Development and evaluation of a cyst wall protein 2-encoding Giardia transmission-blocking DNA vaccine

The high number of Giardia cyst polluting surface water, in conjunction with the inadequacy of current water treatment practices in ensuring the complete removal of giardial cysts, has led our group to propose the use of CWP2-based transmission-blocking vaccines as a mean to control environmental contamination by reducing the spread of infective Giardia cysts in nature. In this PhD thesis, three key studies were performed with the ultimate goal of developing and testing a CWP2-expressing transmission-blocking DNA vaccine in the mouse model of the disease.The study presented in manuscript I examined for the first time the host's local immune response to giardial cyst antigens (Ags) during the acute and elimination phases of a primary G. muris infection. The cyst Ags' specific immune response (which comprised a CWP2-specific component) was indistinguishable from the host's local immune response to trophozoite Ags. This local immune response included the production of a balanced IL-4/IFN-gamma cytokine response as well as intestinal IgA and IgG antibodies. Furthermore, this study showed for the first time the presence of local antibodies capable of interfering with the process of trophozoite encystation. These findings led us to advance the possibility that elimination of cyst shedding in a primary infection may be partly due to intestinal immune responses targeting encysting trophozoites.The study in manuscript II reported the expression of rCWP2 in transfected HEK-293 cells. Therein we showed that cwp2-transfected mammalian cells packaged the full-length CWP2 into membrane-bound vesicles and trafficked it via the secretory pathway, in a manner similar to encysting trophzoites. Analysis of CWP2 truncations expressed in transfected HEK-293 demonstrated that both the N- and C-termini were responsible for the secretion of CWP2. Based on these observations, we concluded that giardial CWPs can be successfully expressed in mammalian cells in addition to proposing the use of transfected HEK-293 cells as a heterologous model system to investigate the signal peptides involved in the intracellular trafficking of proteins in Giardia trophozoites.Finally, the study presented in manuscript III investigated the possible use of Salmonella typhimurium mediated bactofection for the delivery of the CWP2-encoding transmission-blocking DNA vaccine. The study showed that mice receiving the S. typhimurium-delivered CWP2-encoding oral DNA vaccine produced CWP2-specific intestinal immune responses similar to the CWP2-specific immune responses produced by Giardia-infected animals at the elimination phase of primary giardiasis. In addition, the administration of the CWP2-encoding transmission-blocking DNA vaccine resulted in significantly reducing the duration of cyst shedding in addition to reducing the total number of cysts shed.Taken together, these findings constitute original contributions to advancing our knowledge on the cyst Ag-specific component of the host's local immune response during an infection. Secondly, the findings show the usefulness of using transfected HEK-293 cells for performing comparative studies on protein trafficking with Giardia cells. Finally, the data also demonstrate the efficacy of STM1-mediated bactofection for delivering a CWP2-encoding DNA vaccine which led to interference with cyst formation in vivo