vaccinia WR strain [21]. The National Center for AIDS/STD Control and Prevention (China) developed an AIDS vaccine candidate using DNA priming and rVV Tiantan boost with env and gag-pol of CRF07_B’/C as inserts. It has been approved by the SFDA for a phase I trials. Volunteer recruitment started in Beijing in November of 2007 [23,24]. Unfortunately, highly attenuated recombinant vaccinia viruses can induce only limited mucosal immune responses specific to foreign antigens, even at high doses. Enhancing these immune responses or reducing the necessary dose, would render the vaccine easier to make and safer for patients. Regarding obstacles to mucosal immunization, the first line of opposition that a vaccine encounters is the long-lined mucosal epithelium. There have been some reports that measles and Junin viruses infect the polarized epithelium and are released in a polarized way [25,26,27]. We performed this study to evaluate interactions between recombinant vaccinia viruses and epithelial cells and to determine whether epithelial cells released HIV antigens in a polarized way. Upon finding that they did, we performed more tests to see what influence this polarized release has on antigen delivery and specific immune responses. Here we report that recombinant HIV vaccinia virus can infect the mucosal epithelium in vitro and in BALB/c mice; both the progeny vaccinia virus and HIV antigen are preferentially released from the apical surfaces and colchicine treatment causes unpolarized antigen expression, delivery, and enhanced specific immune responses. These results provide guidance for immune enhancement and dose reduction in HIV vaccination. Materials and Methods Cell lines Caco-2 human colonic carcinoma cell line and Vero cell line were obtained from American Type Culture Collection (Rockville, MD, U.S.). Caco-2 cells were maintained in DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco), 12.6 mM HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin (Gibco, 1% P+S), and 16nonessential amino acids (MEM-NEAA, 10 mM, 100X, Gibco). Vero cells were maintained in an otherwise identical DMEM medium containing NEAA. Both were incubated at 37uC in a humidified atmosphere of 5% CO2. Mice Female BALB/c mice, 6–8 weeks old, were purchased from the Hunan SJA Laboratory Animal Co., Ltd. and maintained in the Animal Center (Wuhan Institute of Virology, Chinese Academy of Sciences) under specific pathogen-free (SPF) conditions. Female mice were chosen because previous studies have suggested that nasal priming does not work well in male mice [28]. All experiments were approved by the Wuhan Institute of Virology Experimental Animal Ethics and Welfare Committee, the permit number is A2008-019. Viral preparation rVTT-gagpol vaccinia virus was provided by Dr. Zhi-wei Chen (Director of HKU-AIDS Institute) and constructed by Dr. Ke Zhuang (College of Life Sciences, Wuhan University). The virus was homologously recombined with the gagpol gene from HIV-1 B9 and a gfp gene to replace most of the vaccinia virus HA gene. It was propagated in Vero cells. Cells were frozen and thawed three times and centrifuged at 2000 rpm for 20 minutes. Supernatants were aspirated, aliquoted, and stored at 270uC. Viruses were purified and enriched for immunization. Large- scale propagation of viruses from Vero cells and the supernatant of Unpolarized Release Enhances Mucosal Responses frozen and thawed cells was added to the 36% (m/v) sucrose solution and centrifuged at 18,000 g for 80 minutes at 4uC. The deposits were suspended, aliquoted at about 16109 PFU/ml in PBS, and stored at 270uC. The viral titer was determined by plaque assay of Vero cells. Immunization Mice were primed and boosted with 107 PFU/mouse of rVTT- gagpol (12 ml) at 0 and 4 weeks and euthanized at week 6 (Fig. 1A). For tests of viral and antigen distribution, mice in the colchicine group were i.n. inoculated with colchicine (Duchefa, 20 mg/ mouse, 1.25 mg/ml, 16 ml) 72 hours before rVTT-gagpol (107 PFU/mouse, 12 ml) immunization. In the control group, PBS replaced colchicine (Fig. 1B). Regarding specific antibody response tests, mice in the colchicine group were i.n. primed by rVTT-gagpol (107 PFU/ mouse, 12 ml) and colchicine (40 mg/mouse, 10 mg/ml, 4 ml) and then i.n. boosted with colchicine (4 mg/mouse, 1 mg/ml, 4 ml) 24 hours before immunization with rVTT-gagpol (107 PFU/ mouse, 12 ml) at week 4. They were euthanized at week 6 (Fig. 1C). I.n. immunization was performed under anesthesia by 0.06– 0.07 mg/g pentobarbital. There were 4–7 mice in each group. Collection of serum and mucosal samples Before euthanasia, vaginal washes were collected. Forty microliters of PBS was aspirated into the vaginal tract using 200-ml tips. The fluid was blown and aspirated 10 times and washed twice. Then serum was obtained. For collection of lung bronchial washes (BW), the trachea was exposed surgically and breast diaphragm was opened for subsequent lung expansion. A very small cut was made on the trachea with small surgical scissors. A 1-ml tip covered with a 10-ml tip from a 1-ml pipette was used to aspirate 500-ml PBS with 1% BSA. The apparatus was carefully inserted into the trachea with the tip toward to lung. This was blown and aspirated three times, slowly, to completely wash the whole lung and bronchia. Both vaginal washes and BW were centrifuged at 2000 rpm for 10 minutes before being stored at 270uC. Kinetics of antigen distribution in vivo Mice were immunized and euthanized 1, 12, 24, 48, 72, or 96 hours later. The collecting method was similar to that used previously [29]. Heparinized (20 U/ml) blood was collected and centrifuged at 2000 rpm. Nasal washes and lung bronchial washes were collected in 1 ml DMEM with 1% (P+S) and 10% FBS. The method of collecting bronchial washes is described as above. For nasal washes, the initial steps were the same as those used for bronchial washes, but after the small cut was made on the trachea, a 1-ml tip covered with a 10-ml tip from a 1-ml pipette was carefully inserted into trachea with the tip toward the nostril, and solution was to made to flow out of nostrils. It was washed twice. The nasal turbinate and lungs were separately homogenized in 1 ml DMEM with 1% (P+S) and 10% FBS. Washes and homogenized samples were centrifuged at 2000 rpm for 10 minutes. The vaccinia viral titer and p24 concentration were determined by plaque assay and ELISA, respectively. Immunohistochemical staining of p24 in mice After immunization, mice were bled and euthanized. The nasal turbinate and lungs were collected and fixed in 10% (v/v) formaldehyde for 6–12 hours. The decalcification of nasal turbinate was performed in cooled 5% (v/v) nitric acid in a microwave oven [30]. Decalcification solution was maintained at PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24296