mice was injected into 7–, 13–, and 25–d-o offspring from non- immunized mothers. Mice were then immunized at 7–and 25–d-o with 5 mg LAMP/gag DNA. Injection of IgG from immune mice (IgG-LG) led to a significant decrease in the anti-Gag IgG Ab response after offspring immunization (Figure 5A). There was also a decrease in the cytotoxic T cell response towards the H2-Kd immunodo- minant epitope in vivo (Figure 5B) when compared to immunized offspring treated with IgG from non-immune mice (IgG-NI). The decreased Ab response that occurred when IgG was injected from immune mice suggested that Ag-specific Abs might inhibit the LAMP/gag vaccine response in offspring by forming a complex between Ag and IgG. However, treatment with IgG did not alter the number of IFN-c SFCs that were generated in response to MHC class I or class II epitopes (Figure 5C). These results showed that treatment with IgG from LAMP/gag-vaccinated mice decreased the humoral and CTL responses in immunized neonates, whereas other immune cell functions were not affected. Effect of LAMP/gag DNA maternal immunization on later T and B cell immune responses on offspring Although maternal immunization with LAMP/gag DNA inhibits the humoral and cellular immune responses in offspring, it is unknown whether maternal immunization affects the late immune response. The humoral and cellular responses were thus evaluated PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e31608