Intranasal Immunization with an Archaeal Lipid Mucosal Vaccine Adjuvant and Delivery Formulation Protects against a Respiratory Pathogen Challenge

Archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) is a safe mucosal adjuvant that elicits long lasting and memory boostable mucosal and systemic immune responses to model antigens such as ovalbumin. In this study, we evaluated the potential of the AMVAD system for eliciting protective immunity against mucosal bacterial infections, using a mouse model of intranasal Francisella tularensis LVS (LVS) challenge. Intranasal immunization of mice with cell free extract of LVS (LVSCE) adjuvanted with the AMVAD system (LVSCE/AMVAD) induced F. tularensis-specific antibody responses in sera and bronchoalveolar lavage fluids, as well as antigen-specific splenocyte proliferation and IL-17 production. More importantly, the AMVAD vaccine partially protected the mice against a lethal intranasal challenge with LVS. Compared to LVSCE immunized and naïve mice, the LVSCE/AMVAD immunized mice showed substantial to significant reduction in pathogen burdens in the lungs and spleens, reduced serum and pulmonary levels of proinflammatory cytokines/chemokines, and longer mean time to death as well as significantly higher survival rates (p<0.05). These results suggest that the AMVAD system is a promising mucosal adjuvant and vaccine delivery technology, and should be explored further for its applications in combating mucosal infectious diseases

CD9 Tetraspanin Interacts with CD36 on the Surface of Macrophages: A Possible Regulatory Influence on Uptake of Oxidized Low Density Lipoprotein

CD36 is a type 2 scavenger receptor with multiple functions. CD36 binding to oxidized LDL triggers signaling cascades that are required for macrophage foam cell formation, but the mechanisms by which CD36 signals remain incompletely understood. Mass spectrometry analysis of anti-CD36 immuno-precipitates from macrophages identified the tetraspanin CD9 as a CD36 interacting protein. Western blot showed that CD9 was precipitated from mouse macrophages by anti-CD36 monoclonal antibody and CD36 was likewise precipitated by anti-CD9, confirming the mass spectrometry results. Macrophages from cd36 null mice were used to demonstrate specificity. Membrane associations of the two proteins on intact cells was analyzed by confocal immunofluorescence microscopy and by a novel cross linking assay that detects proteins in close proximity (<40 nm). Functional significance was determined by assessing lipid accumulation, foam cell formation and JNK activation in wt, cd9 null and cd36 null macrophages exposed to oxLDL. OxLDL uptake, lipid accumulation, foam cell formation, and JNK phosphorylation were partially impaired in cd9 null macrophages. The present study demonstrates that CD9 associates with CD36 on the macrophage surface and may participate in macrophage signaling in response to oxidized LDL

Generation of a Convalescent Model of Virulent Francisella tularensis Infection for Assessment of Host Requirements for Survival of Tularemia

Francisella tularensis is a facultative intracellular bacterium and the causative agent of tularemia. Development of novel vaccines and therapeutics for tularemia has been hampered by the lack of understanding of which immune components are required to survive infection. Defining these requirements for protection against virulent F. tularensis, such as strain SchuS4, has been difficult since experimentally infected animals typically die within 5 days after exposure to as few as 10 bacteria. Such a short mean time to death typically precludes development, and therefore assessment, of immune responses directed against virulent F. tularensis. To enable identification of the components of the immune system that are required for survival of virulent F. tularensis, we developed a convalescent model of tularemia in C57Bl/6 mice using low dose antibiotic therapy in which the host immune response is ultimately responsible for clearance of the bacterium. Using this model we demonstrate αβTCR+ cells, γδTCR+ cells, and B cells are necessary to survive primary SchuS4 infection. Analysis of mice deficient in specific soluble mediators shows that IL-12p40 and IL-12p35 are essential for survival of SchuS4 infection. We also show that IFN-γ is required for survival of SchuS4 infection since mice lacking IFN-γR succumb to disease during the course of antibiotic therapy. Finally, we found that both CD4+ and CD8+ cells are the primary producers of IFN-γand that γδTCR+ cells and NK cells make a minimal contribution toward production of this cytokine throughout infection. Together these data provide a novel model that identifies key cells and cytokines required for survival or exacerbation of infection with virulent F. tularensis and provides evidence that this model will be a useful tool for better understanding the dynamics of tularemia infection