Unpolarized Release Enhances Mucosal Responses Figure 3. Infection, expression, and distribution of rVTT-gagpol in polarized Caco-2 monolayer in vitro. Sixteen days after polarization, Caco-2 cell monolayers were infected by rVTT-gagpol at MOI 1 from the apical surface. At various times, monolayers were fixed and prepared for electronic microscopy observation, as shown in images (A), (B), (C), and (D). For infection and distribution kinetics, the p24 content and virus titers of the samples were determined by ELISA and plaque assay, respectively, as shown in images (E) and (F). In detail: (A) Caco-2 monolayer on filter at 48 hours post infection (scale bar: 2 mm). (B) Immature vaccinia virus (x) at 72 hours post infection (scale bar: 1 mm). (C) HIV VLP ( ) about100 nm in diameter at 72 hours post infection (scale bar: 0.5 mm). (D) HIV VLP ( ) and mature vaccinia virus ( ) about 200 nm in short diameter and about 400 nm in long diameter released from apical surface (scale bar: 0.5 mm). (E) p24 distribution. Treated samples were treated with 1% TritonX- 100 before testing to release the internal p24 of VLP. (F) rVTT virus distribution. Kinetics experiments were performed in triplicate, three for each noted point in time. Each electron microscopy observation was performed twice. doi:10.1371/journal.pone.0024296.g003 antigen on mice inoculated with rVTT-gagpol (Figs. 4G, H, I, and J). At 24 hours, there was a little p24 in the epithelium of nose (Fig. 4G). At 72 hours, there was more p24 but large amounts remained in the epithelium and only a small amount was found in lamina propria (Fig. 4H). In the lungs, little p24 was observed at 24 hours (image not shown). A certain amount of p24 was observed in the epithelia of the lungs at 72 hours and some was delivered to the alveolar cells (Fig. 4J). The p24 immunohistochemistry results were considered representative of viral distribution. rVTT-gagpol was found capable of infecting the epithelial cells of the nose and lungs. Progeny virus and p24 were constantly released into the lumen, though only a little reached the lamina propria. These results correspond with those of our in vitro experiments. Viral infection and expression were detected in the respiratory system but not in the blood. As far as a vaccine development is concerned, it was expected that antigen would be delivered primarily into the whole body but not outside it. The limited efficiency of antigen delivery was found to be due to polarized distribution in the epithelium. This also suggests that the polarized release is a barrier to the induction of an efficient immune response. One approach to improve the situation would be to find a way to induce unpolarized release of the virus and HIV antigen. Effects of colchicine on unpolarized distribution of rVTT and HIV antigen in vitro and in mice Polarized budding relies upon the cytoskeleton [26,35]. When its microtubules are depolymerized, proteins that ordinarily target the apical surface can end up elsewhere [36]. Colchicine, a drug that inhibits microtubule polymerization, can cause unpolarized release of the Junin virus in polarized Vero cells [26]. We used colchicine to inhibit polarized release as follows: After Caco-2 cells were allowed to polarize for 14–16 days, colchicine was added apically 72 hours before rVTT-gagpol infection. This group is here called the polarized + colchicine group. Another polarized group was infected with rVTT-gagpol only. The unpolarized groups consisted of infected, unpolarized cells. Caco-2 cells were cultured in transwell for 3 days, and rVTT-gagpol was then inoculated. The data presented in Figure 5 were calculated as the sum of that detected at 48 hours and 72 hours. Compared to the polarized-only group, the unpolarized and polarized + colchicine groups showed relatively unpolarized distributions of p24 and virus (Fig. 5). In the unpolarized group, the total amount of virus was significantly higher than in the polarized group. Progeny viruses were released from both the apical and basolateral surfaces (Fig. 5B). In the colchicine-treated group, polarized budding was disturbed. The trend of distribution was similar to that of the unpolarized group. Compared to the polarized group, the amount of virus in the cells was higher, the amount in the basolateral medium was 20 times higher, and the amount in the apical medium was lower (Fig. 5B). p24 distribution showed a similar trend. In the colchicine-treated group, TER decreased in the 3 mm transwell (Fig. 5C) but increased in the 0.4 mm transwell at low concentrations (0.08, 0.4 mg/ml). It decreased at high concentrations (2 mg/ml) (data not shown). In both the 3 mm (Figs. 5A, B) and 0.4 mm transwells (data not PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24296