colchicine to increase its effectiveness. We showed that colchicine acts by disrupting polarized release in vitro (Caco-2 cell layers) and in immunized mice (Figs. 5 and 6). In vitro, the 3-mm pore in the transwell membrane was used to make sure that rVTT was released only from the basolateral surface. The 0.4-mm transwell pore was able to block basolateral release, showing only 1/103 as many basolaterally released viruses as samples equipped with 3-mm pores. The release of p24 antigen was not significantly different (data not shown). This implies that, due to the large size of the vaccinia virus, it passed through 3-mm transwell pores more easily than through 0.4-mm pores. In mice, colchicine treatment increased viral infection and expression of p24 in the nose, nasal washes, and lamina propria (Fig. 6) with some damage to the epithelium. This is consistent with the situation in vitro, in which cell layers turned out some plaques but maintained the integrity of the epithelial layer relatively well. One explanation of how colchicine treatment was able to increase the rates of infection and expression may be this: Colchicine influenced dissemination between cells during the second period of infection. During rVTT-gagpol infection of the epithelial cells, progeny viruses were released from the apical surface, from which they infected neighboring cells, again via the apical surface (Fig. 8A). Because transportation relies on microtubules, depolymerization of those microtubules by colchi- cinie caused some vesicles to target the basolateral surface (Fig. 8B) [36]. The basolaterally targeted vesicles fused with the upper two thirds of the lateral surface, not just the region immediately beneath the tight junction [36]. In addition, vaccinia virus more readily infects differentiated human airway epithelia from the basolateral surface than from the apical surface [38]. In this way, colchicine treatment rendered infected cells much more likely to infect other cells. Our results demonstrated that the unpolarized release of virus and antigen increased specific antibody responses. Other studies have described the impact of colchicine on immune responses. Colchicine can act as an immune suppressor at high doses (100 mg/ml) and an immune enhancer at low doses (0.5–1 mg/ml) [44]. It can increase immune responses when co-administered with antigens, enhancing immune response by about a factor of two [44]. In this study, the doses were 80 mg/ml in vitro and 4–40 mg/ Unpolarized Release Enhances Mucosal Responses mouse (1000–10,000 mg/ml). Previous reports indicate that both would result in immune suppression. We found that colchicine does not act as an immune enhancer under these conditions. In addition, immune enhancers can more easily enhance specific immune responses to protein antigens than to live viral vectors. This is because live viral vectors activate the natural immune response on their own. In our research, the unpolarized release of antigen was clear (Figs. 5 and 6) and the immune effect was more intense and widespread (Fig. 7). The increase in specific antibody responses were mainly due to the unpolarized release and not to the slightly nonspecific enhancement of immune responses. In this study, anti-p24 gag antibodies served as a representatives of the mucosal humoral response. Although they cannot display traditional neutralization, they are very important to HIV vaccines. First, p24-specific IgA neutralizes HIV in cells intracellularly. This was proven in measles by one member of our team (unpublished data). She found that viral replication could be inhibited by binding of the internal protein M of measles by specific IgA in epithelial cells. This phenomenon was named intracellular neutralizing. Second, the p24 gag genes are relatively conservative, so their specific IgA is able to target more than one subtype of HIV. That of env cannot because its genes mutate readily. This study explored the interactions between rVTT-gagpol and mucosal epithelial cell layers and their impact on antigen delivery and specific immune responses. Results demonstrate that rVTT- gagpol can infect epithelial cells to express p24 and that progeny virus and p24 are mainly released from the apical surface and both in vitro and in mice. The characteristic polarized distribution restricts the delivery of antigen and thus specific immune responses. To improve upon this, colchicine was use to depolarize distribution. This facilitated antigen expression, delivery, and specific immune responses. It has been reported before that polarized release of virus can restrict infection [45], but no research has yet been performed on the relationship between polarized release, antigen delivery, and specific immune responses. These results provide an explanation for the low efficiency of rVTT-gagpol-induced mucosal immunity and supply some guidance for vaccine design with an eye toward increasing immune responses and decreasing dose. First, researchers may Figure 8. rVTT-gagpol released from (A) the apical surface and (B) the basolateral surface (referenced and modified from [35]). doi:10.1371/journal.pone.0024296.g008 PLoS ONE | www.plosone.org 12 September 2011 | Volume 6 | Issue 9 | e24296