Unpolarized Release of Vaccinia Virus and HIV Antigen by Colchicine Treatment Enhances Intranasal HIV Antigen Expression and Mucosal Humoral Responses Yan Zhang1, Jingyi Yang 1 , Rong Bao 1 , Yaoqing Chen 2 , Dihan Zhou 1 , Benxia He 1 , Maohua Zhong 1 , Yaoming Li1, Fang Liu1, Qiaoli Li1, Yi Yang 1 , Chen Han 1 , Ying Sun 1 , Yuan Cao 1 , Huimin Yan1,2* 1 Mucosal Immunity Research Group, the State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China, 2 The State Key Laboratory of Virology and Modern Virology Research Center, College of Life Sciences, Wuhan University, Wuhan, China Abstract The induction of a strong mucosal immune response is essential to building successful HIV vaccines. Highly attenuated recombinant HIV vaccinia virus can be administered mucosally, but even high doses of immunization have been found unable to induce strong mucosal antibody responses. In order to solve this problem, we studied the interactions of recombinant HIV vaccinia virus Tiantan strain (rVTT-gagpol) in mucosal epithelial cells (specifically Caco-2 cell layers) and in BALB/c mice. We evaluated the impact of this virus on HIV antigen delivery and specific immune responses. The results demonstrated that rVTT-gagpol was able to infect Caco-2 cell layers and both the nasal and lung epithelia in BALB/c mice. The progeny viruses and expressed p24 were released mainly from apical surfaces. In BALB/c mice, the infection was limited to the respiratory system and was not observed in the blood. This showed that polarized distribution limited antigen delivery into the whole body and thus limited immune response. To see if this could be improved upon, we stimulated unpolarized budding of the virus and HIV antigens by treating both Caco-2 cells and BALB/c mice with colchicine. We found that, in BALB/c mice, the degree of infection and antigen expression in the epithelia went up. As a result, specific immune responses increased correspondingly. Together, these data suggest that polarized budding limits antigen delivery and immune responses, but unpolarized distribution can increase antigen expression and delivery and thus enhance specific immune responses. This conclusion can be used to optimize mucosal HIV vaccine strategies. Citation: Zhang Y, Yang J, Bao R, Chen Y, Zhou D, et al. (2011) Unpolarized Release of Vaccinia Virus and HIV Antigen by Colchicine Treatment Enhances Intranasal HIV Antigen Expression and Mucosal Humoral Responses. PLoS ONE 6(9): e24296. doi:10.1371/journal.pone.0024296 Editor: Christian Scho¨ nbach, Kyushu Institute of Technology, Japan Received May 18, 2011; Accepted August 4, 2011; Published September 15, 2011 Copyright: 2011 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was financially supported by grants from the National Natural Science Foundation of China (Nos. 30471584 and 30670097) and the Ministry of Science and Technology of the People’s Republic of China (973 Program, Nos. 2005CB522903 and 2012CB518904; the National Key R&D Program, No. 2007BAI28B04; and the National S&T Major Project on Major Infectious Diseases, Nos. 2008ZX10001-010, 2008ZX10001-015, 2012ZX10001-008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hmyan@wh.iov.cn Introduction AIDS (acquired immune deficiency syndrome) remains the great unconquered threat to global public health. An effective HIV vaccine is urgently needed. Ninety percent of AIDS cases are transmitted through mucosal routes, such as sexual contact [1]. Targeting immune responses to the site of viral entry can protect the body and help clear the viral reservoir before HIV dissemination [2,3]. There is evidence that HIV-specific S-IgA could neutralize HIV in the cervicovaginal lavage fluids of uninfected partners of highly exposed persons [4,5,6,7,8]. The SIV-specific CTLs, which are induced by immunization with an attenuated SHIV, were found to be associated with protection against vaginal challenge in rhesus macaques [9]. Most researchers believe that a vaccine against HIV-1 would need to induce a neutralizing antibody, CD4+ Th, and competent CD8+ CTL responses at the initial site of viral entry. Systemic vaccination is not sufficient to generate effective compartmentalized mucosal immunity [2,10]. However, inducing robust mucosal immune responses is difficult. Several factors contribute to successful mucosal immunity [11,12,13]. The key factors are as follows: (i) effective delivery of the antigen to the mucosal immune induction site; (ii) enhance- ment of the mucosal immune responses via safe mucosal adjuvants; (iii) regimen and route of immunization that induce protective responses at the desired mucosal site, preferably systemically; and (iv) adequate vaccine formulation [14]. The vaccinia virus (VV) has been used as a smallpox vaccine. It helped eradicate smallpox worldwide. As a live vaccine vector, it was used for the prevention and eradication of many infectious diseases due to its broad host range, large foreign DNA capacity, lack of carcinogenicity, and ability to induce the expression of foreign antigens in eukaryotic cells [15,16,17]. The recombinant wild vaccinia virus is generally avoided for safety reasons, particularly with regard to young children and immune-compro- mised individuals [18,19]. As a result, in China, Chen and his colleges developed a series of highly attenuated vaccinia virus Tiantan strains by decreasing the neurotoxicity of the virus [20,21,22]. Preliminary experiments demonstrated that the highly attenuated vaccinia virus could induce both humoral and cell- mediated immune responses [20]. One intranasal immunization of this virus protected animals from challenge with the pathogenic PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24296