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WP1 Anti-trypanosome Nb library generation
WP1involves the generation of Nb libraries against all 4 major African trypanosome species needed for the execution of all the other WPs. There is little or no risk that this WP will not be successful. So far the Nb generation technique has proven to be very effective in the past, and in the 10 year history of VIB's research effort, no antigens were ever encountered that failed to elicit a good camel antibody response. In addition, we have already generated a library of Nbs against a cloned T. brucei parasite, (Stijlemans et al. J Biol Chem. 2004 Jan 9;279(2):1256-61).
WP2 Nb-Dipstick development for diagnosis of trypanosomiasis
WP2involves the generation of a dipstick assay for the 4 major African trypanosome species. So far, we have successfully generated a proof-of-principle dipstick for the clone-specific detection of T.brucei. In order to use the test for a broad range T.brucei detection, we need to develop new Nbs. It remains to be seen if a Nb dipstick will have a broad enough recognition range to detect all human infective T.brucei species present in the field. However, circumventing the need for 2 independent cross-recognizing nanobodies is embedded into WP3. A second potential problem facing a dipstick trypanosomiasis assay is the fact that parasite levels in human blood are expected to be low, especially during T.b.gambiense infections. However, given the extremely high concentration of repetitive surface proteins on the trypanosome, the presence of 100 parasites per ml would bring it in the working range of other dipstick diagnostic tests, and would be within the same range as other more complicated diagnostic assays.
WP3 Development of Nb-based magnetic bead diagnostic tools
WP3involves the development of a trypanosomiasis diagnostic assay that utilizes just a single anti-parasite Nb, coupled to magnetic beads. This allows for a fast and easy method of 'fishing' parasites in larger volumes of blood. The technique allows the concentration of parasites with a factor of 1000, before actual parasite detection is performed by microscopy. Obviously, the technique offers advantages over the dipstick approach, in that the parasite itself is visualized after concentration. On the other hand it requires a microscopy infrastructure, which might be considered a disadvantage over a dipstick. If successful, this technique will bring parasite detection within the detection range of the much more demanding PCR diagnostic techniques. A collaboration between FIND and VIB was initiated in July 2007 to optimize the technique proposed here, and allow an accelerated take-off of NANOTRYP.
WP4 Field evaluation of Nb-based HAT diagnostic tools
WP4relies entirely on WP2 and/or WP3 to be successful, as it deals with testing the newly developed HAT diagnostic tools under field conditions. In the extreme case were both WP2 and WP3 would fail, this WP would have to be abandoned. However, this scenario is unlikely due to the sound scientific base of the two 'mother' packages.
WP5 Nb-drug targeting
WP5is the only work packages that deals purely with Nb-based anti-trypanosomiasis treatment as a proof of principle in experimental trypanosomiasis. It relies completely on the generation of cross-recognizing Nbs for all 4 major African trypanosome species, and as such is building on WP1. Building on our previously published data (Baral et al. Nat Med. 2006 12(5):580-4), WP5 proposes to couple anti-trypanosome drugs to a battery of nanobodies in order to improve drug targeting and reduce systemic drug toxicity. In the first approach, encapsulation of existing drugs into Nb-coupled ?-cyclodextrin will be performed. In addition, WP5 will focus on the targeting of trypanocidal peptides by Nbs,. For the coupling of the peptide and the Nb, two independent techniques will be used in parallel, i.e. (i) Chemical coupling, and (ii) Recombinant generation of chimeric constructs.
WP6 Nb-tools for diagnosis and treatment of T.b.rhodesiense and T.b.gambiense infections
WP6will be crucial to evaluate the diagnostic potential of Nbs in experimental models for human trypanosomiasis. In addition, WP6 will serve to vaildate WP5. In WP6, one of the potential risks would be that Nb chimeras become immunogenic to mammalian subjects, so that multiple injections are compromised by the induction of host anti-nanobody immune responses. Actually, this problem arises in mice and has hampered previous efforts to successfully treat late-stage experimental infections (Nature Med. Baral et al., 2006). However, WP6, in combination with WP1 could involve the testing of modified nanobodies that could have much reduced immunogenicity. Indeed, this joint project could be used to assess the possibility through specific mutagenesis and cloning techniques to adapt selective nanobody sequences to monkey antibody sequences (as is already done by VIB for human Nb applications in other fields).
In addition, the analysis of an experimental model for T.b.gambiense infections in vervet monkeys would also be part of the later continuation of WP6, where Nb-based diagnostic tools would be tested in this slow progressing HAT model.
WP7 Nb-tools for diagnosis of livestock trypanosomiasis
WP7is crucial to evaluate the results of WP5 and WP6 in treatment of animal trypanosomiasis, with a focus on T.brucei, T.congolense, T vivax and T.evansi infections in goat and possibly cattle. Here, the additional concern for cattle treatment is the production scale of the Nb-chimeras.
WP8 Trypanosomiasis awareness building and education
WP8is linked to WP4 and WP8. Wherever new diagnostics will be tested, FIND and UEM will engage the governments and local communities in trypanosomiasis awareness campaigns. This education approach will focus on (i) the need for endemic countries to prioritize trypanosomiasis control, (ii) the risk of the human parasite reservoir for infection and re-infection of other individuals, and (iii) the possible transmission of human-infective trypanosomes from livestock. Although vector control (tsetse fly eradication) is not part of this research proposal, it will be part of the trypanosomiasis awareness efforts.
In addition, WP8 involves the training of researchers, as well as the organization of Nb-technology workshops.