Tunneling Nanotube-like Structures in Giardia duodenalis.

Giardia doudenalis (lamblia, intestinalis) is a protozoan parasite that inhabits the lumen of the upper small intestine of vertebrates, causing chronic abdominal pains and severe diarrhea, symptoms of giardiasis, a persistent and recurrent infection. This characteristic is mainly due to the presence of membrane variant-specific surface proteins (VSPs) that give this parasite the ability to successively infect the host through antigenic variation. Using high-resolution scanning microscopy (HR-SM), we observed the presence, formation, and extension of tunneling-nanotube-like surface structures in Giardia, especially following parasite challenges with VSP antibodies. They were seen all over the parasite surface, both in vitro and in vivo, showing that G. duodenalis nanotube formation occurs in complex environments such as the gut. In addition, we also observed that some of these nanotubes displayed a periodic strangulation that produces 100 nm vesicles that seemed to be released in a process similar to that previously observed in Trypanosoma brucei. The presence of nanotube-like structures in G. duodenalis highlights yet another strategy of cellular communication utilized by these parasites, whether between themselves or with the host cell.

Antibodies to variable surface antigens induce antigenic variation in the intestinal parasite Giardia lamblia.

The genomes of most protozoa encode families of variant surface antigens. In some parasitic microorganisms, it has been demonstrated that mutually exclusive changes in the expression of these antigens allow parasites to evade the host's immune response. It is widely assumed that antigenic variation in protozoan parasites is accomplished by the spontaneous appearance within the population of cells expressing antigenic variants that escape antibody-mediated cytotoxicity. Here we show, both in vitro and in animal infections, that antibodies to Variant-specific Surface Proteins (VSPs) of the intestinal parasite Giardia lamblia are not cytotoxic, inducing instead VSP clustering into liquid-ordered phase membrane microdomains that trigger a massive release of microvesicles carrying the original VSP and switch in expression to different VSPs by a calcium-dependent mechanism. This novel mechanism of surface antigen clearance throughout its release into microvesicles coupled to the stochastic induction of new phenotypic variants not only changes current paradigms of antigenic switching but also provides a new framework for understanding the course of protozoan infections as a host/parasite adaptive process.

Efficient oral vaccination by bioengineering virus-like particles with protozoan surface proteins.

Intestinal and free-living protozoa, such as Giardia lamblia, express a dense coat of variant-specific surface proteins (VSPs) on trophozoites that protects the parasite inside the host's intestine. Here we show that VSPs not only are resistant to proteolytic digestion and extreme pH and temperatures but also stimulate host innate immune responses in a TLR-4 dependent manner. We show that these properties can be exploited to both protect and adjuvant vaccine antigens for oral administration. Chimeric Virus-like Particles (VLPs) decorated with VSPs and expressing model surface antigens, such as influenza virus hemagglutinin (HA) and neuraminidase (NA), are protected from degradation and activate antigen presenting cells in vitro. Orally administered VSP-pseudotyped VLPs, but not plain VLPs, generate robust immune responses that protect mice from influenza infection and HA-expressing tumors. This versatile vaccine platform has the attributes to meet the ultimate challenge of generating safe, stable and efficient oral vaccines.