the capacity of maternal immunization with allergens to dowmodulate the newborn allergic response [24,23,25,26]. Our findings have shown that allergy prevention in offspring whose mothers were immunized is mainly due to the formation of immunocomplexes with allergens and maternal Abs as well as the upregulation of the inhibitory FccRIIb receptor in offspring B cells [25,23]. Notably, one effect of the maternal immunization with LAMP/ gag was the induction of a high percentage of CD4+ CD25+FoxP3+ T cells in 7-d-o neonates despite the reduction of TGF-b1 levels in milk. TGF-b1 is essential for the generation of induced T reg cells [27]; however, it is not clear why only offspring from LAMP/gag-immunized mothers showed increased percent- ages of CD4+CD25+FoxP3+ T cells in the spleen. It is possible that other factors are involved in the modulation of the offspring immune response promoted by the LAMP/gag immunization. In fact, it has been shown that T reg cells regulate inflammation during infections, but a high number of T reg cells can suppress the antiviral response [28]. Furthermore, the cells with phenotype of T reg generated after maternal LAMP/gag DNA vaccination could function in controlling offspring immune activation after immunization. There is evidence that to achieve an efficient immune response in offspring, maternal Abs should be directed against epitopes other than neonatal epitopes, thereby avoiding inhibition the offspring response to vaccination [29]. In this case, we should consider the use of different DNA constructs in maternal and offspring immunizations and find a vaccination protocol that is immunogenic in the newborn and can overcome the inhibition caused by maternal Abs. Considering that the chimeric LAMP/gag DNA raised Ab titers by 50 to 100-fold and that it was more immunogenic during the neonatal period, we evaluated the effects of IV immunization of pregnant mice with LAMP/gag DNA on the immunogenicity of LAMP/gag-vaccinated offspring. Maternal immunization was performed close to birth to avoid high levels of Ab transfer to offspring. Our results showed that IV immunization with LAMP/gag DNA five to seven days before delivery generated T and B cell responses in mothers and offspring, confirming that is possible to use a DNA vaccine to prime both the fetus in utero and the immune system of the mother. Moreover, this approach confirmed that the LAMP/gag DNA construct was able to elicit multiple immune responses compared to conventional gag DNA immunization, particularly T-cell activation. Despite a lower magnitude of the immune response observed with the intravenous DNA immunization, just one dose of the chimeric vaccine by intravenous injection in the last few days of pregnancy was able to prime both maternal and fetal immune systems. The transfer of plasmid to the fetus through the placenta and amniotic fluids has been a concern, as it may cause tolerance of the offspring immune system towards the plasmid- encoded antigen. The current study and those by others have confirmed that this is not the case [30,17,18,31]. Moreover, use of LAMP/gag DNA vaccination during pregnancy could be an advantage by subjecting newborns to fewer doses but enough doses to generate an immune response. Together, our data showed that the chimeric LAMP/gag DNA construct, which targets the Gag antigen to MIIC compartments, more efficiently induced T and B cell responses in mice at various stages in life, compared to the conventional DNA construct expressing gag only. Even in the presence of MatAb, the LAMP/gag DNA, administered in the neonatal period, elicited broad T cell responses and considerable B cell responses. Furthermore, preconceptional immunization with LAMP/gag, inducing MIP-1a secretion (data not show) – natural blocker of infection - and Maternal and Fetal Priming by DNA Immunization increasing T reg cell numbers in the offspring, could be protective against HIV-1 infection by inhibiting viral entry into target cells and reducing CD4+ T cell activation. Moreover, giving the LAMP/gag DNA vaccine during pregnancy primed both maternal and fetal immune systems. Therefore LAMP-1 with other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods. Materials and Methods Mouse immunization 8- to 10-week-old BALB/c mice were purchased from CEMIB (UNICAMP, Campinas, Sa˜o Paulo, Brazil) and bred in our pathogen-free animal facility. The Sa˜o Paulo University Institu- tional Animal Care and Use Committee approved the present study. Plasmids A eukaryotic expression plasmid with the fragment of the HIV-1 HXB2 p55gag gene, nucleotides 1-1503 (GenBank accession number KO3455), was inserted into the mammalian expression vector, pITR [32], which contains a cytomegalovirus promoter and the ITR sequences from adeno-associated virus flanking the expression elements. The mouse LAMP-1 gene (GenBank J03881) was cloned into this vector, resulting in the chimeric LAMP/gag plasmid with the p55gag sequence placed between the LAMP-1 luminal domain and the transmembrane/cytoplasmic tail as previously described [14]. Plasmid DNA was produced by transforming DH5a Escherichia coli (Invitrogen, Carlsbad, CA), and plasmids were purified using an endotoxin-free column (Qiagen Inc., Valencia, CA). Immunization protocols Maternal immunization before conception. Female BALB/c mice were immunized (homologous prime-boost) twice by the intradermal route (ID immunized) at a 20-day interval with 50 mg of one of the following plasmid DNAs: pITR-LAMP/gag (LAMP/gag-LG), pITR-gag (gag-G), or pITR-Lamp (Lamp-L). Mice were mated after the boost. Offspring were immunized with the same vaccine as their mothers. Maternal immunization during pregnancy. Pregnant BALB/c mice received 100 mg of the LAMP/gag or gag DNA vaccine by intravenous (IV) injection 7-5 days before delivery. Neonatal immunization. 7- and 25-day-old (d-o) neonates were ID immunized with 1 or 5 mg of LAMP/gag (LG), gag, or Lamp DNA and subjected to analysis at different ages. Passive transfer of IgG from immunized mice. Neonate mice received intraperitoneal (IP) injections at 7-, 13-, and 25-d-o with 100 mg of purified IgG from immune (LG) and non-immune (NI) mice, similar to the DNA immunization protocol. IgG was purified using the Melon Gel IgG Spin Purification kit according to the manufacturer’s instructions (Pierce, Rockford, IL) and stored at 270uC until use. IgG measurements were performed by enzyme- linked immunosorbent assay (ELISA). Collection of amniotic fluid, fetal and maternal sera and milk To evaluate vertical transmission of Ab, amniotic fluid and fetal serum samples were obtained by caesarean section of full-term pregnant mice (21 days) and stored at 270uC until use. Mothers were bled at the time of parturition and on day 20 postpartum. Milk samples were obtained from the stomachs of 5- or 7-d-o newborns as previously described [33]. PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e31608