Unpolarized Release Enhances Mucosal Responses Figure 6. Comparison of p24 and virus distribution between untreated and colchicine-treated rVTT-gagpol immunization groups in mice. The colchicine-treated group included 6-week-old female BALB/c mice that were i.n. inoculated with colchicine (20 mg) 72 hours before rVTT- gagpol immunization (16107 PFU). The rVTT-gagpol immunization group was given identical treatment except that PBS was used as a substitute for colchicine. Infection and expression kinetics in nose samples, nasal washes, lung samples, and lung bronchial washes were evaluated (A, B, C, and D). Immunoperoxidase staining of p24 antigen in the nose (E, F, G, and H). In detail, p24 distribution in (A) nose samples and nasal washes and (C) lung samples and lung bronchial washes. Virus distribution in (B) nose samples and nasal washes and (D) lung samples and lung bronchial washes. Nose sections of the rVTT-gagpol immunization group after (E) 24 hours and (G) 72 hours. Nose sections of the colchicine-treated rVTT-gagpol immunization group after (F) 24 hours and (H) 72 hours. (x) designates epithelium, ( ) designates lamina propria, and ( ) designates lumen. There were five mice in each group and the images shown are typical of those observed. Magnification, 1006. Two independent experiments were conducted with similar results. One set of results is shown. doi:10.1371/journal.pone.0024296.g006 To ascertain the toxicity and reversibility of the effects of colchicine, we measured body weight and observed the condition of mice every other day after immunization. Body weight decreased after immunization but was restored about 10 days later (data not shown). The general condition of mice, as evaluated through coat smoothness and activity level, corresponded with changes in body weight. In conclusion, colchicine treatment was found to depolarize the release of vaccinia virus and p24 both in vitro and in immunized mice. In mice, it also increased the level of both. Our next task was to examine specific immune responses. Effects of unpolarized release of p24 and rVTT-gagpol in the nasal epithelium on mucosal and systematic immune responses We established a colchicine treatment group to determine the influence of colchicine treatment on specific immune responses. Mice were i.n. administered rVTT-gagpol (107 PFU) and colchicine (40 mg) at the same time. During the week 4, the mice were inoculated with colchicine (4 mg) 24 hours before rVTT- gagpol inoculation. PBS was used as a substitute for colchicine in the control group. During week 6, mice were euthanized and blood and mucosal samples were collected. In the colchicine group, anti-p24 immune responses were significantly stronger than in the control group (Fig. 7). The serum IgG titer was about 5 times that of the controls. The serum IgA was about 5 times higher. The lung bronchial wash IgA titer was about 10 times higher. The vaginal wash IgA titer was about 3 times higher. Generally, the antibody titers increased depending on the location of induction. The mucosal level of IgA increased and systematic effects on IgA and IgG levels were observed. Colchicine (20 mg/mouse and 4 mg/mouse) was given to mice to determine its effects on immune responses. We found that the influence on antibody response at 20 mg was similar to that at 40 mg, but 4-mg doses had no significant effects on anything but serum IgA titer, which showed a lower level of increase than that seen at higher doses (data not shown). The conditions of colchicine treatment for detection of antigen distribution differed slightly from those used for evaluation of immune responses. Although we used the same treatment procedure and noted enlarged corre- sponding immune responses, the results were not stably repro- ducible. The results shown in Figure 7, however, were both reproducible and consistent with the idea that colchicine enhances immune response. There are many factors that might have influenced the immune responses at some point between antigen distribution and immune response. These data suggest that the unpolarized release of vaccinia virus and antigens renders p24-specific antibody responses more intense. Discussion The purpose of the present study was to evaluate the interactions between HIV recombinant vaccinia virus (rVTT-gagpol) and the mucosal epithelium and the relationship between antigen distribu- tion and specific immune responses. Even high doses of most vaccinia virus vaccine candidates cannot induce strong mucosal IgA responses, which are related to protection from viral challenge [29]. Other studies have been performed with doses of rVTT ranging from 26106 to 56106 PFU/mouse [29,34,37]. Only low specific antibody titers were produced [29,34]. Large doses are harder to prepare and can cause side effects. However, i.n. administration of recombinant vaccinia virus alongside HN or F was found to protect mice from challenge by Sendai virus via the respiratory duct [29]. When inoculated via either intranasal or intraoral routes, recombinant vaccinia virus with S protein from SARS-CoV induced 20–100 times the level of Nab in serum than that induced by rMVA [22]. A means to increase the efficiency and to reduce dose size would be useful to researchers, doctors, and patients interested in this vaccine. In vitro experiments in which rVTT immunization was performed via the mucosa, demonstrated that rVTT-gagpol could infect polarized Caco-2 epithelial layers and that progeny virus and expressed p24 were released mainly from the apical surface (Fig. 3). Vermeer et al. also reported that the vaccinia virus infected differentiated human airway epithelia and was released mainly from the apical surface in vitro [38]. Huang et al. reported that SIV budded basolaterally in polarized Vero C1008 cells [39]. Randall et al. reported that, when polarized Vero-pFN cells were infected with VVgag, similar amounts of p24 capsid protein were released into virus particles at the apical and at the basolateral surface. In contrast, when cells are infected with both VVgag and VVenv, 94% of p24 and all gpl20 are associated with particles released into the basolateral medium [40]. These results indicate that the HIV-1 envelope glycoprotein renders virus particles containing the gag protein to be released from the basolateral surface [40]. However, after we co-infected polarized Caco-2 cells with rVTT-gagpol and rVTT-env via the apical surface, no increased associative p24 or VLP p24 was detected in the basolateral medium. We also infected polarized Vero C1008 cells with rVTT-gagpol via the apical surface. The integrity of the cell layers deteriorated 4–12 hours after infection depending on the MOI (data not shown). Before the cell layers broke down, p24 and vaccinia virus were mainly released from the apical surface. After that, more p24 and virus was present in the basolateral medium than in the apical. In contrast, Caco-2 cell layers maintained their integrity after infection: p24 and virus were still released mainly from the apical surface. The reason for this might be that Caco-2 cells are from the epithelial cells of the colon, which is mucosal tissue, while Vero cells are derived from the epithelial cells of kidney. This makes Caco-2 cells more representative of mucosal epithelia in vivo than Vero cells. We verified the interaction in immunized mice. rVTT-gagpol was able to infect respiratory systems, though no viruses or p24 could be detected in the blood (Fig. 4). When mice were immunized with rVTT-gagpol via the vagina, the virus and p24 were restricted to the vaginal system and could not be detected in PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e24296