Summary: Francisella tularensis, the causative agent for tularemia, can infect humans by a number of routes, including vector-borne transmission. However, it is inhalation of the bacterium, and the resulting pneumonic tularemia, that represents the most dangerous form of disease. This is due to the short incubation time (3-5 days), non-specific symptoms, and a high mortality rate (greater than 80%) in untreated individuals. Furthermore, F. tularensis has been weaponized by both the United States and the former Soviet Union making it a viable candidate for use as a biological weapon. Despite over 80 years of research on F. tularensis around the world, very little is understood about the dynamic interaction of this bacterium with the host, especially following aerosol infection. [unreadable] In the last several years my laboratory has provided abundant evidence that one of the primary mechanisms by which F. tularensis successfully infects and replicates in the host is via active suppression of the host immune response in the lungs. We have a developed a reproducible murine model in which mice intranasally infected with 10 CFU to study the dynamic changes and progress of infection. This model has revealed several important points concerning pneumonic tularemia. One of the most important observations is that, unlike more attenuated strains, virulent F. tularensis actively suppresses the host immune response, including pulmonary dendritic cells, during the first few days of infection. Although we have not identified the primary mechanism of suppression there are several host molecules that appear to be involved, including Transforming Growth Factor-beta (TGF-beta), Prostaglandin E2 (PGE2) and Vascular Endothelial Growth Factor (VEGF). More importantly is our new observation that CD14 is a critical player in both uptake and elicitation of inflammation following exposure of cells to F. tularensis. Cells that lack CD14 are still infectable, but fail to produce pro-inflammatory cytokines. Furthermore, these cells become refractory to further stimulation by other microbial components. The specific role and the mechanism in which F. tularensis and its components interacts with CD14 is currently under investigation in the laboratory.[unreadable] In addition to CD14, we have made surprising and important observations involving the host plasminogen system (PAS) and its manipulation by virulent F. tularensis. Using both in vitro and in vivo systems we have shown that F. tularensis bind plasminogen and then converts it to the active serine protease plasmin in the presence of the host enzyme urokinase plasminogen activator (uPA). These plasmin coated bacteria readily degrade immunoglobulin in vitro. Importantly, we also observed that mice lacking uPA, and thus would not allow formation of plasmin coated bacteria, readily control F. tularensis infection, have higher numbers of B cells in specific target organs and develop F. tularensis specific IgG. Together these data provide both important insight into the role of the host PAS during Tularemia infections as well as important new understanding of how uPA might control B cell development and proliferation.[unreadable] One elusive goal in combating pneumonic tularemia is the development of effective non-antibiotic based therapeutics that can provide protection shortly before or after infection. Thus, the overall goal of this project is to develop an easily administrated therapeutic that could be quickly distributed following a natural outbreak of terror event. [unreadable] Previous reports have suggested that appropriate immunogens may be able to overcome the immunosuppression invoked by Francisella and enable the host to effectively eradicate the bacterium. For example, LPS purified from the attenuated F. tularensis Live Vaccine Strain (LVS) injected 3 days prior to a lethal LVS challenge protects all infected animals. A similar phenomenon has been observed following injections of CpG nucleic acid motifs or cationic-lipid DNA complexes (CLDC). While it has been previously shown that LVS LPS does not protect against infections with fully virulent F. tularensis, it was not known if CpG or CLDC can engender protective immunity against similar strains. Over the past year we have shown that CLDC alone does not protect against infection with virulent F. tularensis. However, when crude membrane protein preparations from LVS are combined with CLDC prior to administration to mice greater than 75% of mice are protected from lethal F. tularensis infection. In collaboration with Juvaris Biotherapeutics, we are identifying the specific mechanism(s) by which CLDC+MPF controls F. tualrensis infections in vitro and in vivo. Also, these data have resulted in the filing of a provisional patent in which I am the primary inventor of CLDC+MPF as a therapeutic. [unreadable] In addition to understanding the way in which F. tularensis manipulates the host innate immune response we are investigating host components required for development of a protective adaptive response. To date, the only vaccine available (although not licensed in the United States) is an attenuated, Type B strain of F. tularensis known as Live Vaccine Strain or LVS. However, there are a number of problems associated in the use of this vaccine including an unpredictable phase shift in its LPS which renders the bacterium completely ineffective against pneumonic tularemia. In collaboration with Dr. John Belisle (Colorado State University) we are testing acellular vaccines derived from LVS. Using crude sub-cellular fractions we have been able to generate protection against low dose aerosols nearly equivalent as that observed in animals vaccinated with LVS. Furthermore, we are currently identifying correlates of immunity for survival of pneumonic tularemia using these vaccines combined in adjuvants designed to skew the immune response in a polarized fashion. This approach will allow us to identify specific requirements in the host without the complications of using genetically modified mice.[unreadable] One of the most challenging aspects of pneumonic tularemia is the very low dose (10 CFU) inoculum associated with lethal disease. This low number of bacteria make it nearly impossible to adequately address early interactions of bacteria with host cells during the first few days of infection. To gain a better understanding of how F. tularensis might modulate early immune responses we developed an intradermal model of Tularemia in which bacteria are injected directly into the ear pinna of mice. This site of infection allows us to more accurately assess which cell types are directly infected with the bacterium, what host components are required for dissemination and how the bacteria modulates the immune response to cause lethal disease. While this injection site is not the same as the pulmonary environment it will provide us with important clues as to how the host interacts with the bacterium on a cellular level during the first 48 hours after infection. These studies will eventually enable us to better test and examine events in the pulmonary environment.