“I chose the Ph.D. program in Microbiology and Immunology at Wake Forest University primarily based on two aspects the small class size within the department and the genuine care for the development of the students as scientists from the faculty. The medical school setting and the overall enthusiasm received during the application process were also important deciding factors.”

Development of a Cell-Permeable Form of Flagellin for Use as an Adjuvant in Vaccines

The generation of a humoral response is important for the clearance of extracellular pathogens, whereas, cytotoxic T lymphocytes (CTL) are critical for the clearance of intracellular pathogens and tumors.  Recently, investigators have used protein transduction domains (PTDs) to efficiently translocate tumor antigens across cell membranes and thus facilitate antigen processing via the endogenous pathway.  To develop flagellin as an adjuvant for vaccines that are designed to generate CTL, we constructed recombinant flagellins containing either an internal PTD sequence of 9 arginines (flagellin 9xR^int) or 9xR at the carboxy terminus (flagellin9xR^cterm).  Using confocal microscopy, we observed that flagellin 9xR^int was present inside TLR5-negative and positive cell types, whereas flagellin and flagellin9xR^cterm were only present in TLR5-positive cells.  These results indicate that 9xR in the hypervariable region of flagellin permits efficient membrane translocation in a TLR5-independent manner.  The location of the PTD also plays a vital role in its efficiency, as evidenced by the lack of translocation seen with flagellin9xR^cterm.  Since intracellular flagellin promotes cell death in macrophages, we evaluated the effect of 9xR-tagged flagellins on the viability of macrophages and bone marrow derived dendritic cells (BMDC). Unlike flagellin and the flagellin9xR^cterm, the flagellin 9xR^int was found in BMDC and peritoneal macrophages.  However, the internalized flagellin 9xR^int did not induce cell death in either cell type.  In view of the hypothesis that PTD promote uptake of proteins via macropinocytosis, we examined the uptake of flagellin 9xR^int in LPS-matured BMDC since mature dendritic cells exhibit reduced capacity for macropinocytosis.  The mature BMDC, unlike their immature counterparts exhibited a reduced level of internalized flagellin.  This result is consistent with the notion that the flagellin 9xR^int may be taken up by macropinocytosis.  Our working hypothesis is that the internalized flagellin 9xR^int does not induce cell death because an insufficient amount of the protein is available in the cytoplasm to activate the inflammasome.  However, it is possible that the small amount of flagellin that does reach the cytoplasm is sufficient to load MHC class I molecules.  Studies are underway to test these hypotheses.