MHC class I and II epitopes and to p24 was markedly enhanced compared to offspring immunized with Gag (Figure 7B). Moreover, non-immunized offspring from mothers immunized at pregnancy had no vertical Ab transfer when mothers were immunized by IV injection (Figure 7C). Therefore, offspring that received just one dose of the LAMP/gag vaccine had an increased Ab response when compared to the gag-immunized offspring (Figure 7C). Notably, our previous results showed that two vaccine doses are needed and that an individual dose of the LAMP/gag vaccine at 7–d-o is not enough to induce anti-Gag Abs [12]. Thus, the current results suggest that there is a recall response upon in utero priming in offspring immunized with the LAMP/gag vaccine. In conclusion, our data showed that intravenous administration of the DNA vaccine elicited low numbers of IFN-c SFCs and primed both maternal and fetal immune systems. These results opened a possibility of priming the fetal immune system in utero, with the absence of MatAb, although the intensity of cellular and humoral responses generated by this protocol was lower than that observed with preconceptional immunization of the mother followed by neonatal LG immunization. Then, the preconcep- tional protocol represent the best approach to transfer Ab and to elicit higher humoral and cellular immune response on immunized neonate, however if we aim to prime as early as possible the fetus immune system and to avoid the effect of MatAb on the offspring vaccine response, we should consider use the intravenous protocol. Discussion Maternal immunization with LAMP/gag DNA before concep- tion or during pregnancy was assessed to evaluate the neonatal immune response to the DNA vaccine and the possibility for in utero priming, respectively. We have previously shown that administration of the chimeric LAMP/gag DNA vaccine at the neonatal stage is highly immunogenic, as it elicits high levels of anti-Gag Abs and elevated cellular immune responses towards the HIV–gag antigen when compared to immunization with a DNA construct expressing the Gag antigen only [12,13]. However, it was unknown whether maternal immunization with the chimeric LAMP/gag DNA vaccine could affect the offspring response to the vaccine. Our results indicated that maternal immunization with the chimeric DNA vaccine by subcutaneous injection before concep- tion promoted passive Ab transfer that caused a transitory inhibition of the offspring response to the construct. Nonetheless, this inhibition did not impair the long-lasting B- and T-cell responses in the offspring. Moreover, the high levels of Ab that were elicited when maternal immunization occurred prior to conception were circumvented when an individual intravenous dose of the LAMP/gag DNA was administered during pregnancy. The intravenous route of immunization was less immunogenic than the intradermal route, but it was still able to prime both mother and offspring. Immunization with the LAMP/gag DNA vaccine before conception led to the transfer of high levels of anti-Gag IgG1 Abs through the placental/amniotic and breastfeeding routes. IgG1 Abs are the major subclass of Abs produced by adult and neonate BALB/c mice immunized with LAMP/gag DNA [12], and IgG1 Abs were the main Ab subclass that were transmitted to the offspring. Maternal Ab levels lasted up to 60 days in the offspring circulation, indicating that breast milk was an important source of IgG, as weaning takes place at approximately 25 days after birth, and the half-life of IgG is 30 days. The FcRn–IgG Fc receptor, which is expressed in the small intestine of suckling mice and rats, is the main molecule involved in IgG uptake from ingested Maternal and Fetal Priming by DNA Immunization maternal milk [19]. In mice, the yolk sac endoderm constitutively takes up IgG into the fetal circulation [20]. The gag DNA vaccine induced low Ab responses, and consequently, low levels of Ab were transferred to the offspring. Maternal immunization with LAMP/gag DNA decreased the anti-Gag IgG1 and IgG2a Ab levels and the number of anti-Gag IFN-c SFCs in LAMP/gag–immunized offspring when compared to the LAMP/gag–immunized offspring from non-immunized the negative impact of LAMP/gag maternal immunization on the 35-d- o offspring response to the immunization, it did not hinder the broad T-cell recognition of Gag epitopes in the neonates. It is not clear whether the decreased number in IFN-c SFC could interfere in the quality of the total anti-Gag immune response; the decrease in the IFN-c SFC numbers against the class I and II MHC epitopes _ represented by the immunodominant epitopes and not by all of the recognizable epitopes _ by ELISPOT assay do not predict the whole functionality of these Ag-specific T cells. These results should be considered when reviewing how the assays were performed, the ELISPOT assays for determination of IFN-c SFCs represent an in vitro recall response to the stimulating peptides, whereas IgG Ab levels and the cytotoxic assay use in vivo or ex vivo parameters as a readout. Nevertheless, six months after neonatal immunization with LAMP/gag DNA, no inhibitory effect was detected on the anti-Gag IFN-c response to class I or II Gag peptides. This result demonstrates that despite the active suppression in early responses, the generation of T-cell memory responses was intact. However, a boost with LAMP/gag DNA was necessary to elicit an anti-Gag Ab response in six-month-old offspring. As expected, a B-cell late response was generated only with the LAMP/gag immunization and after the 3rd boost, probably due to the higher CD4 T cell activation and T-B cell cooperation. The presence of maternal anti-Gag Abs in neonates is likely the main inhibitory mechanism for immunizing offspring, as these Ab can form immune complexes with the Gag protein when it is transduced upon LAMP/gag DNA immunization. In fact, when neonates were injected with purified IgG from immune mice in conjunction with the LAMP/gag DNA immunization, they showed an inhibition of the humoral and in vivo cytotoxic T cell responses to the H-2dclass I immunodominant peptide. Interestingly, the IgG injections did not interfere with the IFN-c response to the Gag peptides or the p24 protein as compared to the LAMP/gag- immunized mice that received IgG from non-immune mice. The in vivo cytotoxicity of CD8+ T cells is not dependent on IFN-c as recently shown in a murine model of prime-boosting using DNA and recombinant adenoviruses against Chagas disease; here, protection against the disease was reduced in IFN-c2/2 mice even in the presence of specific CTLs [21]. However, it is still possible that other regulatory mechanisms independent of maternal Abs contribute to the attenuation of the offspring immune response to the vaccine. Several mechanisms by which maternal factors influence the immune response in offspring have been described and suggested, including the following: (i) formation of immune complexes with the antigen, which impairs neonatal priming [8]; (ii) anti-idiotypic maternal Ab interaction with immature B and T cells in primary lymphoid organs, or negative or positive modulation of plasma and T cell differentiation [22]; (iii) maternal transfer of cytokines that are able to control cell activation by downregulating the expression of co-stimulatory and activation molecules on dendritic cells and T and B lymphocytes [23,6]; (iv) interaction of maternal Ab immune complexes with the B-cell inhibitory receptors that mediate negative regulation of B-cell function [23]; and (v) generation of T regulatory cells [6]. Our group has been exploiting PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e31608