Bronchus-Associated Lymphoid Tissue (BALT) and Survival in a Vaccine Mouse Model of Tularemia Damiana Chiavolini1, Javier Rangel-Moreno 2 , Gretchen Berg1, Kate Christian1, Laura Oliveira- Nascimento1,3, Susan Weir1, Joseph Alroy4, Troy D. Randall2, Lee M. Wetzler1* 1 Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, United States of America, 2 Division of Allergy, Immunology and Rheumatology, University of Rochester Medical School, Rochester, New York, United States of America, 3 School of Pharmaceutical Sciences, University of Sa˜o Paulo, Sa˜o Paulo, Brazil, 4 Department of Pathology, Tufts University School of Medicine, Cummings School of Veterinary Medicine and Tufts Medical Center, Boston, Massachusetts, United States of America Abstract Background: Francisella tularensis causes severe pulmonary disease, and nasal vaccination could be the ideal measure to effectively prevent it. Nevertheless, the efficacy of this type of vaccine is influenced by the lack of an effective mucosal adjuvant. Methodology/Principal Findings: Mice were immunized via the nasal route with lipopolysaccharide isolated from F. tularensis and neisserial recombinant PorB as an adjuvant candidate. Then, mice were challenged via the same route with the F. tularensis attenuated live vaccine strain (LVS). Mouse survival and analysis of a number of immune parameters were conducted following intranasal challenge. Vaccination induced a systemic antibody response and 70% of mice were protected from challenge as showed by their improved survival and weight regain. Lungs from mice recovering from infection presented prominent lymphoid aggregates in peribronchial and perivascular areas, consistent with the location of bronchus-associated lymphoid tissue (BALT). BALT areas contained proliferating B and T cells, germinal centers, T cell infiltrates, dendritic cells (DCs). We also observed local production of antibody generating cells and homeostatic chemokines in BALT areas. Conclusions: These data indicate that PorB might be an optimal adjuvant candidate for improving the protective effect of F. tularensis antigens. The presence of BALT induced after intranasal challenge in vaccinated mice might play a role in regulation of local immunity and long-term protection, but more work is needed to elucidate mechanisms that lead to its formation. Citation: Chiavolini D, Rangel-Moreno J, Berg G, Christian K, Oliveira-Nascimento L, et al. (2010) Bronchus-Associated Lymphoid Tissue (BALT) and Survival in a Vaccine Mouse Model of Tularemia. PLoS ONE 5(6): e11156. doi:10.1371/journal.pone.0011156 Editor: Samithamby Jeyaseelan, Louisiana State University, United States of America Received March 21, 2010; Accepted May 27, 2010; Published June 16, 2010 Copyright: 2010 Chiavolini 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 supported by National Institutes of Health/National Institute of Allergy and Infectious Diseases grant U19 AI056543. Laura de Oliveira Nascimento had financial support from CAPES Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de N´ıvel Superior and CNPq - Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnolo´ gico. 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: lwetzler@bu.edu Introduction Francisella tularensis is a gram-negative bacterium, and the cause of a severe pneumonic disease known as tularemia. Although the number of cases of respiratory tularemia is relatively low worldwide, the potential for using this organism as a biological weapon has encouraged the search for an effective vaccine. Nasal immunization is a promising alternative to classical parenteral vaccination, because it is non-invasive and capable of eliciting both systemic and local immune responses. In addition, this vaccination route is known to be more immunogenic at the mucosal level than the oral and vaginal routes [1,2]. Another advantage is that it requires smaller amounts of antigen to induce an optimal immune response [3,4]. Nevertheless, the development of mucosal vaccines is generally limited by the lack of effective mucosal adjuvants [5,6]. With regard to intranasal vaccines against tularemia, live organisms have mostly been tested via this route, conferring variable levels of protection against challenge with virulent Francisella. Examples of these immunogens are LVS, attenuated Francisella novicida strains and mutants of the virulent SchuS4 strain [7–10]. Although the live vaccine strain (LVS) of F. tularensis derived from a virulent type B strain has been used for vaccination, it is no longer approved for human use because the basis for its attenuation still remain obscure [11]. Attractive and safe alternatives to substitute live organisms are subunit vaccines, though their use against tularemia has not been fully investigated. Even more attractive could be the use of subunit vaccines for nasal immunization, to induce mucosal protection against tularemia in a safer and potentially more effective way, although this approach has not been widely explored. Our group has previously shown that lipopolysaccharide (LPS) from F. tularensis in combination with porin B (PorB) purified from Neisseria meningitidis elicited 70% protection from bacterial challenge, when given subcutaneously [12]. Other groups have reported that mice immunized with F. PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e11156