Unpolarized Release Enhances Mucosal Responses Figure 2. Humoral immune responses to high doses of rVTT-gagpol after i.n. inoculation. Mice were i.n. inoculated with rVTT-gagpol at weeks 0 and 4 and euthanized during week 6. Sera, lung washes, and vaginal washes taken during week 6 were collected to ascertain anti-p24 IgG and IgA by ELISA. There were seven mice in each group. Anti-p24 (A) serum IgG, (B) serum IgA, lung wash IgA, and (C) vaginal wash IgA are displayed above. Three independent experiments were conducted with similar results. The results of one representative experiment are shown. doi:10.1371/journal.pone.0024296.g002 and released from the apical but not the basolateral surface. The p24 in VLP was evaluated using 1% TritonX-100. Results suggested that increased levels of cellular and apical p24 were released from the apical surface as well. Basolateral VLP p24 still could not be detected. For total p24, about 10 ng/well was found to be present in the cells, about 1–8 ng/well was present in the apical medium, and none was detected in the basolateral medium. Viral titer was determined by plaque assay. The virus was found to replicate at about 106 PFU/well in cells and was released from the apical surface at about 104–105PFU/well. This is about 1000 times the level in the basolateral medium. Even when the medium was changed every 48 hours, the kinetics showed similar trends (data not shown). After rVTT-gagpol infection, polarized Caco-2 cell layers assembled progeny vaccinia viruses, expressed HIV genes, and released viral products from the apical surface. Whether the situation would be the same in vivo and what impact this preferential release might have on specific immune responses remained to be determined. To this end, we studied the interactions between rVTT-gagpol and the epithelium in BALB/c mice. Locations of rVTT-gagpol replication and HIV antigen expression in nasally immunized mice To identify the amount and location of HIV antigen, mice were i.n. immunized with rVTT-gagpol and euthanized 1, 10, 24, 48, 72, or 96 hours later. Then plasma, nasal washes, lung bronchial washes, and homogenized nose and lung tissue samples were collected. Levels of virus and p24 in these samples were detected by plaque assay and ELISA, respectively. Results showed that after mice were i.n. inoculated, the virus replicated and p24 was expressed in respiratory system but not in the blood (Figs. 4A, B, C, D, E, and F). Viruses entered the nose tissue. Viral levels decreased to their lowest point (102 PFU) at about 10 hours and then increased through the first and second days, reaching a peak at 72 hours (about 56106 PFU). At the same time, viruses were released into the lumen of the nose, where viral levels reached their lowest point (about 8 PFU) at 24 hours and their highest point (about 103 PFU) at 72 hours (Fig. 4B). p24 expression and distribution took on similar trends. The largest amount of expression was achieved, about 70 ng per mouse, at 72 hours (Fig. 4A). Viruses were able to reach the lungs when the volume of i.n. administration exceeded 10 ul. Then the viruses replicated and p24 was expressed in the lungs (Figs. 4D and C). The lowest virus and p24 levels were detected at 24 hours and the highest at 72 hours. Viral titers were found to reach 106 PFU, lower than that in nose tissue. For p24, the amount reached 30 ng. If the volume of i.n. administration was 8 ul, fewer viruses reached the lungs and peak viral titer only reached 105 PFU per mouse (data not shown). However, neither virus nor p24 was detected in the blood (Figs. 4E and F). This shows that infection and expression were restricted to the respiratory system. In order to determine the exact location of p24 antigen in the nose and lungs, we performed immunoperoxidase staining of p24 PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24296