Schistosoma mansoni tetraspanning orphan receptor - SmTOR

Schistosomiasis is a neglected tropical disease affecting more than 207 million people worldwide with an estimated number of 700 million people at risk in 74 endemic countries. There are five species of schistosoma that are known to infect humans by contact with its larval stage, the cercariae: Schistosoma mekongi, S. intercalatum, S. mansoni, S. japonicum and S. haematobium, the latter three being the main schistosome species that affect humans. Although the infection can be treated by using chemotherapeutics, it is hardly possible to influence strongly the risk of re-infection and a vaccine against schistosomiasis is a desirable goal.
Schistosome parasites need to adapt to survive at various points of their complex life cycle. In the vertebrate definitive host, this involves the avoidance of the host immune system in general and the battle against the host complement system participating in the first line of immune defenses against the invading parasite in particular. There are several complement regulators found on schistosomes. We are interested in complement regulation at the level of the formation of the C3 convertase by a complement C2 binding receptor. This C2 binding receptor was first characterised in one of the human schistosoma species S. haematobium and denominated ShTOR for trispanning orphan receptor. After the S. mansoni genome had been sequenced, we had a closer look at the ShTOR receptor homologue in this species.
The aims of the project were to characterise the S. haematobium TOR receptor homologue SmTOR in S. mansoni (PART I), to test if the first extracellular domain 1 of SmTOR binds C2 and to define its antigenicity (PART II) and to define the immunogenicity of SmTORed1 in mice and test it as a vaccine candidate against schistosomiasis in a murine vaccination and challenge model (PART III). In line with the structure of the project outline presented above, the work presented in this thesis can be summarised as follows:
At first, we found that SmTOR is a tetraspanning receptor expressed in the tegumental membranes of schistsosomes. As compared to the truncated receptor versions described before, it possesses a longer extracellular domain 1 still comprising the C2 binding motif. Its expression was highest in the infectious stage of the parasite, in the S. mansoni cercariae. SmTOR might play an important role in skin penetration of S. mansoni larvae and it is an interesting target for vaccination since it is an early antigen on schistosoma cercariae and has a complement inhibitory activity.
We then overexpressed SmTOR extracellular domain 1 (rSmTORed1) in E. coli, which was purified by immobilised metal affinity chromatography. We were able to show that full length recombinant SmTORed1 binds C2. We moreover detected specific antibodies against rSmTORed1 in sera of patients infected with S. mansoni and also in some normal human sera. Specificity of antibody to rSmTORed1 was ensured by pre-incubation of sera with the
Halo-tagged version of SmTORed1 immobilised on a solid support. SmTORed1 N-terminally fused to HaloTag and the corresponding control constructs were produced in E. coli as well.
Thirdly, we tested the immunogenicity of rSmTORed1 in two different mouse strains, BALB/c and C57BL/6 using muramyl dipeptide (MDP) and Complete/Incomplete FreundÕs adjuvant (CFA/IFA) as adjuvants. BALB/c mice immunised with rSmTORed1 in CFA/IFA generated the highest titer of specific antibodies to rSmTORed1 and were subsequently tested in an immunisation challenge experimental setup. For this, immunised mice were infected with S. mansoni cercariae and status of infection assessed by adult worm count. Immunised mice showed a 60 % reduction of worm burden when compared to the two control groups.
Taken together, we managed to newly characterise the S. mansoni TOR receptor and to show that it is an interesting anti-schistosome vaccine target to be followed up in additional experiments.