2 hours at 37uC. Mouse anti-p24-HRP antibody (1:1200) was added and incubated for 1 hour at 37uC. The colorimetric conversion of TMB substrate reagent was used for 10–20 minutes at room temperature. The working solutions were as follows: 250 ml solution B +10 ml buffer A +3.75 ml 30% H2O2. Buffer A: 205 mM potassium citrate (pH 4.0). Solution B: 41 mM TMB in DMSO [31]. After each incubation, but not after blocking, plates were washed with 0.05% Tween 20 in PBS. Colorimetric conversions of the substrate were read at 450 nm and 570 nm. OD value = (OD value at 450 nm) 2 (OD value at 570 nm). The p24 standard curve was drawn and a linear regression equation was made. The linear range was 0.005 ng/ml to 0.1 ng/ml. The p24 concentration of the sample was computed using the equation. Polarized Caco-2 monolayers Monolayers were grown in 12-well Corning Costar Transwell plates (Corning Inc., U.S.) with 0.4-mm or 3-mm pore filter inserts. Cells were cultured for 14–16 days after becoming confluent (which took about 2 days). The medium was changed every other day so that would be well polarized. The TER readings were about 600 Ohm/cm2 (as measured by a Millicell-ERS voltmeter; Millipore Corp., U.S.). rVTT-gagpol infection of Caco-2 cells Polarized Caco-2 monolayer cells were infected with rVTT- gagpol apically at MOI (multiplicity of infection) 1. Virus was added directly onto the apical surface and incubated at 37uC in 3% FBS-containing DMEM medium. The same medium was added to the basolateral compartment. Virus was aspirated after 2 hours. The apical surface was washed three times with PBS. Then the cultures were fed fresh medium and returned to the incubator. Infected polarized Caco-2 monolayer cells were sampled at 0, 24, 48, and 72 hours post-infection. Apical, basolateral media were collected. Cells were frozen and thawed three times. Finally, three types of samples from apical media, basolateral media, and cells were aliquoted and stored at 270uC. In the unpolarized group, starter cells were seeded in transwells for 3 days and then infected with rVTT-gagpol. In the colchicine- treated group, colchicine (0.08 mg/ml) was added to polarized Caco-2 cell layers 72 hours before infection. Forty-eight hours after infection, apical and basolateral media were collected and fresh medium was added. All samples were collected at 72 hours. The p24 and viral titer were determined by ELISA and plaque assay, respectively. The virus titer this time was calculated as the sum of that collected at 48 hours and at 72 hours. Experiments were performed double or in triplicate. Electron microscopy Each monolayer was rinsed with PBS and fixed with 2% formaldehyde and 0.1% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2, at 4uC for 2–4 hours [32]. Following fixation, the cells on filters were cut free of the plastic inserts, postfixed in OsO4, contrasted en bloc with 1% uranyl acetate, dehydrated in a graded series of ethanol solutions, and embedded in epon. Sections were cut perpendicularly to the filter, collected on Formvar-coated mesh grids, and examined under a JEM-1230 electron microscope. Statistical analyses All statistical analyses were conducted using GraphPad Prism, version 4. Statistical comparisons were conducted using the 2- tailed unpaired t test. P values ,0.05 were considered significant. Unpolarized Release Enhances Mucosal Responses Results Effects of high doses of rVTT-gagpol on levels of mucosal humoral immune response after intra-nasal immunization To evaluate the mucosal immune responses associated with rVTT-gagpol, BALB/c mice were intra-nasally administered 107 PFU of rVTT-gagpol (Fig. 2). During week 6, after two immunizations four weeks apart, mice were euthanized and antibody levels were determined. In detail, the anti-p24 serum IgG titer was about 7500, the serum IgA titer was about 20, the lung wash IgA titer was about 18, the vaginal wash IgA titer was about 7, and no significant differences from the PBS group were found (Fig. 2). The anti-p24 IgG titer in lung washes was found to be higher than that of IgA (data not shown). However, IgG levels were not stable at the mucosal surface [33]. Vaginal washes were not evaluated for IgG. This showed that high-dose rVTT-gagpol immunization could induce high levels of p24-specific IgG in serum but only low IgA titers, suggesting a weak mucosal immune response. These results were similar to those reported by other groups: In one study, 56106 PFU/mouse of recombinant vaccinia virus was given to mice at 0 and 4 weeks, and these mice were then euthanized and sampled at 6 weeks [34]. The specific IgA in lung washes was shown via OD values and could be converted to about 20 titer. Another group gave its mice 26106 PFU i.n. only once and euthanized them 3 weeks later. Their specific antibody results were as follows: serum IgG 2 10061,370, serum IgA 8.064.2, bronchial wash IgA 24.0626.3 [29]. Although rVTT-gagpol is a replicating virus vaccine, it still needs high doses to induce even small mucosal immune responses after mucosal immunization. In order to find the reasons behind this and ways to enhance immune response and reduce the dose, we studied the interactions between rVTT-gagpol and polarized epithelia. Release of rVTT-gagpol and HIV antigen p24 from epithelial cells in vitro It is known that epithelial cell layers are polarized in vivo. In vitro, mucosal epithelial cell lines cultured on a permeable membrane called a transwell can become polarized monolayers. In this study, the human colonic carcinoma Caco-2 cell line was cultured and developed into polarized cell layers. rVTT-gagpol was inoculated from the apical side. Then small, local cytopathic effects (CPEs) appeared in the cell layer: Some loose connections were formed between neighboring cells but these had no great effect on the integrity of the monolayer. The infected cell monolayer was fixed and processed for electronic microscopy. p24 concentrations and rVTT viral titers in the apical, basolateral, and cell samples were determined at various time points. The polarized cell monolayer was observed under electronic microscopy (Fig. 3A). Some viral factories were present in the cell plasma, in which there were many round immature vaccinia viruses of about 200 nm in diameter (Fig. 3B). Some of the gagpol assembled HIV-virus-like particles (VLP), about 100 nm in diameter, were in the process of being transported (Fig. 3C) and some were outside of the cells’ apical surface (Fig. 3D). Mature vaccinia viruses, about 200 nm in short diameter and 400 nm in long diameter, were observed both inside the cells (not shown) and outside of the apical surface (Fig. 3D). No vaccinia viruses were seen outside the basolateral surface. The kinetics of p24 and virus distribution post-infection are shown in Figures 3E and F. The concentrations of dissociative p24 and p24 in VLP were determined using double antibody sandwich ELISA. Disassociated p24 was found to be expressed in the cells PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24296