Unpolarized Release Enhances Mucosal Responses Figure 7. Comparison of anti-p24 humoral immune responses between untreated and colchicine-treated rVTT-gagpol immunization groups in mice. The colchicine-treated group consisted of five 6-week-old female BALB/c mice that were given i.n. rVTT-gagpol (107PFU) and colchicine (40 mg) at the same time. During week 4, mice were given colchicine boosters (4 mg) 24 hours before rVTT-gagpol. The mice were euthanized during week 6. The rVTT-gagpol immunization group was given identical treatment except that PBS was used as a substitute for colchicine. Serum and mucosal samples were collected to test for the presence of anti-p24 antibody by ELISA. Anti-p24 levels of (A) serum IgG, (B) serum IgA, bronchial wash IgA, and (C) vaginal wash IgA are displayed. T-testing was conducted between the colchicine-treated group and the rVTT- gagpol immunization group. Statistical significance (P,0.05 in an unpaired test) is indicated by an asterisk. Two independent experiments were conducted with similar results. One set of results is shown. doi:10.1371/journal.pone.0024296.g007 blood (unpublished data). Other groups found similar results. Recombinant vaccinia virus has been shown to replicate in the nose and lungs after i.n. inoculation without any virus detectable in the blood. However, after i.p. inoculation, some virus was detected in the blood on days 1–4 [29]. Another study focused on the activity of luciferase in various mouse tissues after immuniza- tion with recombinant MVA through intranasal (i.n.), intravaginal (i.v.), and intrarectal (i.r.) routes. After i.n. immunization, luciferase activity was detected only in the lungs, NALT, and lung-draining lymph nodes but not in other tissues. In an i.r. immunization model, activity was not detected in the rectum, rectal lymph node, or other tissues. Similarly, after i.v. immuni- zation, no activity was detected in the ovary or vaginal lymph node. Activity in the vagina lumen was not tested [41]. In contrast to the vaccinia virus, the vesicular stomatitis virus (VSV) has been found to be released from the basolateral surface after MDCK infection in vitro [42]. After VSV type New Jersey (VSV-NJ) was i.n. inoculated in deer mice, infection of the olfactory epithelium took place on the day 1 and a great deal more virus was observed in the lamina propria on the day 4. Of the juvenile and adult deer mice studied, 49% developed virusemia on days 1, 2, or 3 [43]. These results imply that VSV infects the epithelium first and then spreads to the lamina propria and blood. Unlike VSV, vaccinia viruses are mainly released to the lumen, and it is difficult for them to enter the blood through the lamina propria. Because the polarized release of rVTT-gagpol and p24 antigen limits antigen delivery and specific immune responses, we used PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e24296