Genetic engineering of Francisella tularensis LVS for use as a novel live vaccine platform against Pseudomonas aeruginosa infections.

Francisella tularensis LVS (Live Vaccine Strain) is an attenuated bacterium that has been used as a live vaccine. Patients immunized with this organism show a very long-term memory response (over 30 years post vaccination) evidenced by the presence of indicators of robust cell-mediated immunity. Because F. tularensis LVS is such a potent vaccine, we hypothesized that this organism would be an effective vaccine platform. First, we sought to determine if we could genetically modify this strain to produce protective antigens of a heterologous pathogen. Currently, there is not a licensed vaccine against the important opportunistic bacterial pathogen, Pseudomonas aeruginosa. Because many P. aeruginosa strains are also drug resistant, the need for effective vaccines is magnified. Here, F. tularensis LVS was genetically modified to express surface proteins PilAPa, OprFPa, and FliCPa of P. aeruginosa. Immunization of mice with LVS expressing the recombinant FliCPa led to a significant production of antibodies specific for P. aeruginosa. However, mice that had been immunized with LVS expressing PilAPa or OprFPa did not produce high levels of antibodies specific for P. aerugionsa. Therefore, the recombinant LVS strain engineered to produce FliCPa may be able to provide immune protection against a P. aeruginosa challenge. However for future use of this vaccine platform, selection of the appropriate recombinant antigen is critical as not all recombinant antigens expressed in this strain were immunogenic.

The presence of interleukin-27 during monocyte-derived dendritic cell differentiation promotes improved antigen processing and stimulation of T cells.

Dendritic cells (DCs) are potent antigen-presenting cells necessary to establish effective adaptive immune responses. The cytokine environment that exists at the time of DC differentiation may be an important but often ignored determinant in the phenotypic and functional properties of DCs. Interleukin-27 (IL-27) is a unique cytokine that has both inflammatory and immune suppressive activities. Although it can both promote and oppose activity of different T-cell subsets, mostly anti-inflammatory activity has been described toward macrophages and DCs. However, the specific effect of IL-27 during DC differentiation and how that may change the nature of the antigen-presenting cell has not been investigated. In this report, we show that IL-27 treatment during monocyte-derived DC differentiation enhanced the ability to process antigens and stimulate T-cell activity. DCs differentiated in the presence of IL-27 showed enhanced acidification of latex bead-containing phagosomes that was consistent with elevated expression of vacuolar-ATPases. This resulted in inhibition of intracellular growth of Staphylococcus aureus. In addition, the levels of MHC class II surface expression were higher in DCs differentiated in the presence of IL-27. Production of IL-12 was also significantly increased during S. aureus infection of IL-27-differentiated DCs. The net effect of these activities was enhanced CD4(+) T-cell proliferation and T helper type 1 cytokine production. These findings are important to a wide number of immunological contexts and should be considered in the development of future vaccines.

Mycobacterium tuberculosis infection of human dendritic cells decreases integrin expression, adhesion and migration to chemokines.

Tuberculosis (TB) remains a major global health problem accounting for millions of deaths annually. Approximately one-third of the world's population is infected with the causative agent Mycobacterium tuberculosis. The onset of an adaptive immune response to M. tuberculosis is delayed compared with other microbial infections. This delay permits bacterial growth and dissemination. The precise mechanism(s) responsible for this delay have remained obscure. T-cell activation is preceded by dendritic cell (DC) migration from infected lungs to local lymph nodes and synapsis with T cells. We hypothesized that M. tuberculosis may impede the ability of DCs to reach lymph nodes and initiate an adaptive immune response. We used primary human DCs to determine the effect of M. tuberculosis on expression of heterodimeric integrins involved in cellular adhesion and migration. We also evaluated the ability of infected DCs to adhere to and migrate through lung endothelial cells, which is necessary to reach lymph nodes. We show by flow cytometry and confocal microscopy that M. tuberculosis-infected DCs exhibit a significant reduction in surface expression of the ß(2) (CD18) integrin. Distribution of integrin ß(2) is also markedly altered in M. tuberculosis-infected DCs. A corresponding reduction in the αL (CD11a) and αM (CD11b) subunits that associate with integrin ß(2) was also observed. Consistent with reduced integrin surface expression, we show a significant reduction in adherence to lung endothelial cell monolayers and migration towards lymphatic chemokines when DCs are infected with M. tuberculosis. These findings suggest that M. tuberculosis modulates DC adhesion and migration to increase the time required to initiate an adaptive immune response.

Identification of phosphorylation sites on the E3 ubiquitin ligase UBR5/EDD.

UBR5 (ubiquitin protein ligase E3 component n-recognin 5)/EDD (E3 ligase identified by differential display) is an E3 ubiquitin ligase that is a potential biomarker for poor prognosis for recurrent, platinum-resistant ovarian cancer. UBR5 has a role in the DNA damage response and many such proteins are regulated by phosphorylation. UBR5 is a 309 kDa nuclear phosphoprotein that we previously identified as a substrate of the MAP kinase ERK2. With its 477 potential phosphorylation sites, little is known about UBR5 phosphorylation and how it may regulate protein function. Currently, thirty-four sites of phosphorylation on UBR5 have been reported in the literature, mostly identified by large scale proteomics studies of tissues or of cells after various treatments; however, no studies have specifically targeted the identification of UBR5 phosphorylation sites. In this study, we used Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to obtain a total sequence coverage of 64.3% from combining tryptic and GluC digests on UBR5 isolated from transfected COS-1 cells. We identified 24 sites of phosphorylation, 18 of which are novel sites. This data enhances our knowledge of UBR5 phosphorylation and provides a framework for the study of how phosphorylation affects UBR5 function.