Ab response Anti-Gag IgG Ab levels were assessed by ELISA) as previously described [12]. Briefly, 96-well microplates (Greiner, German) were coated with 5 mg/mL HIVIIIB lysate (ABI, Rockville, MD) and incubated overnight at 4uC. After blocking with PBS-0.5% gelatin for 1 h at 37uC, the plates were washed three times with PBS and incubated with serially diluted sample for 2 h at 37uC. After washing with PBS-0.5% Tween, the plates were incubated with biotinylated Ab anti-c1 (Southern Biotech, Birmingham, Alabama, USA) for 1 h at 37uC followed by the addition of streptavidin peroxidase (Sigma, St. Louis, MO, USA). The reaction was developed with tetramethyl benzidine (Calbiochem Corporation, San Diego, CA, USA), and the absorbance was read at 450 nm in an ELISA microplate reader (Bio-Rad, USA). Isolation of mononuclear spleen cells Spleens were obtained aseptically and mashed through cell strainers (BD-Biosciences, San Diego, CA, USA), and mononu- clear spleen cells (MSCs) were isolated after centrifugation in Ficoll-Hypaque solution (Sigma, St. Louis, MO, USA). After two washes, the cell pellet was diluted in RPMI 1640 containing 10% fetal calf serum (FCS, HyClone III, Logan, UT, USA). Viability was greater than 95%. Gag-specific IFN-c ELISPOT assay Enumeration of IFN-c–producing splenic T cells was performed by ELISPOT assay (BD-Biosciences) as previously described [12]. Briefly, 96-well microplates with polyvinylidene fluoride mem- brane support (Millipore, Bedford, MA, USA) were incubated with anti-IFN-c for 18 h at 4uC, washed and blocked with RPMI containing 10% FCS for 2 h at room temperature. Cells (56105 cells/well) were plated in the presence of medium alone, hamster monoclonal anti-mouse CD3 (1 mg/mL,BD-Biosciences),10 mg/ mL HIV-1 Gag epitope restricted to H-2d MHC class I AMQMLKETINEEAAE197–211,10 mg/mL HIV-1 Gag epitope restricted to H-2d MHC class II VDRFYKTLRAEQASQ297–308 (NIH AIDS Research and Reference Reagent Program), 2 mg/mL p24Gag from HIV-1 (kindly provided by Prof. Luis Carlos Ferreira, Institute of Biomedical Sciences, University of Sa˜o Paulo), or 10 mg/mL of 25 pools containing five HIV-Gag peptides (15-mers of the HIV-1 HXB2gag peptide), totaling 123 peptides, for 18 h at 37uC in 5% CO2. The plates were washed, incubated with biotinylated anti-IFN-c followed by incubation with avidin-peroxidase, and developed with the 3-amino-9- ethylcarbazole substrate (Calbiochem). Spots were quantified with an ImmunoSpot Imager using the ImmunoSpot 3.2 software (CTL ImmunoSpotH S4 Analyzer, C.T.L., Cleveland, OH, USA). The reaction was considered positive when the number of spot- forming cells (SFCs) was equal to or higher than 10 SFC/106 cells. All results were expressed as the mean number of SFCs per 106 MSCs. In vivo analysis of CTL activity Ten days after immunization with 5 mg of the DNA vaccine, offspring received target cells as previously described with modifications [12]. Briefly, MSCs from non-immunized (NI) BALB/c mice were used as target cells, and these cells were stained with high (10 mM) or low (1 mM) concentrations of carboxyfluorescein succinimidyl ester (CFSE, Molecular Probes, USA) in RPMI medium and incubated for 30 minutes at 37uC in the dark. The population stained with high concentrations of CFSE was pulsed with 2 mg of the class I peptide, AMQMLK- ETI197–205, for one hour at 37uC. Target cells with low and high Maternal and Fetal Priming by DNA Immunization concentrations of CFSE were washed and mixed at a 1:1 ratio in PBS. Approximately 36107 cells in 200 mL of PBS were IV injected into immunized mice (IM). Splenocytes from IM were collected after 18 hours, and transferred target cells were evaluated by flow cytometry based on high and low intensities of CFSE staining. The lysis percentage was determined by the following calculation: percentage of specific lysis = 1002 {[(% CFSEhigh cells IM/% CFSElow cells IM)/(% CFSEhigh cells NI/% CFSElow cells NI)]6100}. Immunophenotyping of CD4+CD25+FoxP3+ T cells MSCs were incubated with PerCP-conjugated anti-CD4 (BD- Biosciences), PE-conjugated anti-CD25 or isotype control for 30 min at 4uC. After washing, the cells were fixed with 4% paraformaldehyde for 10 min at 4uC. Cells were then incubated with PBS–0.5% saponin (Sigma) and FITC-conjugated anti-FoxP3 (eBioscience, San Diego, CA). After washing, the cells were analyzed by flow cytometry (BD FACSCalibur, Cell Quest PRO). A minimum of 250,000 events was collected from the lymphocyte gate. Measurement of TGF-b1 in milk samples An ELISA for TGF-b1 (Promega, Madison, USA) was performed to measure concentrations in milk samples according to the manufacturer’s recommendations. The detection limit of TGF-b1 was 32 pg/mL. Statistics Values for all measurements are expressed as the mean 6 SEM. Comparison between two groups was performed by the Mann- Whitney test; differences were considered significant when the p value was ,0.05. Three or more groups were analyzed using the Kruskall-Wallis test with Dunn’s post-test. The data were analyzed using the GraphPad Prism 3.0 software package (GraphPad, San Diego, California, USA). Supporting Information Figure S1 Transfer of anti-Gag Ab to offspring. Female BALB/c mice were primed and boosted with 50 mg of Lamp (L), gag (G), or Lamp/gag (LG) DNA and mated one day after the boost. (A) Serum samples from 0- (fetal), 30-, 60- and 90-d-o offspring were obtained by caesarean section from full-term pregnant mice, and offspring were bled monthly; (B) breast milk samples (1:20) were obtained 5 days after delivery, and anti-Gag Ab levels were determined by ELISA using HIV-1 lysates. The results of 4–6 animals per group were expressed as the mean 6 SEM. *p#0.05 and **p,0.01 when compared to Lamp. (TIF) Figure S2 Maternal Lamp/gag immunization reduces the IFN-c response in immunized offspring. Offspring from mothers immunized with either gag (G) or Lamp/gag (LG) were immunized with 1 mg of (A) LG or (B) G DNA. Spleen cells from 35-d-o offspring were cultured with 25 pooled of HIV-1Gag peptides. The figure represents 4–5 assays per group (2–3 animals per group). Bars represent the mean 6 SEM. *p#0.05 and **p,0.01 when compared with immune offspring from non- immunized (NI) mothers. (TIF) Acknowledgments We thank Vilma dos Anjos Mesquita for the dedicated animal care, Prof. Luis Carlos Ferreira (Institute of Biomedical Sciences, University of Sa˜o PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e31608