Final Report Summary - NANOTRYP (Exploiting Nanobodies in development of new diagnostic tools and treatment methods for Trypanosomiasis.)
Executive Summary:
Trypanosomiasis is a disease with a devastating socio-economic impact in Sub-Saharan Africa through the direct infection of humans and livestock. The NANOTRYP project aimed at addressing the problems encountered in the fight against trypanosomosiasis, i.e. (i) the lack of specific diagnostic tools that can be used as test-of-cure, (ii) the high drug toxicity of current treatment schemes, and (iii) the limited understanding of the disease by local communities of endemic areas. NANOTRYP was initiated using nanobody (Nb) tools whose efficacy already had been proven in the laboratory. Based on this knowledge, the main objectives of NANOTRYP were to: (1) Generate a number of Nanobody (Nb) libraries that are capable of recognizing all four major African trypanosome species, (2) Develop new, rapid, and easy to use Nb-based trypanosomiasis diagnostic tools based on parasite detection, (3) Asses the use of anti-trypanosome Nbs as drug targeting molecules, and (4) Develop a forum for exchange of knowledge between European partners and partners from disease endemic countries.
To reach the objectives outlined above, the project was divided into seven scientific work packages, one training and education work package and one management work package.
Work packages 1 to 3 were designed to deliver various libraries of Nanobodies and a Nb-based test kit for trypanosomiasis. The Nbs generated were able to bind highly conserved antigens of T. brucei, T. evansi, T. congolense and T. vivax. Nbs were obtained from various vaccination protocols as well as from naturally and experimentally infected camelids. Rather than preselecting certain antigens as preferred candidates, the consortium opted for a strategy in which Nbs were made against soluble extracts of trypanosomes, hence increasing the change of picking up unexpected candidates such as conserved intracellular targets. Following the selection of promising Nbs, a first test format was designed under the format of an ELISA. In a next step, the consortium attempted to translate the ELISA format into a lateral flow design, using gold-labeled Nbs for detection. Unfortunately, this step proved more difficult than anticipated and reproducibility of the coupling procedure was not optimized by the end of the project. However, a proof-of-principle RTD was developed together with the Korean based company SD (Standard Diagnostics). The collaboration with SD resulted from the activities planned in WP4 that was initially designed to test, validate and implement a Nb-based RTD for trypanosomiasis in the field. Having been unable to standardize the envisaged final test format, the latter WP only had a limited outcome.
As mentioned above, the fight against trypanosomiasis is also hampered by the high toxicity of existing drug. To increase the therapeutic index of existing drugs, and hence lower the toxicity at an effective dose, we proposed to use the Nbs developed in WP1 as drug targeting molecules, aiming at an increased efficacy of target specific drug delivery. As a model concept, work was performed using a well establish T. brucei rodent model, and pentamidine as a trypanocide. The results of WP5 showed that the envisaged strategy gave a ten-fold increase in treatment efficacy, when Nb drug targeting was applied.
In order to assess the future usability of Nb tools in the field, Work Packages 6 and 7 were developed using non-human primates and livestock animals. The first WP allowed the characterization of models for both T. gambiense and T. rhodesiense, two human infective trypanosomes. Obtained results are crucial for future diagnostic and drug delivery strategies, as the past has proven that neither in vitro, nor rodent models are suitable for this when considering the encephalitic late stage of human sleeping sickness. The latter WP delivered a wealth of experimental and field samples, needed for further diagnostic development, and allowed the specificity and sensitivity assessment of a T. conglonese specific Nanobody-ELISA. Both WPs were coupled to WP8, designed to provide a tool for training and awareness campaigning. The NANOTRYP consortium as whole can be proud of (i) having organized two intense training schools in Kenya and Mozambique and (ii) having delivered 4 PhDs and 7 Master thesis graduations towards the end of the programme.
Project Context and Objectives:
When NANOTRYP was initiated, the consortium proposed four objectives that ultimately would lead to the generation of tools allowing an improvement in diagnosis of both HAT (Human African Trypanosomiasis) and AAT (Animal Africa Trypanosomiasis). These objectives included:
1. Generate a number of Nanobody (Nb) libraries that are capable of recognizing all four major African trypanosome species.
2. Develop of a Nb-based trypanosomiasis diagnostic tools based on parasite detection. This could complement or replace the current agglutination assays that are based on anti-parasite antibody detection. The nanobody technique proposed avoids expensive, labour intensive molecular techniques that impose extensive requirements on infrastructure and equipment maintenance.
3. Assessment of the use of anti-trypanosome Nbs as drug targeting molecules as proof of principle for the treatment of experimental trypanosomiasis.
4. Develop a forum for exchange of knowledge in which researchers from participating developing countries can get first-hand experience in the further development of Nb-technology, and in which new trypanosome diagnostic and treatment campaigns are linked to HAT and livestock trypanosomiasis awareness programs.
Major measurable outcomes that had been put forward at the start of NANOTRYP were:
• A collection of nanobodies that will recognize the HAT causing parasites T.rhodesiense T.gambiense as well as surface molecules of the parasites T.brucei T.congolense T.vivax and T. evansi, the causative agents of animal trypanosomiasis. These Nanobodies would subsequently provide a new way forward to diagnostic development and eventual trypanosome-specific drug delivery.
• Working and training visits of members of the African participant teams to the laboratories of the European partners.
• The organization of two workshops in applied diagnostic and treatment of trypanosomiasis in the African partner countries.
• Conducting of trypanosomiasis awareness campaigns in parallel to all field diagnostics and treatment efforts.
NANOTRYP builds largely on the established fundamental Nanobody research done with respect to experimental trypanosomiasis research in mice. It focuses on taking concepts to field conditions, in order to deliver new diagnostic and treatment methods for trypanosomiasis. To date, two main problems exist in the fight against trypanosomiasis. First, trypanosomiasis diagnosis (particularly in the case of HAT) relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT), an assay that detects host antibodies that cross-react with a laboratory prepared trypanosome protein complex. Due to its high rate of unconfirmed results (Radwanska et al., Am J Trop Med Hyg. 2002 67(6):684-9), a second confirmatory assay is needed for all positive tests. In addition, as CATT measures antibody titers, it cannot be used as test-of-cure and neither is it suitable for use in endemic areas where treatment is common (as in both cases antibodies remain in the circulation long after parasites have been cleared). New and better diagnostics should solve this problem. Second, at the level of trypanosomiasis treatment, only a limited number of drugs have been registered. Based on the increased appearance of drug-resistance, and the unacceptable level of side effects (up to 10% mortality in case of Melarsoprol treatment) it is clear that there is an urgent need to engage in activities that seek to develop new anti-trypanosome compounds to eradicate HAT and animal trypanosomiasis. Hence, NANOTRYP aimed at using nanobodies in the development of new methods for diagnosis and treatment of trypanosomiasis.
Nbs are small antibody fragments with unique protein or antigen recognizing properties. They are derived from conventional camelid antibodies through recombinant gene technology.
All antibodies from vertebrate animals are constitutively composed of two identical heavy and two identical light chains. More than fifteen years ago, by serendipity, one of the research groups at the Vrije Universiteit Brussel (VIB - the coordinating partner) discovered the occurrence of bona fide antibodies devoid of light chains in Camelidae (Hamers-Casterman et al., Nature. 1993 363(6428):446-8). These so-called heavy-chain antibodies (HCAbs) bind antigen solely with one single variable domain with high affinity, and are referred to as VHHs or nanobodies because their size is in the nanometer range. As such, the Nb is the smallest intact antigen-binding entity that can be obtained. Methods were developed to clone the Nb repertoire of an immunised dromedary (or llama) in phage display vectors, and to select the antigen-specific Nbs from these ‘immune’ Nb libraries (Lauwereys et al., EMBO J. 1998 17(13):3512-20). The optimised protocol is fast and straightforward. In addition, the method is far superior to the selection of antigen binders from conventional antibodies. Moreover, Nbs are economically produced by bacterial, fungal or plant expression systems to a high yield and possess excellent biophysical properties (e.g. specificity, solubility, stability, long shelf-life). As nanobodies are produced as recombinant molecules, it is also relatively easy to produce larger nanobody complexes, generating for example bivalent or bivalent-Fc coupled nanobodies. In addition, in order to reduce possible immunogenicity of nanobodies when injected into non-camelids, they can be customized by altering specific amino acid sequences. For example, for use in man, nanobodies are ‘humanized’ to reduce the risk of the induction of anti-nanobody antibodies in the circulation. While NANOTRYP is not expected to generate anything new, beyond the current state-of-the-art insights at the level of Nb generation, it does provide the opportunity to transfer the knowledge of how to generate anti-parasite Nbs to members of the participating African research communities. Given the relative ease with which Nbs can be generated, this transfer of knowledge can later result in a whole range of new applications, originating from the views and needs of these partners.
Over the last decade, VIB has been involved in the development of new approaches to HAT and livestock trypanosomiasis diagnosis. Indeed, RT-PCR methods as well as PNA-FISH techniques for differential diagnosis of T.rhodesiense and T.gambiense (Radwanska et al. Am J Trop Med Hyg. 2002 67(6):684-90, Radwanska et al. J Clin Microbiol. 2002 40(11):4295-7., Radwanka et al. Am J Trop Med Hyg. 2002 67(3):289-95.) were pioneered in collaboration with VIB, and were based on the earlier discovery by VIB of a species-specific trypanosome resistance genes (i.e. SRA, De greef et al. Ann Soc Belg Med Trop. 1992;72 Suppl 1:13-21 , Xong et al. Cell. 1998 Dec 11;95(6):839-46.). However, considering the burden that these molecular biology techniques pose at the level of equipment infrastructure and maintenance, as well as the high costs involved in routine PCR screening under field conditions, these innovations were not explored beyond the ‘proof-of-principle’ in screening for HAT. In addition, molecular biology approaches for the preventive screening of HAT causing parasites in the African livestock herd are considered to be unaffordable on a large and systematic scale. Hence, in an alternative approach, VIB has generated fluorescent anti-trypanosome Nbs that greatly facilitate parasite detection in blood of experimentally infected animals. This technique was shown to be successful for T.b.brucei T.b.rhodesiense T.b.gambiense T.evansi and T.vivax trypanosomes. However, as parasite levels in human infections are often 1000-times lower than those levels found in experimental mouse infections, the minimal detection limit of fluorescent parasite visualisation techniques remains a problem in field conditions. Hence, the project proposed here will focus on Nb-based antigen capturing diagnostic methods that can drastically increase parasite detection limits.
Before the initiation of NANOTRYP, all Nb work done with respect to the diagnosis of trypanosomiasis had been executed at a laboratory scale, involving well-characterized, cloned parasite stabilates. In order to field-validate the diagnostic tools, two partners with key positions in assessing HAT and livestock trypanosomiasis respectively participated in NANOTRYP.
The first partner is the Foundation for Innovative New Diagnostics (FIND). FIND is an independent Swiss Foundation dedicated to the development of diagnostic tests for poverty related infectious diseases of public health importance. Prior to its participation in NANOTRYP, FIND has used other diagnostic programmes (such as their tuberculosis programme) as a model to develop a rigorous and systematic approach to the needs-driven development, evaluation, demonstration and access of diagnostic technologies. This is done in partnership with academic and research institutes, ministries of health in endemic countries, and with biotechnology companies in the private sector, in the framework of contractual agreements that ensure eventual access to affordable tools in the public sector of developing countries. A critical obstacle to the development of improved assays for HAT is access to patients, quality diagnostic and clinical data, carefully collected and stored reference materials, and to sustained field programs with the capacity for long-term follow-up. The partnership with WHO, ministries of health and national research institutes in endemic countries has enabled the establishment of screening sites for collection of biological materials, and a specimen bank owned by the WHO. Clinical trial sites were also being strengthened through a memorandum of understanding between FIND and the WHO. The establishment of a specimen bank has guaranteed more efficient use of limited resources, limited the need for field trials, promotes product comparisons and facilitated quality control. This as tol be used as a first step in the evaluation of diagnostic tests developed through the project. One of the key requirements in the HAT diagnostics programme at FIND is the development of an access strategy for diagnostics that ensures that these tools reach the population at risk in a sustainable and affordable manner. The strategy that FIND has developed includes advocacy at the level of the African Union, ministries of health, finance and agriculture of endemic countries, and affected communities. The second partner of NANOTRYP crucial for Nb diagnostic development is the University Edwando Mondlane in Mozambique (UEM)). UEM selected to validate Nb trypanosomiasis diagnostic tools in on livestock infections. At UEM, regular field diagnosis screenings for trypanosomiasis are performed, covering most of the trypanosome affected area of Mozambique. Hence, within the context of this project, Nb-tools were assessed in a completely uncontrolled environment.
Besides problems with accurate HAT and AAT diagnostics, problems with anti-trypanosome treatments hamper the eradication of trypanosomiasis. To date, anti-trypanosome treatment is hampered by (i) a limited choice of effective compounds, and (ii) severe treatment-related side effects due to the limited specificity of the available drugs. Starting with existing anti-trypanosome nanobodies, and including at a later stage newly generated nanobodies, NANOTRYP aimed at generating chemical chimeras in which nanobodies are used to specifically target drug compounds towards the membrane of the trypanosome. This part of the project was to rely on the state-of-the-art expertise of the Spanish partner FIBOA. This partner engaged in efforts to use an encapsulated Nb-targeted delivery system for existing drugs, mainly aiming at improving the balance between efficacy and toxicity, and as such enhancing the drug quality of existing compounds. Indeed, site-specific delivery is a recognised strategy for improving the therapeutic efficiency and safety of drugs, and nanobodies have already proven to be successful in this context (Stijlemans et al. Biol Chem. 2004 279(2):1256-61). The primary purpose of drug delivery systems is to deliver the necessary amount of drug to the target site efficiently and precisely. In addition, a fundamental aspect of these delivery systems is that they should work without modifying the desired biological features of the drug. Considerable efforts have focused on developing new strategies for controlled drug release systems based on biodegradable polymers such as cyclodextrins, which are mainly used as complexing agents to increase the aqueous solubility of poorly water-soluble drugs and to increase bioavailability and stability of drugs (review in Brewster and Loftsson. Adv Drug Deliv Rev. 2007 Jul 30;59(7):645-66). Indeed, cyclodextrins allow the formation of non-covalent complexes with ‘guest’ drug molecules, altering the physicochemical properties of the drug, and improving their water solubility. Hence, at FIBAO the aim will be to couple the advantages of nanobody-mediated drug delivery to the benefits of cyclodexrtin-controlled drug delivery methods. Important here is that VIB has already proven that coupling an anti-trypanosome nanobody to the human ApoL1 lytic domain increase lytic activity of the ApoL1 protein a 100 fold. However, as ApoL1 is the only human serum defence molecule that acts against a wide range of trypanosomes, modification of this molecule combined with wide spread use could result in a disastrous outcome in terms of ApoL1 resistance build-up. Hence, as an alternative strategy, NANOTRYP focused on encapsulation of exsisting drugs and peptides with unique anti-trypanosome properties.
As indicated above, the Nb-ApoL1 anti-trypanosome treatment approach provided the basis for the drug-targeting strategy of NANOTRYP. However, previous work only used targeting strategies in an experimental mouse model only. In order to evaluate the possibility of Nb-based drug treatment in the field, this project incorporated research on the treatment of trypanosome infected non-human primates. Human African Trypanosomiasis is a disease in which the crossing of the trypanosome into the central nervous system is the cause of several complications at the level of diagnosis and treatment. To date, little is known about the immunobiological mechanisms that are involved in this aspect of the disease, partly because of the fact that experimental rodent infection models do not offer the right settings to mimic HAT in this aspect. For example, basic information such as timing of parasite entry into the brain in HAT is still not known and is not possible to assess in rodent models. Hence, in order to address the question of parasite diagnostics as well as anti-trypanosomiasis treatment, experimental research models need to extend beyond the conventional laboratory rodent models to mimic more realistic HAT-associated problems. At the Institute of Primate Research in Kenya (IPR), trypanosome research is conducted using a vervet monkey (Chlorocebus aethiops syn. Cercopithecus aethiops) model that can be used to mimic several aspects of HAT. Including IPR as a research partner in this proposal allowed for the testing of Nb-based diagnostics in function of disease progression, and was to allow the testing of Nb-based drug therapy during these different disease stages as well.
In contrast to HAT, livestock trypanosomiasis is not hallmarked by neurological complications, but by severe infection-associated immunopathology (anaemia) and very low levels of circulating parasites. The participation of UEM in NANOTRYP provided experimental as well as field models for livestock trypanosomiasis, allowing the testing of Nb-based diagnostics in realistic conditions.
Finally, the NANOTRYP project also provided the build-up of research capacity in the participating African research institutes, as well as at the organization of a trypanosomiasis awareness campaign at all places where field trials would be performed.
First, the NANOTRYP project included the use of nanobodies in the T. brucei rhodesiense research model currently being studied at IPR. However, NANOTRYP also supported a sustainable research project in the field of T.b.gambiense trypanosomiasis, as very little is known to-date about the disease kinetics of this slow-progressing HAT variant. The project allowed IPR to engage in a longer-term fundamental research program and as such contributed to the goal of building sustainable research capacity in trypanosomiasis affected countries, through the support of the local research community. This aspect of research strengthening benefited from the research training of IPR members at both VIB and FIBAO.
Second, while HAT has a tremendous detrimental direct effect on the human population of sub-Sahara Africa, animal trypanosomiasis causes an equally severe problem as it renders vast areas of up to 60% of Africa’s grazing land unsuitable for livestock production. Hence, the UEM partner from Mozambique, where several livestock infective trypanosome species are present, participated in the development of new Nb-based strategies to fight this economic problem. Also, training of UEM researchers at VIB and FIBAO served the purpose of strengthening the local research community. In addition, UEM engaged in raising the community awareness about trypanosomiasis, with special focus on combining field work and education with respect to disease transmission, control and treatment.
In addition to the training at VIB and FIBAO of staff from IPR and UEM, 2 scientific workshops aimed specifically at transferring the Nb-knowledge to a wider public within the African research community.
Third, with the participation of FIND, NANOTRYP created a direct link to an established HAT prevention and treatment programme. Indeed, the FIND/HAT programme, executed jointly with WHO and with funding from amongst others the Bill & Melinda Gates Foundation, has links with industry, academic and research institutes in developed and endemic countries. While the proposed project fitted perfectly within the FIND diagnostics development pipeline, it also served to further expand the scope of FIND activities within the awareness programme to include the important component of livestock reservoirs.
Training in diagnosis and management of HAT by FIND was crucial for accelerate control of HAT in sub-Sahara Africa. Advocacy with ministries of health, agriculture and finance will also in the future encourage allocation of national resources to HAT control and ownership by affected countries. Companies developing diagnostic tests for HAT are being lobbied by FIND to manufacture them at prices that are affordable by the public sectors of endemic countries. These are crucial elements of a successful global access plan. In addition, the role that livestock play in disease epidemiology is now being highlighted, to ensure that livestock disease control is made an integral component of HAT control programs. Advocacy highlights the importance of HAT and livestock trypanosomiasis at international, national, and community levels, and a global access strategy. Here, NANOTRYP did not only focus on the development and implementation of Nanobody-based tools developed within the programme, but through the awareness campaigning tasks took into account the bigger picture of both HAT and AAT problems, including all available information on existing control strategies as well.
Project Results:
Overall Strategy
At the start of NANOTRYP, the main goals of the project were defined as:
1. the development of new, easy-to-use, inexpensive and reliable Nb-based diagnostic tools,
2. the build-up of a knowledge exchange and capacity building program that would enhance the access of Nb-technology by members of the African (trypanosomiasis) research community, and increase efforts in disease awareness programme.
In order to attain these goals, NANOTRYP is subdivided into 9 individual work packages, with one work package (WP1) dedicated to the nanobody generation itself, and one work package (WP9) dedicated to project management.
The generation of Nb libraries against all 4 major African trypanosome species was needed for the execution of all the other WPs. By the time NANOTRYP was initiated, the Nb generation technique was proven to be very effective and already the generated a Nbs against a cloned T. brucei parasite had been published (Stijlemans et al. J Biol Chem. 2004 Jan 9;279(2):1256-61). However, taking the use of Nbs to a next level, we aimed within NANOTRYP to design a rapid diagnostic test for the 4 major African trypanosome species. Prior to the start of the project, the VIB partner had 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, NANOTRYP first needed to develop new Nbs, with a detection range broad enough to detect all human infective T.brucei species present in the field as well as animal trypanosomes. In terms of sensitivity, the requirement for a useful Nb-based RTD would be a detection limit of 100 parasites per ml. From the start it was clear to all NANOTRYP participants that any antigen detection test would always be inferior with respect to sensitivity, when compared to an antibody detection test. However, at the level of specificity and it usefulness as test-of-cure, the antigen detection test is far more advantageous.
In order be successful in the development of a field applicable RTD for trypanosomiasis, NANOTRYP relied on the pre-existing contacts between the FIND, Switzerland and SD, Korea. Initially, the aim was to involve a commercial partner in the format transformation of a laboratory scale Nb-based diagnostic tool such as an ELISA, to a dipstick-like device for field use. SD has proven in the past to hold crucial knowledge in the field of conventional antibody-based dipstick development. In this domain they already collaborated with FIND. In the context of NANOTRYP, SD attempted to transfor their antibody technology into Nanobody technology.
When NANOTRYP was initiated, the project also included to use of Nbs for trypanosome-specific drug targeting of existing drugs, aiming at improving their therapeutic index and hence reducing toxicity associated with treatment. Only one Work Package was dedicated to this task, and the work was limited to a single well established rodent laboratory model for experimental tryopanosomiasis, using pentamidine as trypanocide. This proposal was building on our previously published data (Baral et al. Nat Med. 2006 12(5):580-4)
When dealing with trypanosomiasis, one crucial aspect of the research is the understanding of the host-pathogen interaction. To date, most academic knowledge of trypanosomiasis is based on either in vitro work, or experimental mouse models. While the first omits the relevant host interaction altogether, the second studies a host-parasite interaction that often does not reflect the relevant issue for the natural host. One example of this is the study of anemia in rodent trypanosomiasis models, while anemia is not a severe disease associated problem in HAT. The second is the encyphalicit complications of HAT, for which there is no good experimental rodent model. Hence, NANOTRYP included the International Primate Research centre (IPR, Kenya) as a partner is order to all the development of relevant models for HAT, which in a later stage would be crucial for diagnostic testing during the late stage of the disease. NANOTRYP did not include experimental testing of Nb-drug targets in non-human primates. During all experiments associated to NANOTRYP, animals were treated with a well established anti-trypanosome treatment according to protocols approved both in the home institution as well as by the institutions of the EU partners. Along the same research line, also infections in livestock anomals were included in the NANOTRYP research programme. For this purpose, UEM provided the bulk of the material, i.e. samples for both experimentally and naturally infected larger animals.
Finally, at the start of NANOTRYP, a list of deliverables was made, that allowed the follow-up of progress of the project. These mainly included the libraries of Nbs against the 4 main pathogenic species of trypanosomes (D1.1-D1.4) a Nb-based RTD (dipstick format) for these trypanosomes (D2.1-D2.4) The production of a commercial RTD for HAT, including the validation evaluation and implementation (D4.1-D4.4) a chimeric Nb-drug compound as well as a Nb-peptide construct with trypanocidal activity, validated in an experimental mouse model (D5.1 5.2) testing of Nb immunogenicity in non-human primates (D6.1) and the set-up of models for diagnostic testing in these animals for both T. rhodesiense and T. gambiense (D6.2- 6.4) optimized diagnostic method for animal trypanosomiasis (D7.1) initiating activities of awareness campaigning, training and education which included writing of an evaluation report of HAT awareness campaigning (D8.1) writing of an evaluation report of AAT awareness campaigning (D8.2) writing of a paper on the current status of trypanosomiasis diagnosis and new EC developments (D8.3) the organization of a Mid-term EC symposium on trypanosomiasis (D8.4) and the delivery of a number of joint master and PhD degrees (D8.5). Finally, three management deliverables were proposed that included the project management plans and quality assurance, the Consortium Agreement signature, and the Annual Project Reports including Financial summary report (D9.1-9.3).
Important to mention is that during the execution of NANOTRYP, a number of alterations were implemented as a result of discussion with the EU midterm evaluation panel.
• First, it was decided to drop the initial deliverable D3.1 which was a magnetic Nb-based antigen capturing tool. Based on the results obtained during the first year of NANOTRYP, this approach was considered too complicated and error-prone for later filed implementation.
• Secondly, it was proposed to limit the work on Nb-drug delivery to a single rodent model for T. brucei infections, and not to attempt to develop a strategy for larger animals or non-human primates. This resulted in term in a refocus of D6.2-6.4 in which initially a Nb-drug treatment modality was foreseen. The initial argumentation of the Mid-term review panel (that included a DNDi member) was the fact that a Nb-based compound would not suite the Target Product Profile for HAT which is an oral drug. However, two reasons were provided by the NANOTRYP consortium to persuade the panel to allow the continuation of the proof-of-concept work, being: (1) a large HAT reservoir, in particular in East Africa where the most acute form of HAT occurs is found in livestock animals, for which oral treatment is NOT the way the go, and (2) in case of late stage HAT with T. rhodesiense, oral treatment is often impossible as victims are too weak to eat, drink of swallow medication. Hence also in the this case the TPP as defined by DNDi is not ideal.
• Third, D8.5 initially only focussed on the delivery of one joined VIB/FIBAO PhD, and the Mid-term review panel asked to broaden this effort. Hence this deliverable was expanded resulting in the delivery of 4 PhDs and 7 Master thesis graduations towards the end of the NANOTRYP programme.
Results obtained in individual Work Packages
WP1: Nanobody generation
Original Objectives: The objective of WP1 is the generation of extended batteries of nanobodies directed against surface exposed epitopes of T.brucei T.congolense T.vivax and T.evansi parasites. In a first approach, Nbs will be generated against all 4 major African trypanosome species. In an additional approach, cross-reactive nanobodies, recognizing more than 1 species will be identified.
Results: NANOTRYP generated multiple libraries of binders against all parasites targeted. For T. brucei 6 libraries were made, for T. congolense and T. evansi 3 libraries were made and for T. vivax, two libraries were made, all based on material obtained from alpaca vaccinations. In addition, after the knowledge of Nb generation was transferred from VIB to both FIBAO and IPR, both partners initiated the construction of a library based on lymphocyte mRNA derived from camels. FIBOA used material that originated from a naturally infected camel identified on the Canary Islands (Spain), while IPR experimentally infected and treated a camel, allowing obtaining an anti-T. congolense response in a natural host.
T. brucei was the first parasite to be targeted by the NANOTRYP project. Having obtained a number of Nanobodies capable of binding intact parasites, it was shown that simple immunofluorescent microscopy using these Nbs made it much easier and more reliable to correctly score the infection status of suspected samples. In a similar approach, NANOTRYP showed that specific Nbs can be used in FACS to score suspected samples for infection. This was done for various clones of human infective T. b. gambiense and T. b. rhodesiense parasites.
Figure 1: T. b. gambiense brucei (A,C) and T. b. brucei (B) were stained with ALEXA488 fluorescent tagged Nb63(A), N69(B) and Nb72(A). NbBruc2.2 was subsequently used in FACS on T. brucei MITat 1.1 MITat 1.4 and MITat 1.5 infected blood samples (red = negative control).
Finally, after the screening of 6 libraries constructed using different T. brucei variants, two promising Nanobodies were selected that showed good cross-reactive binding on all samples tested. These Nbs (Figure 2 below) were transferred to WP2. Both Nb863 and Nb971 tagted conserved proteins. Screening on various purified VSG molecules (Variant Surface Glycoprotein) showed that they were not targeting a conserved region of these components of the trypanosome coat.
Figure 2: After screening of 6 libraries constructed using different T. brucei variants, two promising Nanobodies were selected that showed good cross-reactive binding on all samples tested
For T. congolense, 3 libraries were constructed and very stringent parameters were used to select for a Nb that would be suitable for binding all T. congolense parasites available to us in a laboratory setting. Several Nbs were found to be useful for fluorescent parasite detection as shown in Figure 3. However, finally only a single Nb was withheld that met all our criteria (Nb474) for sandwich Ag-capturing development. This Nb was subsequently transferred to WP2.
Figure 3: Assessment of T. congolense Tc13 specificity of 3 Nbs of the Tc2 library in an on live T. congolense parasites omitting any cold-step incubation.
T. vivax library construction was performed having only a single laboratory stock as our target. From this library 11 Nbs were obtained of which most could be used in fluorescent detection of parasites in infected blood samples. All these Nbs were transferred to WP2 aiming at developing a sandwich Ag capturing test.
For T. evansi a first library was constructed using a vaccination protocol in which a mix soluble protein extracts, obtained from 5 different T. evansi stocks, was used. With this strategy, 4 Nbs were obtained that were capable of binding a 6th T. evansi parasite that was not part of the initial vaccination protocol. Perfomring various ELISAs in which the detection limit for the 4 different Nbs was assessed, only Nb392 was withheld for further use in WP2. At the same time, Nb392 was also produced as a GFP fusion construct for the use in fluorescent microscopy and FACS. Following the generation of Nb392, we engaged in the construction of a new anti-T. evansi library with a slightly different approach. Rather than using a mix of antigens for every vaccination boost, we purified T. evansi soluble protein extracts and subsequently use one specific extract fo a single vaccination/boost only, hence exposing the alpaca to 6 different ‘parasites’ during the subsequent weeks of the protocol. Interestingly, this strategy resulted towards the end of the NANOTRYP proposal in 16 different Nb families that will be be used in future strategies.
Figure 4. Left panel: Solid-phase ELISA coating serum and lysates from T.evansi STIB 816, T.evansi 0101399B, T.evansi 150399B, T.evansi 120399C and T.vivax (negative control). Right panel: Solid-phage ELISA coating two-fold serial dilution of antigen (T.evansi STIB816) and subsequent recognition by Nb392.
One of the goals of NANOTRYP WP1 was to generate a Nb-based Ag capturing system. While for a long period of time it seemed that this goal would not be attained, in the end ant anti-T. evansi Nb392 appeared to target a very conserved protein that is part of the flagella of the parasite. Hence this Nb was tested in fluorescence microscopy for binding to various parasites. Figure 5 shows identical staining patters on T. brucei, T. congolense, T. vivax and T. evansi. Future identification of the exact target may hence contribute to the development of a truly cross-recognizing Ag capturing device for the diagnosis of trypanosomiasis.
Figure 5: Binding of Nb392 to fixed permeabilzed T. evansi parasites (left panel) and T. congolense parasites (right), indicting the recognition of a highly conserved flagellar protein. Staining on T. brucei and T. vivax (both not shown here) gave a similar result.
Conclusion: WP1 was mostly executed according to the initial planning. During the progress made, vaccination protocols were altered mainly with respect to the challenge strategy during the subsequent vaccination boosts. Form both the T. congolense work and the T. evansi work most valuable lessons were learned that will guide future work. Using mixed prtein extracts derived from several non related stabilates improves the change of obtaining Nbs with good binding characteristics against consrevd targets. However, in the case of this project, only one truly cross-reactive Nb was obtained, suggesting that immunodominance of a limited number of compounds results in a biased outcome. Obtaining only one Nb does not allow the construction of a sandwich system (the aim of WP2), but it allowed to identify the target which in the future now can be used for a much more targeted immunization strategy. This in turn will greatly enhance the chance of finding a second binder that will allow the construction of a sandwich system. Using different trypanosome stocks for vaccination and selection of binders, as was done for T. congolense, resulted in a single Nb that could be used in a sandwich system. Indeed as Nb474 appears to recognize a target with repeated epitopes it was successfully transferred to WP2. In a third approach (used for T. evansi) successive alpaca vaccinations were performed with each time a different trypanosome variant. This resulted in the selection of 16 families of cross-recognizing Nbs. Whether or not all of those are ssuitable for transfer to WP2 is still under investigation. In contrast to the success with T. congolense and T. evansi, the generation of applicable anti-T. vivax Nbs was less successful. The main issue encountered here is the fact that in general, T. vivax cannot be propagated in laboratory rodents, making it impossible to have access to protein extracts of various isolates that have been properly characterized. Hence, using different isolates for vaccination and Nb selction was not an option here. In addition, it appears that the surface organization of T. vivax must be quite radically different from other trypanosome as we were able to use T. vivax as a ‘negative’ control for specificity during the selection of binders against all other parasites. The molecular reason for this is has not been elucidated at this stage.
For the generation of anti-T. brucei nanobodies (including binders for the human infective T. b. gambiense and T. b. rhodesiense), the most successful approach was the generation of a Nb library starting form material obtain from an experimentally infected camel (using T. evansi for infection – subsequently drug cured before any infection-associated pathology signs became apparent). This strategy resulted in two Nb-pairs that were successfully transferred to WP2.
A final remark for WP1 that is important to reiterate on is the fact that even when Nbs that are not suitable for the development of a sandwich Ag-capturing tool, they can still be very efficiently used in immunofluorescent detection of trypanosomes. This was shown in NANOTRYP both for the collection an anti-T. vivax Nbs, as well as the cross-recognizing anti-T. evansi Nb.
WP2: Development of a Nb-based lateral flow assay
Original Objective: The objective of WP2 is to use the batteries of species-specific nanobodies from WP1, and select couples of Nbs to make specific dip-stick tools for the detection of the 4 major African trypanosome species
Results: WP2 started with the transformation of an existing VSG-specific Nb-sandwich system (that was used as a proof of principle to put the NANOTRYP consortium together) into a dot-blot system. A dot-plot is the laboratory format that closes resembles an RTD for field use. As this initial system was however unable to detect human infective T. b. gambiense and T. b. rhodesiense parasites, this WP swiftly moved on to indentify Nbs that were capable detecting Both HAT parasites, and subsequently tested the Nbs identified in ELISA (see above WP1 Figure 2) in a dot-blot setting.
Figure 6: Sandwich Dot-blot and ELISA system using parasite lysate as antigen. Left panel (A): T. b. rhodesiense lysate captured with various His-tagged Nbs (as indicated) and detected with a mix of both Nb61 and Nb64 biotin-tagged Nbs. Left panel (B): idem. For T.b.gambiense. Right panel: ELISA with T. b. gambiense lysate using His-tagged Nbs for coating and biotin-tagged Nbs for detection
After having obtained convincing data showing that several Nb-sandwich systems were capable of capturing parasite antigen in infected samples, an MTA was drafted with Standard Diagnostics (SD, Korea), i.e. the commercial partner identified by FIND (see WP4) that would engage in actual RTD development. At SD, Nbs were processed according to internal protocols in order to make them suitable tools for antigen capture and detection. In short, 16 different Nb-capturing/Ag-detection combinations were tested (Figure 7)
The test format designed followed the conventional later flow Ag capturing design that has been successfully commercialized by SD for a whole range of diseases, using conventional antibodies rather than Nanobodies.
Results obtained are visualized below:
Figure 7: Various combinations of Nbs were used for coating and detection, with results boxed in yellow providing the information that the combination Nb971/971-gold provided sensitive and specific Ag recognition. This is the same Nb pair that was indentified in WP1 – see Figure 2 – as the best Nb combination in ELISA.
Unfortunately, despite the initial hopeful results, SD realized that the Nb-gold conjugate used for detection was unstable and that production using a standard protocol yielded unacceptable batch-to-batch variations. After several rounds of discussion and two visits by the VIB partner to SD, it was decided that two technical issues hampered further development, i.e. the fact that (i) both capturing and detecting Nbs in this system bind the same epitope, resulting in saturation of the Ag in the first step of the assay, leading to subsequent lowering of capturing capacity, and (ii) that Nb-gold conjugation needs to be optimized, most lileky using a protocol that is not part of the standard production methods at SD. The last trail runs at SD were performed in April 2013, towards the end of the 6 month extension period. In the near future, we will initiate alternative protocols, involving most likely a new partner, in order to solve the technical problems encountered at SD.
Later flow test development for T. congolense, T. evansi and T. vivax were not initiated, but in an alternative way forward, NANOTRYP did develop a highly sensitive Nb-based ELISA system for the detection of T. congolense infections as part of WP2 and also WP7. Using Nb474 for both capturing and detection, we were able to construct a system that could be used for both diagnosis of active cases, and could be used as test-of-cure.
Figure 8: VIB tested the Nb474 ELISA system on a large range of well characterized T. congolense laboratory strains as well as in an experimental infection setting performed at the Onderstepoort Veterinary Institute in South Africa. Here, T. vivax and T. brucei infections were both used as negative controls
Following the results obtained in Figure 7, the Nb474 system was extensively tested together with the UEM and IPR partners (see WP7).
Conclusion: As outlined above, NANOTRYP did not deliver a final format for a Nb-based diagnostic lateral flow test for trypanosomiasis. The preliminary results obtained by Standard Diagnostics (SD, Korea) presented in Figure 6 were hopeful (in particular as they confirmed the VIB ELISA results of Figure 2) but technical problems with the colloid-gold detection system used in conventional RTDs could not be resolved in a timely manner. One of the main consequence of this ‘failure’ is the fact that a large portion of WP4 – the testing in a field setting of the developed device – was not executed. Looking back on the project, the fact that SD was unable to transform the Nb-based ELISA into a dipstick was impossible to anticipate, as from the start we did have successful preliminary data from a prototype VSG specific dipstick developed by VisionBiotech, South Africa. Taken that both SD and VisionBiotech are now owned by the same industrial group, moving back to South Africa with the project was not an option. One lesson learned during NANOTRYP is the fact that the engagement of SD into the project happened far too late into the project. On one hand, more than a year of three-way negotiations between FIND, SD and VIB were needed to come to a proper MTA. Secondly, SD initiated the RTD development only after a suitable Nb pair for HAT had been identified. From an SD perspective, this is understandable as optimizing the technology in advance with an irrelevant Nb-pair would have been a lost investment if later-on we would have been unable to deliver a suitable Nb-pair. From The NANOTRYP side however, it would have provided more time to solve issues with the Nb-gold coupling, a technique that once resolved should be pretty standard for all Nbs. Finally, only when the full SD RTD development strategy was revealed (this happened after the failure to produce a prototype), it became clear that the successful ELISA sandwich system did not fully meet the conceptual requirements for a lateral flow design. Indeed, while the detection of a multimeric target with repeated epitopes allows in ELISA for the use of a Nb homo-sandwich setup, resulting in high sensitivity and specificity, a lateral flow design needs to be constructed around the use of two Nbs targeting different epitopes of the same target, i.e. the design of a Nb hetero-sandwich system is a must. On the positive site, the combined WP1 and WP2 packages showed that it is possible to develop a Nb-based sandwich system that can be used successfully trypanosomiasis detection. In the immediate future, we will now concentrate on different ways to solve the Nb-detection system in a RTD context, and secondly, we have now designed a new Nb selection strategy to fish out hetero-sandwich pairs right from the start of the procedure. While NANOTRYP has come to an end, the WP2 package has provided us with extremely valuable information for future efforts. The NANOTRYP consortium remains fully convinced that development of new diagnostic tools are crucial for the future fight against trypanosomiasis, and that an Ag-detecting tool in the end is the way to go, as only such assay can be used for (i) active case diagnosis, (ii) test-of cure after treatment or drug failure detection, and (iii) diagnosis of re-infection after successful treatment.
WP3: Development of a Nb-based magnetic bead assay
Original Objectives: The objective of WP3 is to develop an alternative trypanosome detection method as back-up assay for the dipsticks developed in WP2. Here the same Nbs from WP1 will be coupled to magnetic beads, allowing the development of an alternative magnetic-bead detection method for all 4 major African trypanosome species.
Results: During the first reporting period of NANOTRYP, various Nbs were used to set up a magnetic bead capturing assay. The assay is based on the principle of using Nb-coated paramagnetic beads for incubation with target blood samples, containing parasite antigens. Subsequently, beads are collected using a Pickpen® device, washed and Ag-binding is measured using a colorimetric substrate conversion reaction similar to those used in ELISA. After optimizing washing conditions as presented below, the assay sensitivity was brought to 100 parasites/sample. While this sensitivity is good enough for diagnostic screening tool, the entire procedure of development turned out to be too laborious to properly be performed under realistic field conditions.
Figure 9: Pickpen® capturing of T. brucei AnTat 1.1 using a variety of incubation and washing conditions and a combination of the pan-reactive Nb33 and Nb1 from Library 1. Virtually identical results were obtained with Nb7 and Nb8 from the same library.
Conclusion: Taken the technical difficulties of performing this assay under optimal conditions, in particular the requirement for large volumes of washing buffer, prompted the consortium to halt this WP. This decision was approved by the Mid-term reviewing panel that assessed the NANOTRYP program in September 2010.
WP4: Diagnostic field evaluation
Original Objectives: To evaluate T. brucei specific dip-stick and magnetic bead assays, demonstrate their use, and develop a strategy for their access to the population at risk
Result: The first task of WP4 was to indentify a commercial partner willing to transform a laboratory scale Nb-diagnostic tool into a field applicable format. Based on the experience of FIND in other projects, Standard Diagnostics (SD, Korea) was contacted. After three-way negotiations involving VIB, FIND and SD, and MTA was signed between the VIB and SD and the most promising Nb pairs obtained in WP1 and WP2 were transferred (see above, results WP2). However, due to the technical problems encountered during lateral flow development (see above, results WP2), the subsequent tasks allocated to WP4, i.e. the validation, evaluation, demonstration and implementation were not executed.
Conclusion: Unfortunately, the main tasks of WP4 with respect to bringing a Nb-based diagnostic tool to the field were not fulfilled due to the lack of a proper functioning test prototype. The efforts to develop such prototype will continue in the future.
WP5: Nanobody-drug coupling
Original Objectives: The aim of WP5 is to develop a Nb-based targeting strategy for site-specific drug delivery, in order to improve current anti-parasite therapies. Two different approaches will be employed: i) coupling existing anti-trypanosome drugs to Nbs; and ii) coupling trypanolytic peptides to Nbs to target the parasite.
Results: During the NANOTRYP project, a nanobody based drug delivery system consisting in a cyclodextrin molecule linked to the six histidines tail present in the carboxiterminal end of nanobody NbAn33 was generated. Next step was the occlusion a trypanocidal drug into Nb33-cyclodextrin complex without affecting its trypanosome binding capacity. For this purpose, two trypanocidal drugs were synthesized and occluded into Nb::CD conjugate: nitrofurazone and tionitrofurazone. Both are hydrophobic nitroaromatic compounds. By drug occlusion assays into Nanobody:cyclodextrin conjugate we determined that the occlusion rate of both Nifurtimox and Nitrofurazone was 40%. Tionitrofurazone occlusion rate was lower but improved after increasing the ionic strength. We have analyzed the trypanocidal activities of nitrofurazone and tionitrofurazone complexes (figure 10) and both complexes kill trypanosomes in vitro.
Figure 10: Growth curves of bloodstream AnTat 1.1 in the absence (circles) and the presence of Nb:CD:Nitrofurazone and Nb:CD:Tionitrofurazone complexes (squares) and Nb:CD conjugate (triangles).
However, two were the major limitations of the Nb::CD trypanocidal drug delivery system: i) Its specify for the occlusion of hydrophobic drugs. ii) Its limited loading capacity. Only one drug molecule per Nb::CD conjugated and according to the occlusion rate for nitrofurazone and tionitrofurazone about 60 % of Nb::CD molecules are not loaded with drug. In order to overcome these limitations we decided to alter the the original plan. Two different polymeric nanoparticles coated by a Nb that specifically target T. brucei cell surface (NbAn33) and loaded with the trypanocidal drug pentamidine have been synthesized. The natural polymers chitosan and PLGA have used to generate noparticles of chitosan and PLGA termed NbAn33-pentamidine-chNPs and NbAn33-pentamidine-PLGANPs respectively. Both drug carries displayed a potent in vitro trypanocidal activity, as the half-maximal inhibitory concentration (IC50) of pentamidine-loaded, nanoparticles was 14 and 7 fold lower 7 respectively than free pentamidine (P<0.0001) (figure 11).
Figure 11: Trypanocidal activity of pentamidine formulations. A) Gray column: IC50 value for free pentamidine. Red column: IC50 value for pentamidine-chNPs. Blue column: IC50 value for NbAn33-pentamidine-chNPs. Errors bars indicate SD from 3-9 independent experiments. Fold reductions are indicated in the graph. B) Green: nanobody-PLGA-pentamidine nanoparticles; Purpure: PLGA-pentamidine nanoparticles; Blue: pentamidine.
We next tested the trypanocidal activity of the Nb::pentamidine-chitosanNP. The minimal full curative dose of pentamidine in a mouse model of acute infection of T. brucei was previously established as four doses of 2.5 mg∙kg-1 administrated daily by i.p. injection in four consecutive days, starting upon detection of parasites in blood (day 3 after infection) (Fig. 11b). When mice were treated with a 10-fold lower dose of pentamidine (4 x 0.25 mg∙kg-1) the parasites disappeared from the peripheral blood after the third dose. However, the infection relapsed and the mice began to die at day 22 after infection, curing only 20% of the treated animals (Fig. 12). Having established the suboptimal pentamidine curative dose, we treated mice with equal dose of pentamidine loaded into NbAn33-NPs. Clearance of parasites from this group was complete after the first dose and the treatment successfully cured 100% of the animals (Fig. 12). Infected mice were treated with the same pentamidine dose loaded into NPs non-coated by the nanobody. In this group, parasites disappeared from the blood after the first dose, however 40% of the treated mice succumbed to the infection (Fig. 12). Next, a dose 100 times lower than the minimal curative dose of pentamidine was tested. At that low concentration (4 x 0.025 mg∙kg-1), free pentamidine did not cure mice from trypanosome infection. Remarkably, treatment with 4 doses of NbAn33-pentamidine-chNPs at 0.025 mg∙kg- cured all treated mice. However, the same low dose of pentamidine loaded into chNPs non-coated by the NbAn33, , did not cure mice, with 100% of the treated animals dying from the infection.
Figure 12: Therapeutic effect in T. b. brucei acute infection mouse model. Survival (Kaplan-Meier plot) of female C57BL/6J mice infected with T. b. brucei AnT1.1 (inoculum 1x104 parasites). The treatment started once the parasites were detected in blood, at 3 day after infection and consisted in a daily dose in four consecutive days. Treatment with pentamidine, pentamidine-chNPs (pentamidine-loaded PEGlycated chitosan nanoparticles), NbAn33-pentamidine-chNPs (nanobody-coated pentamidine-loaded PEGlycated chitosan nanoparticles), NbAn33-chNPs empty (nanobody-coated non pentamidine-loaded PEGlycated chitosan nanoparticles) and vehicle (physiological serum).
All know mechanisms associated to pentamidine resistance are dependent on drug uptake associated to surface membrane proteins (transporters and aquaglycerolporins). A tripanocidal drug vehicle consisting in pentamidine loaded nanoparticles coupled to a nanobody that bind to the surface of the parasite, like Nb33, sould overcome drug resistance associated to pentamidine uptake. To test this hipotheis we generated a T. brucei Antat 1.1 cell line resistant to 25 ng/ml pentamidine by slowly increasing drug concentration added to the culture medium. We named this cell line T. brucei R25. We next compare the IC50 of penamidine (Fig. 13), pentamidine loaded nanoparticles and pentamidines loaded NPs coated with Nb33 in our resistant cell line. As observed in wild type cells, the IC50 obtained with both Nb::NPs was significantly lower compared to pentamidine alone (12.5 nM, 5.9nM and 87.6 nM respectively).
Figure 13: IC50 reduction of the described formulations.
The second proposed approach was to develop a Nb-based anti-trypanosomiasis treatment through coupling trypanolytic antimicrobial peptides (AMPs) to specific nanobodies that target trypanosomes. In the initial proposal one of the strategies proposed was to couple lytic peptides to Nbs by chemical methods. However conjugation with nanobodies may affect the lytic capacity of the peptides at the surface membrane. To circumvent this problem a protease cleavage site was added to the end of each synthetic peptide. Thus, all synthetic lytic peptides employed carried at their carboxy-terminal end a tail of five residues containing the Cathepsin B cleavage sequence. Cathepsin B is a lysosomal protease that is also present in T. brucei. If the Nb-lytic peptide conjugate do not kill the parasite by cell membrane disruption when they are on the cell surface, the complex of surface protein-Nbs-(Cathepsin B cleavage site)-lytic peptides will be internalised by the very active endocytic pathway. Upon delivery to the lysosome, the lytic peptides/nanobodies/receptor complexes will be processed by Cathepsin B. Released lytic peptides will then be free to insert into and disrupt the lysosomal membrane causing cell death.
First, the trypanocidal activity of two AMPs, temporin A and temporin 1Lc were assayed. Both Nb::Temporin bioconjugates were able to reduce the IC5O of the AMP about 4-fold lower than temporines (figure 14).
Figure 14. Effect of AMPs on T. brucei bloodstream forms viability. Curve dose response (viability percent versus Temporin 1Lc concentration).
Conclusion: WP5 was mostly executed according to the initial planning. During the progress made, two different polymeric nanoparticles coated by a Nb that specifically target T. brucei cell surface (NbAn33) and loaded with the trypanocidal drug pentamidine have been synthesized. The natural polymers chitosan and PLGA have been used to generate noparticles of chitosan and PLGA termed NbAn33-pentamidine-chNPs and NbAn33-pentamidine-PLGANPs respectively. Both drug carries displayed a potent in vitro trypanocidal activity, as the half-maximal inhibitory concentration (IC50) of pentamidine-loaded, nanoparticles was 14 and 7 fold lower respectively than free pentamidine (P<0.0001). Furthermore, the curative dose of pentamidine-loaded nanobody-nanoparticles was 100-fold lower than pentamidine alone in a murine model of acute African trypanosomiasis. Crucially, pentamidine-loaded nanoparticles displayed undiminished in vitro and in vivo activity against a trypanosome cell line resistant to pentamidine.
We have also generated two anti-trypanosome nanobodies carrying each one an antimicrobial peptide (temporin A and temporin 1Lc) covalently attached via Cathepsin B cleavage sites. Both Nb::peptide conjugates were active in vitro against T. brucei bloodstream forms.
WP6: Nb-tools for diagnosis and treatment of T.b.rhodesiense and T.b.gambiense infections
Original Objective: The objective of WP6 is to take the result from WP2, WP3, and WP5 to more field-related trypanosomiasis models. Here, a monkey model for T. brucei (rhodesiense), in which blood-brain-barrier complications may hamper drug efficacy, will be assessed. WP2 and WP3 will be applied in the sense that both a T.brucei specific dipstick assay and a trypanosome magnetic bead detection assay will be assessed in this infection model. WP5 will be evaluated as new treatment methods for experimental trypanosomiasis, in a model that is much closer to the human situation than the usual mouse model. In this WP, Nbs could also be subjected to host-specific mutagenesis to reduce possible immunogenicity that will be checked for at the start of this WP..
Results:
Determination of Immunogenicity of Nb in mammalian hosts
For chemotherapy of HAT, it is envisaged that the Nb-drug conjugates will need to be repeatedly (but with short intervals) injected into infected subjects. Thus, their immunogenicity requires evaluation, under conditions in which several subsequent daily injections would be administered. The current study evaluated the immunogenicity of NbA33, a recently generated nanobody for use in HAT studies under NANOTRYP project. The study was carried out in vervet monkeys (Chlorocebus aethiops) which were to be used as the ultimate animal model for treatment and diagnostic tests during the project.
Three groups of experimental animals were used divided and inoculated with various antigens thus;
• Group 1: Inoculated with NbAn33 at 50ug intramuscularly (IM) for 3 consecutive days
• Group 2: Negative control, phosphate buffer saline (PBS) administered at 1ml for 3 days
• Group 3: Positive control, infected with Leishmania donovani promastigotes
The monkeys were sedated and sampled for blood at weekly interviews for 5 weeks. This regime was meant to simulate the treatment protocol for diminazene aceturate as carried out during experimental infection treatments in this lab animal model. Peripheral mononuclear lymphocytes (PBMCs) were harvested and blast assays carried out while serum was analysed for anti-Nb stimulation.
The mean ODs of the monkeys treated with NbAn33 at all the sampled days were not significantly different from those inoculated with PBS buffer. However monkeys inoculated with L. donovani had a consistent elevation of OD values to significantly higher between 7 and 35 days post-inoculation in comparison to either PBS or NbAn33 groups (Fig 15a and Fig 15b).
Figure 15a: Mean OD values in vervets testing for immunogenicity against NbAn33
Figure 15b: Blast assays in vervets testing for immunogenicity against NbAn33
Conclusion:
The study showed that NbAn33 have undetectable immunogenicity in mammalian hosts and therefore it may be safe as a vehicle for targeted delivery of HAT drugs.
Diagnosis and treatment of early and late stage Tb rhodesiense infection in vervet monkey model
Vervet monkeys were experimentally infected with T. b. rhodesiense IPR 001 isolate. The animals were monitored for disease through ear prick (Ndungu 1994; Sayer & Schimdt, 1983), sampled at weekly intervals under sedation for blood (for serum separation and haematology) and cerebrospinal fluid (CSF). At 28 days after infection the animals were treated with diminazene aceturate (DA). It is assumed that treatment at this stage clears trypanosomes from the haemolymphatic system and allows those in the CSF to thrive, become neuroinvasive and precipitate late stage disease (meningoencephalitis). The animals were monitored post DA-treatment for late stage disease clinically and by immunological assays. The experiment was stopped by humane euthanasia of the experimental animals approximately 51-56 days when relapse parasitaemia alongside salient clinical signs of paresis and paralysis were observed in the animals. Non-infected control animals subjected to similar experimental procedures were also included in the study. All the experimental procedures were approved by the Institutional Ethical Review Committee (IRC) of IPR, including an oversight International Advisory Board (IAB) with a membership drawn from Europe and USA. Two MSc students were enrolled for this task.
Analyses for immunospecific IgM and IgG was carried out using enzyme linked immunosorbent assays (ELISA), cytokines IL-6 and IFN-γ were assayed using flow cytometry as well as ELISA whereas albumin and total protein were quantified using colorimetric protein assays.
The four experimentally infected animals had a typical clinical presentation of the disease with patent parasitaemia 4 days after infection which peaked at 9 days after which the parasitaemia fluctuated with typical waves of parasitaemia. Late stage disease was established by demonstration of typical meningoencephalitic lesions including perivascular lesions (Figure 16)
Figure 16a: perivascular cuffing in the brainstem of a vervet monkey infected with Tb rhodesiense
Figure 16b: perivascular cuffing in the cerebellum of a vervet monkey infected with Tb rhodesiense
Detailed immunological analysis further showed a significant elevation of IFN-gamma and IL-6 in serum of experimentally infected animals as depicted in the Figure 17. These molecules were not detectable in the CSF during early stage disease.
Figure 17a: Serum IFN-γ spike 7dpi in early stage HAT
Figure 17b: Serum IL-6 spike 21dpi in early stage HAT
Characterisation, diagnosis and treatment of Tb gambiense in a NHP model
The T.b. gambiense is responsible for over 90% of total cases of HAT (Malvy et al., 2011). The infection is characterized by low parasitaemia with no specific clinical symptoms especially during the early stage when trypanosomes are confined to the hemolymphatic system (Inojosa, 2006). This has limited the use of standard diagnostic techniques, with an estimated 20 to 30% of patients being undiagnosed (Robays et al., 2004). The serological test—card agglutination test for trypanosomiasis (CATT)—for T. b. gambiense has varying sensitivities and cannot decisively differentiate between active and cured cases (Lejon, 2010). The parasitological detection techniques have limited sensitivity hence it is possible that some of these unconfirmed CATT-seropositive individuals are actually infected (Garcia et al., 2000). Vast improvements have been made by the development of a rapid diagnostic test (www.finddiagnostics.org accessed 6 September 2013). In order to study the pathogenesis of this chronic form of the disease, several attempts have been made at characterising a laboratory animal model, with limited success. Two attempts have been made in this project to characterise a nonhuman primate model of Tb gambiense, in two different species (vervets and Sykes monkey).
Alongside the immunological characterisation, molecular detection of the parasite DNA in various tissues and fluids was made. The loop mediated isothermal amplification (LAMP) technique was first described by Notomi et al. (2000). It amplifies DNA using an enzyme with strand displacement activity under isothermal conditions using four to six primers. LAMP has been used extensively in HAT studies. In the diagnosis of T. brucei using the PFR A protein (Kuboki et al., 2003), T. gambiense using ITS2 gene (Thekisoe et al., 2007), Trypanozoon subgenus using RIME (Njiru et al., 2008) and T. rhodesiense using the SRA gene (Njiru et al., 2008). LAMP has several advantages over other detection formats. These include: rapidity, high sensitivity, specificity, several product detection formats and amplification at isothermal conditions.
DNA was isolated from the blood, urine, cerebrospinal fluid and saliva samples of Sykes monkeys obtained at regular intervals during the study. T .b. gambiense primers described by Njiru et al (2011). The LAMP reaction was performed for 80 mins in a LoopAmp Turbidimeter® using purified trypanosome DNA from the T. b. gambiense IL 3253
Body Compartment Duration of infection when test was Positive
PCR LAMP
Blood/serum 7 – 123 dpi 7 – 168 dpi
CSF - 168 – 175 dpi
Urine - 28 – 105 dpi
Saliva 14 – 35 dpi 14 – 112 dpi
Parasitaemia was detectable between 7 and 98 dpi
CSF parasites were not detected by microscopy for the entire duration of the study
The overall detection rate of parasite DNA in all the samples was low. Whole blood has a higher detection rate (14%) as compared to the other samples- urine (8%), CSF (6%) and saliva (2%). The parasites could not be detected in the later stages of the disease in any of the samples. This may imply the infection in the animals had resolved. A similar study in the vervet model reported higher detection rates in all the four sample groups.
Conclusion:
The following conclusions can be made from the studies carried out under WP6:
1) NbAn33 has undetectable immunogenicity in mammalian hosts and therefore it may be safe as a vehicle for targeted delivery of HAT drugs
2) The clinical disease, biochemistry and immunological analysis of Tb rhodesiense nonhuman primate model was characterised.
3) Partial characterisation of the Tb gambiense model has been accomplished leading to the detection of parasites DNA in urine and saliva opening possibilities of non-invasive samples for diagnosis
WP7: Treatment testing in cattle and goat
Original objective: The objective of WP7 is to take the result from WP2 and WP3 to larger animal models, and in particular assess the use of Nb-based tools for the diagnosis infected goats, focussing on T. brucei, T.congolense T.vivax and T.evansi while using the tools from WP5, treatment for T. brucei infections will be assessed as well.
Results:
Collection of sera and field isolates
Eleven field missions were carried out aiming at the collection of serum samples and Trypanosoma isolates from naturally infected animals. Eight field missions were done in Matutuíne District, Maputo Province (100 Km South of the City of Maputo) and three in the districts of Nicoadala and Inhassunge, Zambézia Province (1600 Km North of the City of Maputo). The field work was executed at dip-tanks or corridors. From each animal, blood was collected from the coccygeal vein, using two 7 ml vacuum tubes (with and without anti-coagulant [EDTA]). All the samples were parasitologicaly analysed in the field, using the micro-haematocrit centrifugation technique, “Buffy-coat”. All the parasitological positive samples were inoculated into mice or in goats in the case of T. vivax, pure or mixed infections. All the samples were PCR analysed (18S PCR –RFLP) in the Biotechnology Center-UEM for molecular species specific identification. The serum was prepared from the tubes without anti-coagulant and stored and labelled according to the parasitological and molecular results. All serum samples from negative animals in both assays were discarded.
During the field missions 1048 bovines were screened and a panel of 124 reference sera (103 T. congolense positive; 7 T.vivax; 14 T. congolense and T. vivax mixed infections) was established. Furthermore, two hundred cattle serum samples were collected from a Trypanosoma free area of Maputo Province (Boane District), to constitute a panel of bovine negative sera. From the field diagnosed positive animals a collection of 45 isolates (43 T. congolense and 2 T. vivax) was added to the cryobank.
Experimental Infections
All experimental infections were performed in fly proof conditions. Five goats and 50 bovines were infected with T. congolense and three goats with T. vivax . Before each experimental infection 20 ml of blood were collected from jugular vein into plain tubes to prepare the pre-infection serum sample (approximately 10 ml). For the experimental infection, stabilates from a specific isolate were removed from the cryobank, thawed in water at 38oC and and injected I.P. in two Balbc mice. The infected mouse blood was diluted in PSG, to a concentration of 104 trypanosomes/ml, and subsequently I.V. inoculated in an experimental goat or bovine. For the T. vivax infections the process of isolate amplification after retrieval from the cryobank was done, using goats. During the course of the experimental infection the parasitaemia and the PCV was determined by micro-haematocrit centrifugation technique and followed daily until patency. After patency the above mentioned parameters were checked weekly. A serum sample (5 ml) was prepared weekly and each animal was followed until ninety days post-infection. All the serum samples were appropriately labelled and stored at -80oC.
The biological material collected up to now combined with the present availability of nanobodies will allow for the evaluation of any nanobody- based diagnostic assay. As part of the NANOTRYP effort to evaluate the potential of a Nb-based diagnostic tool, Steven Odongo (VIB) visited UEM and tested a Nb ELISA that had been optimized previously at the VIB. The results obtained show that the Nb474/474 combination ELISA developed in WP1/2 can be used to detect trypanosomiasis infections. The specificity of the assay is sufficient to discriminate T. congolense trypanosomiasis form other common bovine infections such as theileria and babesia. T. vivax Trypanosomose was not detected with the assay, making the Nb474/474 a specific detection tool for T. congolense. While testing the ELISA, the storage requirements for test samples were addressed as well. Storage for up to 2 weeks at 4°C of infected samples did not hamper the correct identification of infections. However, samples that were stored for up to 3 weeks at 4°C started to show an unacceptable level of false negative results. Under field conditions this is not be considered to be an issue as samples would most likely be tested within 24 hours after isolation.
In addition to the UEM sample collection, the Nb474/474 ELISA was also tested on a sample collected obtained from IPR, Makerere University (Uganda), Onderstepoort Veterinary Institute (South Africa) as well as a collection that was obtained through a more recent collaboration with GALVmed. In the latter collection, samples pre- and post-treatment were included, showing that within 48h after treatment, a negative ELISA score was obtained.
Conclusion: Combined, in all cases similar results were obtained showing that the Nb approach proposed in NANOTRYP is a successful, and offers the potential to be developed as a test-of-cure. However, as indicated in WP4, for the next step, i.e. development of an field applicable RTD, the Nb474 target has to be identified and a second detecting Nb has to be generated in order to allow the use of a hetero-sandwich system.
Figure 18: Cattle sera characterized by buffy coat technique (BCT) were cross-examined with T. congolense Nanobody®_ELISA (Nb_ELISA). A,B: Local cattle reared in trypanosome endemic regions of Mozambique (right) or Kenya (left) were bled and whole blood examined for trypanosomes by BCT followed by cross-examination of their sera for T. congolense by Nb_ELISA. C: Performance of Nb_ELISA evaluated on sera of cattle experimentally infected with T. congolense (IL1180). High OD450nm are consistent with sera where T. congolense infection is involved indicating the test’s ability to detect T. congolense infection. More so the sensitivity of Nb ELISA on experimentally infected cattle was 87%. D: Assessment of Nb_ELISA for possible cross-reactivity with Theileria, Babesia, or Anaplasma. Nb_ELISA was performed on hemolyzed whole blood confirmed positive for the named protozoan parasites. Negative (-ve) were whole blood from naive cattle. The Nb_ELISA was scored as positive 2/3 trypanosome samples, 5/44 theileria samples, and negative on babesia and anaplasma samples. Therefore, apparent cross reactivity observed with theileria could be attributed to mixed infection with T. congolense that was missed by BCT. These positive samples will be cross-checked by PCR.
WP8: Awareness campaigning, education and training
Original Objectives: The aim of WP8 is to ensure that the project results are disseminated to the affected community. This WP includes training of students in HAT/AAT endemic countries, training of PhD students as well as generating education material for local communities. Finally, also an EU mini-symposium envisaged the communication of FP7 research efforts to the wider public.
Results:
In Uganda, FIND awareness campaigns were carried out by Gulu University in the T.b. gambiense region in the north of the country. The specific objectives of the campaigns included a) understanding the local community’s perception of the disease, b) appraising the community’s level of understanding of the symptoms of HAT and nagana, transmission, diagnosis, treatment and control, and c) sensitizing leaders and communities living in these areas so that they seek early diagnosis and treatment for both human and livestock diseases, in order to remove reservoirs of infection. Familiarization and interpersonal meetings were held with key district and selected sub-county officials, to brief them on the purpose of the project and get their understanding on the severity of the problem. Community mobilization campaigns at the district, sub-county and at community levels focussed on the importance of early diagnosis of HAT and Nagana, the animal-human infection relationship, understanding the ecology of tsetse flies and their role in transmission of the diseases, local community perception of sleeping sickness and Nagana, and community coping strategies and adaptations. Advocacy materials that were developed and distributed included a) posters on trypanosomiasis and how it is spread, b) posters on actions in control of trypanosomiasis, c) Question and Answer booklet (Frequently Asked Questions) for community mobilization, d) leaflets in the various local languages in the endemic areas highlighting how trypanosomiasis is spread and control methods and T shirts, e) radio talk shows in the two dialects zones of Adjumani and Nwoya – Amuru, and f) training workshop for village Health Teams.
The Ministry of Health (MOH) of Uganda and the National Livestock Resources Research Institute (NaLIRRI) focused on the T.b. rhodesiense region in the south east and central Uganda. In addition to using advocacy materials that were similar to those used in northern Uganda, the MOH held advocacy workshops in primary schools and distributed a Comic Book of HAT that was developed by the WHO. They also held workshops with senior government officers and parliamentarians, resulting in a significant increase in budgetary allocation for African Trypanosomiasis control by the government, within the Annual budget. A similar outcome was also reported by Tanzania.
In Tanzania, awareness campaigning was carried out by both the Ministry of Health and Social Welfare (MOHSW) and the Ministry of Livestock and Fisheries Development (MOLFD). Various government institutions involved in tsetse and trypanosomiasis research and control came together and established the Network for Mapping African Trypanosomiasis in Tanzania (NetMATT). Institutions that became members of the network included a) Ministry of Livestock Development and Fisheries (Coordinator), b) National Institute of Medical Research (NIMR), c) Tsetse and Trypanosomiasis Research Institute (TTRI), d) Ministry of HeaIth and Social Welfare (MOHSW), e) Institute of Resource Assessment (IRA) - University of Dar es Salaam (UDSM), f) Tanzanian National Parks Authority (TANAPA), g) Tropical Pesticide Research Institute (TPRI), and h) Sokoine University of Agriculture (SUA). The activities that NeTMATT undertook included a) developing tools to guide surveillance, intervention and the targeting of resources, b) consolidating mapping data to support the FIND/PATTEC HAT advocacy programme, c) using reports and arising publications as tools for advocacy, to guide further research and leverage funding, d) strengthening data collection and mapping of African trypanosomiasis in Tanzania through collaboration between various stakeholders (including government ministries, research institutions and universities), e) facilitating sharing of data between institutions, and f) improving the quality of trypanosomiasis data analysis by enhancing the amount and quality of mapping and spatial analysis that is undertaken using the data. The outcome of the activities relating to characterization of health centres was series of Geographic Information System (GIS) maps showing the health centres where HAT has been reported during the past 10 years, and the changes in numbers of cases by year.
A key observation was a dramatic fall in the number of cases of HAT, indicating that elimination of the disease in this country is imminent.
While livestock trypanosomiasis is widespread in Nigeria, cases of HAT have in recent years only been reported in Delta State, which is in the south of the country. Nigeria has intensified surveillance and control of both diseases in the entire country, with the aim of eliminating them. During the reporting period, FIND supported Nigeria to intensify awareness campaigning, and to actively screen communities in Delta State for HAT.
In addition to the efforts coordinated by FIND, UEM dedicated a significant effort to awareness campaigning in the Matutíne District in the South of Mozambique which borders the North-East region of Kwazulu-Natal in South Africa. These two geographical areas are relatively isolated from the main belt of trypanosomiasis in East and Southern África and represent the south most distribution of the genus Glossina in África. Glossina brevipalpis and G. austeni are the only species present and Trypanosoma congolense, T. vivax and T. brucei brucei and T. simiae are the species associated with livestock trypanosomiasis in this region. T. congolense is undoubtedly the most important species, with prevalences higher than 30% reported in some locations. Livestock production, mainly represented by cattle and goats, is considered an important activity for the local rural families and trypanosomiasis is generally perceived as a major constraint for the improvement of herd production and productivity. The campaign launched at Matutuíne District, targeted especially school children and cattle owners and had as main objective to increase the awareness towards trypanosomiasis by creating an environment conducive to dialogue, simple but technically accurate messages, and making use of adequate material.
Launch of awareness campaign
The campaign was launched in the presence of the Permanent Secretary of the District, the Heads of the five District Administrative Areas, the head of District Services for Economical Activities , the Provincial Director of Education, the State Veterinary at District level and Technicians, including the livestock technicians from the five District Administrative Areas, the representative of the Provincial Director of Agriculture, The representative of the Director The Biotechnology Center – UEM (CB-UEM), two researchers from CB-UEM and representatives from the main local Communities.
All the activities were filmed and photographed by the “Centro de Comunicação e Imagem – UEM”. Moreover, a full text on the NANOTRYP promoted campaign appeared in the Mozambican main daily newspaper “Notícias”, in the UEM newsletter and the campaign was further advertised by a half hour interview in Mozambican main TV channel “TVM 1”.
Dissemination materials
Two types of posters, both printed in Portuguese and in the local language Xi-Ronga, 250 of each type were put up in all the schools shown in the Table below, and at the headquarters of the five District Administrative Areas. Six thousand leaflets in Portuguese and one thousand in Xi-Ronga, containing the main messages on tsetse and trypanosomiasis, were also made and delivered to all students, teachers and livestock owners targeted by the campaign.
Schools in which awareness campaigns were conducted
School name Number of students District Administrative Area
EPC Bela-Vista 980 Bela-Vista
EPC Mudada 272
EPC Salamanga 463
EPC Tinonganine 205
Instituto Agro-industrial de Salamanga 200
EPC Nsime 346 Catembe-Nsime
EPC Zitundo 139 Zitundo
EPC Gala 33
EPC Catuane 211 Catuane
EPCNdlala 225
EP1 Mhala 93 Machangulo
EP1 Ngomene 123
EPC Ndelane 206
EPC Machangulo 246
EP1 Mabuluco 41
EPC Nhonguane 117
Main activities of the campaign
Seminar with the teachers
One hundred and twelve teachers participated in the campaign. A twenty five minutes seminar with the teachers was held in all the schools. A power point presentation containing exactly the same content of the leaflets and the posters was made. Just before the seminar a pre-exposure questionnaire was administered to the students. It is expected that at school level the teachers will assume an active role in conveying the message to the students.
Seminars with the cattle owners
A seminar with the presentation of a power point containing exactly the same content of the leaflets and the posters was conducted. Just as in the schools the seminar was preceded by the administration of a pre-exposure questionnaire.
Pre-exposure questionnaire
Immediately before the campaign, a questionnaire on the content of the dissemination material was administered to all members of the target audience (students, teachers and cattle owners). The questionnaire has nine questions with different marks. The difference between the marks and the total of correct answers will be analysed by the appropriate statistical methods and used to measure campaign efficacy.
Post-exposure questionnaire
The same questionnaire prepared for the pre-exposure phase was administered sixty days after the launching of the campaign in each school and dip-tank.
Dip-tanks
Nine dip-tanks, including Felipe, Mudada, Tinonganine, Salamanga, Gala, Zitundo, Catuane, Ndala e Machangulo, were involved in the campaign with the participation of approximately one hundred cattle owners.
Besides efforts to educate local comities and bring awareness of the trypanosomiasis problem to the different stakeholders, NANOTRYP also dedicated a considerable effort to education both at the level of PhD and Master training, as well as research training from both IPR and UEM researchers at the European VIB and FIBAO partner laboratories.
Training of IPR researcher at VIB
Christopher Kinyanjui Kariuki (IPR) visited VIB for two consecutive training periods of three months each (Jan-March 2011 and Oct.-Dec. 2011). During this period, Mr Kariuki developed a set of Nanobodies that can be used as tools for the work ongoing at IPR, in which the T. brucei. rhodesiense IPR001 parasite stock is being used. A detailed progress report of the work performed was included in the previous progress report / description of WP1.
This work formed part of Mr Kariuki’s MSc training at Jomo Kenyatta University of Agriculture & Technology (JKUAT), Nairobi. Mr Kariuki returned home to initiate the nanobody generation work leading to immunization of camels for generation and extraction of mRNA. However the next stages of nanobody generation still require heavy investment which IPR may not have, and therefore the collaboration with VUB to access the VIB Nanobody generation Centre is likely to remain alive beyond the life of this project.
Training of UEM and IPR researcher at FIBAO
Mr. Mwadime and Mr. Mucache were successfully training in laboratory techniques that included Immunofluorescence of the Tb brucei parasite Antat 1.1 with the Nb (Nanobody) and fluorochromes e.g. Rodamine, In vitro culture of the Tb brucei. Maintenance of culture parasites and working with in vitro based systems, IC 50 and Alamar Blue Assays involving, the Nb, Nb tagged to cyclodextrin, Pentamidine, Pentamidine + Nicotinamide and Pentamidine delivered in a variety of Nanocapsules such as Chitosan and PLGA, Endocytosis measurement of nanoparticles loaded with fluorochromes by FACS analysis, and finally Protein molecular weight determination methods including SDS and use of a Nanodrop™, General lab techniques such as microscopy and parasitemia.
Training of UEM researcher at VIB
Hermogenes Mucache (UEM) visited VIB for a training period of three months (May-Aug. 2010). During this period, Hermogenes participated in the development of a set of NANOBODIES that can be used as tools for the detection of T. evansi. A detailed progress report of the work performed was included in the previous reporting document / description of WP1. At the end of the last reporting period, a second student form UEM joinded VIB, i.e. Paula Alberto Macucule. She joined the laboratory at VIB to get acquainted with basic techniques in library panning and Nb selection.
Joined PhD training program VIB/FIBAO
A joint PhD was initiated at the start of the NANOTRYP project, and results obtained are included in the report on WP1 and WP2. The PhD (of Florencia Le Greca) covers the work on T. vivax. The PhD has passed through a first round of defence at VIB and will be finalised at the end of August 2013.
Addition training efforts
During the mid-term evaluation, a comment was made that pointed to the fact that the exchange of a single PhD student between two of the partner laboratories cannot be considered a ‘PhD training Program’. However, it should be mentioned here that the NANOTRYP budget only incorporated a budget for 1 student. Nevertheless, in the last reporting period we have extended the PhD training to include several more students.
• Juan Diego Unciti Broceta (FIBAO) spend a 2 month training period at VIB (May-June 2011), focussing on the generation of a Nb library against the non-variant OpdB T. brucei antigen. After having received this training, Juandi returned to FIBOA and initiated the generation of a specific library for T. evansi.
• Steven Odongo (Uganda) joined the NANOTRYP project at the start of 2011 and has been focussing at the development of a Nanobody sandwich system for T. congolense. This project includes (i) the generation, selection and production of Nbs, and (ii) the use of Nbs in the development of an ELISA based diagnostic tools, that could later be transformed into a dip-stick (lateral flow) format
• Fyei Obishakin (Nigeria) joined the NANOTRYP project in late 2010, and focuses on the development of a Nanobody-based diagnostic tool for T. evansi. His PhD has a dual focus that includes the assessment of immunopathology of T. evansi infections as well as the development of diagnostic tools for the infection, and hence about 50% of his project is directly associated with NANOTRYP.
Directly related to NANOTRYP is the fact that other research teams working on other parasitic diseases have gained interest in Nanobody technology and have asked for training for PhD students. Within a European context it is worth mentioning that the COST BM0802 action (Cooperation for Science and Technology) has been used to exchange both a PhD and Master student between th VIB and the University of Alcala, Spain (Prof Antonio Jiménez Ruiz), with the aim of developing Nbs as molecular tools for studying the fundamental biology of Leishmania. In turn, this collaboration has lead to two additional collaborations (within the COST action) with the University Medical Centre of Leiden, The Netherlands (Dr. Shahid Khan), and the University of Tubingen, Germany (Prof. Michael Duszenko).
Conclusion:
Besides the obvious achievements of the NANOTRYP education efforts, the key achievements of the awareness campaigns included:
• Broad coverage - the awareness campaigns were carried out using different forums that varied from country to country, including among others, presentations of projects at international, regional and national conferences, community workshops, news articles in the local press, TV interviews and documentaries, road shows, banners and brochures. These approaches ensured that the campaigns impacted all the levels that were targeted.
• The awareness and ownership of HAT problem at local, national, regional and international levels was enhanced. This is reflected by a decision by the WHO to identify HAT as one of the diseases targeted for elimination by the year 2020. Meanwhile in early 2012, a large group of funding agencies and industry partners passed the London Declaration, committing to support efforts to eliminate the disease.
• Strategies for intensified control and elimination of tsetse and trypanosomiasis were better harmonized at the national, regional and international levels. As a result, the prevalence of the disease is currently being reported with higher accuracy than before. Indeed Uganda has now initiated a national project to intensified surveillance and control of T.b. gambiense HAT.
• The ability of health workers to suspect HAT was improved at all levels including public and private clinics and traditional healers in endemic areas. This has resulted in accelerated control of the disease, and could be contributing to the dramatic fall on the number of cases of HAT reported in recent years.
• Many endemic countries have now allocated annual budgets for trypanosomiasis control, while others have increased the annual budgetary allocation.
• Characterization of health facilities in HAT endemic regions has strengthened the countries’ ability to re-allocate resources to priority areas based on the evidence provided. The information generated is also providing guidance on targeting of diagnostics to facilities based on the available capacity at those facilities.
• The capacity of health personnel to utilize available diagnostic tools for HAT was strengthened through training, contributing to better detection of cases and proper management of the disease.
• Education on the importance of accurate diagnosis created an environment for sustainable introduction of novel diagnostic tools in endemic countries. This will be critical as efforts to eliminate HAT are intensified.
• A number of countries are now able to apply early warning systems to predict disease outbreaks, and therefore take appropriate measures to prevent escalation of the disease.
Potential Impact:
An important component of the NANOTRYP project was an awareness campaign on the importance of both human and animal African trypanosomiasis. This activity was mainly executed by FIND and UEM. In addition to this, NANOTRYP also included a large education component for academic trainees. Together, VIB, FIBAO, IPR and UEM organised a multitude of research exchanges and provided the possibility for the 4 PhD programmes to be executed.
With respect to awareness campaigning itself, FIND took the lead, and at the international level, funding agencies were lobbied to commit resources for research and control of the disease. Within the endemic countries, governments were urged to allocate resources for control of the disease within their national budgets. Medical and veterinary personnel were reminded to include the disease in their differential diagnosis, while communities were made more aware of the importance of these diseases.
The focus of the campaigns was communities that are at risk of infection, or who keep livestock in areas infested with tsetse flies, medical personnel working in health centres located in endemic regions, and policy makers in governments of endemic countries. Implementation of advocacy activities was guided by a Strategic Plan on Advocacy (SPA) for African Trypanosomiasis developed by FIND, the Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC) office of the African Union, and endemic countries. Phase one of the Strategic Plan covered the period January 2008 to December 2011. The selected countries appointed contact persons, who worked with PATTEC, and developed country-specific work plans and budgets. The work plans were guided by a country’s capacity to implement activities against specific outputs that were agreed on and included in the SPA.
The key achievements and impacts of the awareness campaigns included:
• Broad coverage - the awareness campaigns were carried out using different forums that varied from country to country, including among others, presentations of projects at international, regional and national conferences, community workshops, news articles in the local press, TV interviews and documentaries, road shows, banners and brochures. These approaches ensured that the campaigns impacted all the levels that were targeted.
• The awareness and ownership of HAT problem at local, national, regional and international levels was enhanced. This is reflected by a decision by the WHO to identify HAT as one of the diseases targeted for elimination by the year 2020. Meanwhile in early 2012, a large group of funding agencies and industry partners passed the London Declaration, committing to support efforts to eliminate the disease.
• Strategies for intensified control and elimination of tsetse and trypanosomiasis were better harmonized at the national, regional and international levels. As a result, the prevalence of the disease is currently being reported with higher accuracy than before. Indeed Uganda has now initiated a national project to intensified surveillance and control of T.b. gambiense HAT.
• The ability of health workers to suspect HAT was improved at all levels including public and private clinics and traditional healers in endemic areas. This has resulted in accelerated control of the disease, and could be contributing to the dramatic fall on the number of cases of HAT reported in recent years.
• Many endemic countries have now allocated annual budgets for trypanosomiasis control, while others have increased the annual budgetary allocation.
• Characterization of health facilities in HAT endemic regions has strengthened the countries’ ability to re-allocate resources to priority areas based on the evidence provided. The information generated is also providing guidance on targeting of diagnostics to facilities based on the available capacity at those facilities.
• The capacity of health personnel to utilize available diagnostic tools for HAT was strengthened through training, contributing to better detection of cases and proper management of the disease.
• Education on the importance of accurate diagnosis created an environment for sustainable introduction of novel diagnostic tools in endemic countries. This will be critical as efforts to eliminate HAT are intensified.
• A number of countries are now able to apply early warning systems to predict disease outbreaks, and therefore take appropriate measures to prevent escalation of the disease.
Awareness campaigns supported with NANOTRYP funds focused on Uganda, Tanzania and Nigeria in collaboration with the governments of those countries and PATTEC. This was done alongside a larger program on advocacy in all countries reporting cases of HAT in Africa, and funded by the BMGF and the DFID. A detailed account of the achievements is reported in the following section.
In Uganda, awareness campaigns were carried out by Gulu University in the T.b. gambiense region in the north of the country. The specific objectives of the campaigns included a) understanding the local community’s perception of the disease, b) appraising the community’s level of understanding of the symptoms of HAT and nagana, transmission, diagnosis, treatment and control, and c) sensitizing leaders and communities living in these areas so that they seek early diagnosis and treatment for both human and livestock diseases, in order to remove reservoirs of infection. At the onset, familiarization and interpersonal meetings were held with key district and selected sub-county officials, to brief them on the purpose of the project and get their understanding on the severity of the problem. Community mobilization and campaigns at the district, sub-county and at community levels focussed on the importance of early diagnosis of sleeping sickness and Nagana in humans and animals, respectively, the animal-human infection relationship, understanding the ecology of tsetse flies and their role in transmission of the diseases, local community perception of sleeping sickness and Nagana, and community coping strategies and adaptations. Advocacy materials that were developed and distributed included a) posters on trypanosomiasis and how it is spread, b) posters on actions in control of trypanosomiasis, c) Question and Answer booklet (Frequently Asked Questions) for community mobilization, d) leaflets in the various local languages in the endemic areas highlighting how trypanosomiasis is spread and control methods and T shirts, e) radio talk shows (30 minutes each) in the two dialects zones of Adjumani and Nwoya – Amuru, and f) training workshops for village health teams on awareness of trypanosomiasis.
The Ministry of Health (MOH) of Uganda and the National Livestock Resources Research Institute (NaLIRRI) focused on the T.b. rhodesiense region in the south east and central Uganda. However, in addition to using advocacy materials that were similar to those used in northern Uganda, the MOH carried advocacy workshops in primary schools and distributed a Comic Book of sleeping sickness that was developed by the WHO. They also held workshops with senior government officers and parliamentarians, resulting in a significant increase in budgetary allocation for African Trypanosomiasis control by the government, within the Annual budget. A similar outcome was also reported by Tanzania. The latest data from these activities indicates that the T.b. gambiense focus may be shrinking northwards, reducing the risk of merging with the T.b. rhodesiense belt. This is important because the two forms of the disease are not distinguishable clinically, yet treatment is different. A merger of the two would therefore pose a challenge in management of the disease.
In Tanzania, awareness campaigning was carried out by both the Ministry of Health and Social Welfare (MOHSW) and the Ministry of Livestock and Fisheries Development (MOLFD). Various government institutions involved in tsetse and trypanosomiasis research and control came together and established the Network for Mapping African Trypanosomiasis in Tanzania (NetMATT). Institutions that became members of the network included a) Ministry of Livestock Development and Fisheries (Coordinator), b) National Institute of Medical Research (NIMR), c) Tsetse and Trypanosomiasis Research Institute (TTRI), d) Ministry of HeaIth and Social Welfare (MOHSW), e) Institute of Resource Assessment (IRA) - University of Dar es Salaam (UDSM), f) Tanzanian National Parks Authority (TANAPA), g) Tropical Pesticide Research Institute (TPRI), and h) Sokoine University of Agriculture (SUA). The activities that NeTMATT undertook included a) developing tools to guide surveillance, intervention and the targeting of resources, b) consolidating mapping data to support the FIND/PATTEC HAT advocacy programme, c) using reports and arising publications as tools for advocacy, to guide further research and leverage funding, d) strengthening data collection and mapping of African trypanosomiasis in Tanzania through collaboration between various stakeholders (including government ministries, research institutions and universities), e) facilitating sharing of data between institutions, and f) improving the quality of trypanosomiasis data analysis by enhancing the amount and quality of mapping and spatial analysis that is undertaken using the data. The outcome of the activities relating to characterization of health centres was a series of GIS maps showing the health centres where HAT has been reported during the past 10 years, and the changes in numbers of cases by year.
While livestock trypanosomiasis is widespread in Nigeria, cases of HAT have in recent years only been reported in Delta State, which is in the south of the country. Nigeria has intensified surveillance and control of both diseases in the entire country, with the aim of eliminating them. Nigeria was supported to intensify awareness campaigning, and to actively screen communities in Delta State for HAT. Active screening was carried out using the CATT test (the screening test in current use), and case confirmation by microscopy. While a significant percentage of people were found positive with CATT, none of them was confirmed by parasitology. This also indicates that the prevalence of HAT in Nigeria is very low, and elimination of the disease in this country is also imminent.
The second NANOTRYP partner that provided a particular valuable contribution to the impact of the programme with respect to affected population was UEM – The University Eduardo Mondlane (Mozambique). From the Eduardo Mondlane University standpoint to evaluate the social impact of the Nanotryp project, aspects associated to the enhancement of the Mozambican Research Group on African Animal Trypanosomiasis, the exposure of Mozambican students and researchers to world class scientific seminars and the involvement of communities affected by animal trypanosomiasis in awareness campaigns should be considered.
Boosting Trypanosomiasis Reasearch in Mozambique
The Nanotryp project contributed directly to the increase in the research activities in the area of animal trypanosomiasis in Mozambique. Young researchers participating in the project had the opportunity of training both at national level and in the laboratories of participating institutions in Belgium and in Spain. In Mozambique the researchers were actively involved in intensive field activities combined with the establishment and validation of modern techniques like PCR and PCR-RFLP for Trypanosoma species identification. At the laboratories of European participating institutions these researchers had the opportunity to familiarize themselves with the theory and practice of nanobody technology as well as with advance techniques applied to cellular immunology.
It is worth mentioning that, apart from the technical and scientific experience acquired by the Mozambican researchers, in the course of this project a substantial amount of very valuable biological material was collected which will enhance dramatically the probability of future participation of the Mozambican Research Group on African Animal Trypanosomiasis in specialized research networks.
Last but not least, the project has contributed to the boost of researcher’s individual careers and through that, they manage to get hold permanent positions at the university. It is of crucial social importance to train and retain a generation of competent young African scientists in order to achieve a substantial improvement on the continental scientific output, which is directly linked with the possibility of further job creation at African institutions of science and technology.
Nanotryp Scientific Workshop
In the context of the Nanotryp project, a scientific workshop was held in February 2011, at the Veterinary Faculty of Eduardo Mondlane University. The workshop was a unique opportunity to expose students and researchers to world class scientific seminars, addressing a broad spectrum of topics, including Nanobody principles and applications, advances in the pharmacology of typanocidal compounds, immunology and diagnostics. It is worth mentioning the inclusion in the workshop program of practical classes.
This scientific exercise was extremely important not only by the relevance of its contents, but mainly by setting a model of academic excellence in a place where this kind of contribution are greatly required.
Awareness Campaign (see also WP8)
The trypanosomiasis and tse tse fly awareness campaign carried out in the context of WP8 of the Nanotryp project was arguably the activity with greater social impact. The involvement in the campaign of more than 3000 students, approximately 100 primary school teachers and 100 local cattle owners generated a tremendous social movement. In African Animal Trypanosomiasis the activities related with disease control are normally assumed by the government through the veterinary services, generally in collaboration with one or more international agencies. The participation of affected communities has been largely neglected and normally the small holders have lost completely the sense of ownership regarding the main control interventions.
A particular feature of this campaign was to define as main target children from primary schools. In the Mozambican rural areas, particularly in the South, children play a major role in cattle husbandry. Generally, the children are the responsible to take the cattle to the pastures and they are also the ones who bring the animals to the dip-tanks or crush-pen for any type of veterinary intervention.
During the campaign, making use of simple, but technically accurate messages, emphasis was put on the basic concepts of disease ethiology and epidemiology as well as on the rational management of trypanocidal drugs. It is expected a much more proactive involvement of individuals and communities on all aspects of disease management and control.
The third major partner that provided a direct impact for the trypanosomiasis affected community was IPR, the International Primate Research centre, Kenya. Here the main contribution of NANOTRYP has been in the following fields:
1. Technology Transfer and Training
The biggest single gain with a long lasting effect is the training that this project accorded the Kenyan partners. Four MSc students at Kenyan universities were successfully trained by carrying out an aspect during the life of the studies that comprised the tasks on this project thus;
i. Mr Chris Kinyanjui, Jomo Kenyatta University of Agriculture & Technology (2009, JKUAT). Mr Kinyanjui was trained at VUB on nanobody generation using Tb rhodesiense IPR 001 isolate. This training formed his MSc research project. Mr Kinyanjui was mid-way through the program offered a scholarship to pursue molecular biology studies at MSc level in Belgium, which was an offshot of his engagement with VUB. He is due to return home on completion of his studies to resume his position at IPR.
ii. Mr Alex Gaithuma – MSc, (2009,JKUAT), developing the non-human primate model of Tb rhodesiense
iii. Ms Dawn Maranga – MSc (2013, JKUAT) characterizing the immunological profile of the Tb rhodesiense nonhuman primate model. Ms Maranga was later offered a research fellowship following the departure of the first research fellow. This allowed the HAT research project to proceed to its logical conclusion.
iv. Ms Beatrice Gachie – MSc (on-going, JKUAT) characterizing the diagnostic value of non-invasive procedures in development of nonhuman primate model of Tb gambiense
v. Mr Victor Mwadime trained at FIBAO on Nb::drug coupling. He is currently undertaking his MSc studies (University of Nairobi) and is expected to carry out his research project on the basis of this training.
2. Nonhuman primate models of Tb rhodesiense and Tb gambiense
The NHPs model of Tb rhodesiense has been fully established and has taken its place in the IPR HAT research programs. Developed as part of the Nanotryp project, this model attracted two other research grants for preclinical testing of diagnostic products. It is anticipated that this laboratory animal model will be even more useful as a testing tool for treatment and diagnostic tests especially as the clamour for eradication of the disease gains momentum.
The development of a NHP model of Tb gambiense was also supported by Nanotryp. Two series of infections were carried out in vervet as well as Sykes monkeys with encouraging results. Some diagnostic tests based on non-invasive samples such as saliva and urine indicated that these tests are possible considerations for monitoring infection. This would be a vast improvement over the existing tests which require patients to undergo a painful lumbar puncture over a period of two years before cure can be ascertained. Even more important is that this test was able to demonstrate relapse before detection of parasites in blood.
IPR is the biggest Primate Centre in Africa and continues to attract research scientists from all over the world. It is hoped that this development in the field of HAT will provide the necessary leverage needed to attract firms and investigators interested in elucidating more information to plug the existing knowledge gaps.
Following the training at VUB and FIBAO, IPR proceeded to carry out immunization of camels under field settings in Kenya. The camel farm was identified and terms of engagement entered into. The farm is found within camel trypanosomiasis zone (0.121639N 36.630885E). The immunization and subsequent processing of samples for nanobody preparation was a demonstration of the practicality of the training. However the process stopped short of completion due to inability to transfer the end product in Kenya (RNA) to the University of Brussels laboratory where the Nb production would have gone to completion. This implies that the technology transfer has been successful except for the heavy investment to process the materials to full generation of Nb. The reliance on the facility at Brussels is likely to be so in the foreseeable future. The understanding with the farm owners was such that long-term engagement may be addressed as and when needs arise.
The initial aim of this project was to transfer this Nb technology to other tropical diseases of importance under the Kenyan research priority. This has been achieved by initiating the diagnostic potential of Nb against Leishmaniasis a neglected disease. It is anticipated that the import/export hitches between Kenya and Belgium will be sorted to allow the full processing of this initiative.
3. Field evaluation of T. congolense nanobody
IPR provided a platform for testing of nanobodies against the T, congolense which was carried out in a field setting where livestock trypanosomiasis is still a major constraint to animal production. The interaction with farmers was a very fulfilling experience when carrying out sensitization control strategies for application post research. The presence in the field was taken positively and seen that the government still cares about the prevalent problem affecting farmers. It is anticipated that the test results will have a bearing on the way livestock trypanosomiasis is diagnosed in the future. Kenya stands to benefit greatly considering T. congolense is the main cause of cattle, sheep and goats morbidity affecting agricultural productivity in about 60% of arid and semi-arid land surface where livestock is the main source of economic activity.
4. Infrastructure Development: Equipment
Through Nanotryp, IPR benefited from procurement of a haemtology analyser which replaced an old and frequently breaking down equipment. The equipment is centrally placed and therefore available to all users in IPR. This way the maintenance has been transferred from the Nanotryp project to the users through levy of user charge. All data on Nanotryp project was generated through this machine.
The procurement of a fluorescent microscope allowed IPR to carry out varied staining tests on trypanosomes as well as other parasites of important research programs at IPR. The Acridine Orange (AO) staining is gaining ground as the stain to use in order to visualize trypanosomes and other protozoan parasites much faster and easily than traditional laboratory stains. The application of this technology is likely to only be better utilized at IPR in the coming years for training and research purposes. Already tests in Leishmania and malaria parasites have been carried out with encouraging results.
In addition to helping to sustain the scientific infrastructure, the Nanotryp project paid to IPR 12.5% of all grant money received, according to Institutional Policy. The funding went into construction of a new building for group housing animals in experiments as part of the animal welfare improvements (Figure 20). All animals in the experiments were taken through environment enrichment protocols. These infrastructure developments will have a huge beneficial effect on the future of IPR well beyond the life of Nanotryp Project, particularly because IPR has initiated the process of international accreditation by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC International). The new building has been put up with advice from reputable international firm (Arrowmight Inc) which has provided consultancy services in the renovation of several NHP centres in Europe. The dimensions meet the current EU guidelines and exceed the American rules (The Guide).
Figure 20: New building for group housing animals in experiments as part of the animal welfare improvements
In the future, animals will be housed in compartible pairs as the minimum housing requirements. The animal house is built to use as much natural light as possible hence the translucent sunroof sheets and open sides to allow natural air circulation since the animals will be housed within their natural/home setting.
Besides efforts to educate local comities and bring awareness of the trypanosomiasis problem to the different stakeholders, NANOTRYP also dedicated a considerable effort to education both at the level of PhD and Master training, as well as research training from both IPR and UEM researchers at the European VIB and FIBAO partner laboratories.
Training of IPR researcher at VIB
Christopher Kinyanjui Kariuki (IPR) visited VIB for two consecutive training periods of three months each (Jan-March 2011 and Oct.-Dec. 2011). During this period, Mr Kariuki developed a set of Nanobodies that can be used as tools for the work ongoing at IPR, in which the T. brucei. rhodesiense IPR001 parasite stock is being used. A detailed progress report of the work performed was included in the previous progress report / description of WP1.
This work formed part of Mr Kariuki’s MSc training at Jomo Kenyatta University of Agriculture & Technology (JKUAT), Nairobi. Mr Kariuki returned home to initiate the nanobody generation work leading to immunization of camels for generation and extraction of mRNA. However the next stages of nanobody generation still require heavy investment which IPR may not have, and therefore the collaboration with VUB to access the VIB Nanobody generation Centre is likely to remain alive beyond the life of this project.
Training of UEM and IPR researcher at FIBAO
Mr Mwadime and Mr Mucache were successfully training in laboratory techniques that included Immunofluorescence of the Tb brucei parasite Antat 1.1 with the Nb (Nanobody) and fluorochromes e.g. Rodamine, In vitro culture of the Tb brucei. Maintenance of culture parasites and working with in vitro based systems, IC 50 and Alamar Blue Assays involving, the Nb, Nb tagged to cyclodextrin, Pentamidine, Pentamidine + Nicotinamide and Pentamidine delivered in a variety of Nanocapsules such as Chitosan and PLGA, Endocytosis measurement of nanoparticles loaded with fluorochromes by FACS analysis, and finally Protein molecular weight determination methods including SDS and use of a Nanodrop™, General lab techniques such as microscopy and parasitemia.
Training of UEM researcher at VIB
Hermogenes Mucache (UEM) visited VIB for a training period of three months (May-Aug. 2010). During this period, Hermogenes participated in the development of a set of NANOBODIES that can be used as tools for the detection of T. evansi. A detailed progress report of the work performed was included in the previous reporting document / description of WP1. At the end of the last reporting period, a second student form UEM joinded VIB, i.e. Paula Alberto Macucule. She joined the laboratory at VIB to get acquainted with basic techniques in library panning and Nb selection.
Joined PhD training programme VIB/FIBAO
A joint PhD was initiated at the start of the NANOTRYP project, and results obtained are included in the report on WP1 and WP2. The PhD (of Florencia Le Greca) covers the work on T. vivax. The PhD has passed through a first round of defence at VIB and will be finalised at the end of August 2013.
Addition training efforts
During the mid-term evaluation, a comment was made that pointed to the fact that the exchange of a single PhD student between two of the partner laboratories cannot be considered a ‘PhD training Program’. However, it should be mentioned here that the NANOTRYP budget only incorporated a budget for 1 student. Nevertheless, in the last reporting period we have extended the PhD training to include several more students.
• Juan Diego Unciti Broceta (FIBAO) spend a 2 month training period at VIB (May-June 2011), focussing on the generation of a Nb library against the non-variant OpdB T. brucei antigen. After having received this training, Juandi returned to FIBOA and initiated the generation of a specific library for T. evansi.
• Steven Odongo (Uganda) joined the NANOTRYP project at the start of 2011 and has been focussing at the development of a Nanobody sandwich system for T. congolense. This project includes (i) the generation, selection and production of Nbs, and (ii) the use of Nbs in the development of an ELISA based diagnostic tools, that could later be transformed into a dip-stick (lateral flow) format
• Fyei Obishakin (Nigeria) joined the NANOTRYP project in late 2010, and focuses on the development of a Nanobody-based diagnostic tool for T. evansi. His PhD has a dual focus that includes the assessment of immunopathology of T. evansi infections as well as the development of diagnostic tools for the infection, and hence about 50% of his project is directly associated with NANOTRYP.
Directly related to NANOTRYP is the fact that other research teams working on other parasitic diseases have gained interest in Nanobody technology and have asked for training for PhD students. Within a European context it is worth mentioning that the COST BM0802 action (Cooperation for Science and Technology) has been used to exchange both a PhD and Master student between th VIB and the University of Alcala, Spain (Prof Antonio Jiménez Ruiz), with the aim of developing Nbs as molecular tools for studying the fundamental biology of Leishmania. In turn, this collaboration has lead to two additional collaborations (within the COST action) with the University Medical Centre of Leiden, The Netherlands (Dr. Shahid Khan), and the University of Tubingen, Germany (Prof. Michael Duszenko).
List of Websites:
www.nanotryp.eu
P1 Flanders Institute for Biotechnology VIB - Stefan Magez - stemagez@vub.ac.be
P2 ARTTIC in Brussels, SPRL ARTTIC - Paul Crompton - pdc@arttic.be
P4 Institute of Primate Research IPR - Maina N’Gotho - ngothojm@yahoo.co.uk
P5 University Eduardo Mondlane UEM - Luis Neves - nidzi@tvcabo.co.mz
P6 Foundation for Innovative New Diagnostics FIND - Joseph Ndung'u - joseph.ndungu@finddiagnostics.org
P7 Fundacion para la Investigation Biosanitaria de Andalucia Oriental - Alejandro Otero FIBAO - Jose Garcia Salcado - jagarciasalcedo@yahoo.es
Trypanosomiasis is a disease with a devastating socio-economic impact in Sub-Saharan Africa through the direct infection of humans and livestock. The NANOTRYP project aimed at addressing the problems encountered in the fight against trypanosomosiasis, i.e. (i) the lack of specific diagnostic tools that can be used as test-of-cure, (ii) the high drug toxicity of current treatment schemes, and (iii) the limited understanding of the disease by local communities of endemic areas. NANOTRYP was initiated using nanobody (Nb) tools whose efficacy already had been proven in the laboratory. Based on this knowledge, the main objectives of NANOTRYP were to: (1) Generate a number of Nanobody (Nb) libraries that are capable of recognizing all four major African trypanosome species, (2) Develop new, rapid, and easy to use Nb-based trypanosomiasis diagnostic tools based on parasite detection, (3) Asses the use of anti-trypanosome Nbs as drug targeting molecules, and (4) Develop a forum for exchange of knowledge between European partners and partners from disease endemic countries.
To reach the objectives outlined above, the project was divided into seven scientific work packages, one training and education work package and one management work package.
Work packages 1 to 3 were designed to deliver various libraries of Nanobodies and a Nb-based test kit for trypanosomiasis. The Nbs generated were able to bind highly conserved antigens of T. brucei, T. evansi, T. congolense and T. vivax. Nbs were obtained from various vaccination protocols as well as from naturally and experimentally infected camelids. Rather than preselecting certain antigens as preferred candidates, the consortium opted for a strategy in which Nbs were made against soluble extracts of trypanosomes, hence increasing the change of picking up unexpected candidates such as conserved intracellular targets. Following the selection of promising Nbs, a first test format was designed under the format of an ELISA. In a next step, the consortium attempted to translate the ELISA format into a lateral flow design, using gold-labeled Nbs for detection. Unfortunately, this step proved more difficult than anticipated and reproducibility of the coupling procedure was not optimized by the end of the project. However, a proof-of-principle RTD was developed together with the Korean based company SD (Standard Diagnostics). The collaboration with SD resulted from the activities planned in WP4 that was initially designed to test, validate and implement a Nb-based RTD for trypanosomiasis in the field. Having been unable to standardize the envisaged final test format, the latter WP only had a limited outcome.
As mentioned above, the fight against trypanosomiasis is also hampered by the high toxicity of existing drug. To increase the therapeutic index of existing drugs, and hence lower the toxicity at an effective dose, we proposed to use the Nbs developed in WP1 as drug targeting molecules, aiming at an increased efficacy of target specific drug delivery. As a model concept, work was performed using a well establish T. brucei rodent model, and pentamidine as a trypanocide. The results of WP5 showed that the envisaged strategy gave a ten-fold increase in treatment efficacy, when Nb drug targeting was applied.
In order to assess the future usability of Nb tools in the field, Work Packages 6 and 7 were developed using non-human primates and livestock animals. The first WP allowed the characterization of models for both T. gambiense and T. rhodesiense, two human infective trypanosomes. Obtained results are crucial for future diagnostic and drug delivery strategies, as the past has proven that neither in vitro, nor rodent models are suitable for this when considering the encephalitic late stage of human sleeping sickness. The latter WP delivered a wealth of experimental and field samples, needed for further diagnostic development, and allowed the specificity and sensitivity assessment of a T. conglonese specific Nanobody-ELISA. Both WPs were coupled to WP8, designed to provide a tool for training and awareness campaigning. The NANOTRYP consortium as whole can be proud of (i) having organized two intense training schools in Kenya and Mozambique and (ii) having delivered 4 PhDs and 7 Master thesis graduations towards the end of the programme.
Project Context and Objectives:
When NANOTRYP was initiated, the consortium proposed four objectives that ultimately would lead to the generation of tools allowing an improvement in diagnosis of both HAT (Human African Trypanosomiasis) and AAT (Animal Africa Trypanosomiasis). These objectives included:
1. Generate a number of Nanobody (Nb) libraries that are capable of recognizing all four major African trypanosome species.
2. Develop of a Nb-based trypanosomiasis diagnostic tools based on parasite detection. This could complement or replace the current agglutination assays that are based on anti-parasite antibody detection. The nanobody technique proposed avoids expensive, labour intensive molecular techniques that impose extensive requirements on infrastructure and equipment maintenance.
3. Assessment of the use of anti-trypanosome Nbs as drug targeting molecules as proof of principle for the treatment of experimental trypanosomiasis.
4. Develop a forum for exchange of knowledge in which researchers from participating developing countries can get first-hand experience in the further development of Nb-technology, and in which new trypanosome diagnostic and treatment campaigns are linked to HAT and livestock trypanosomiasis awareness programs.
Major measurable outcomes that had been put forward at the start of NANOTRYP were:
• A collection of nanobodies that will recognize the HAT causing parasites T.rhodesiense T.gambiense as well as surface molecules of the parasites T.brucei T.congolense T.vivax and T. evansi, the causative agents of animal trypanosomiasis. These Nanobodies would subsequently provide a new way forward to diagnostic development and eventual trypanosome-specific drug delivery.
• Working and training visits of members of the African participant teams to the laboratories of the European partners.
• The organization of two workshops in applied diagnostic and treatment of trypanosomiasis in the African partner countries.
• Conducting of trypanosomiasis awareness campaigns in parallel to all field diagnostics and treatment efforts.
NANOTRYP builds largely on the established fundamental Nanobody research done with respect to experimental trypanosomiasis research in mice. It focuses on taking concepts to field conditions, in order to deliver new diagnostic and treatment methods for trypanosomiasis. To date, two main problems exist in the fight against trypanosomiasis. First, trypanosomiasis diagnosis (particularly in the case of HAT) relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT), an assay that detects host antibodies that cross-react with a laboratory prepared trypanosome protein complex. Due to its high rate of unconfirmed results (Radwanska et al., Am J Trop Med Hyg. 2002 67(6):684-9), a second confirmatory assay is needed for all positive tests. In addition, as CATT measures antibody titers, it cannot be used as test-of-cure and neither is it suitable for use in endemic areas where treatment is common (as in both cases antibodies remain in the circulation long after parasites have been cleared). New and better diagnostics should solve this problem. Second, at the level of trypanosomiasis treatment, only a limited number of drugs have been registered. Based on the increased appearance of drug-resistance, and the unacceptable level of side effects (up to 10% mortality in case of Melarsoprol treatment) it is clear that there is an urgent need to engage in activities that seek to develop new anti-trypanosome compounds to eradicate HAT and animal trypanosomiasis. Hence, NANOTRYP aimed at using nanobodies in the development of new methods for diagnosis and treatment of trypanosomiasis.
Nbs are small antibody fragments with unique protein or antigen recognizing properties. They are derived from conventional camelid antibodies through recombinant gene technology.
All antibodies from vertebrate animals are constitutively composed of two identical heavy and two identical light chains. More than fifteen years ago, by serendipity, one of the research groups at the Vrije Universiteit Brussel (VIB - the coordinating partner) discovered the occurrence of bona fide antibodies devoid of light chains in Camelidae (Hamers-Casterman et al., Nature. 1993 363(6428):446-8). These so-called heavy-chain antibodies (HCAbs) bind antigen solely with one single variable domain with high affinity, and are referred to as VHHs or nanobodies because their size is in the nanometer range. As such, the Nb is the smallest intact antigen-binding entity that can be obtained. Methods were developed to clone the Nb repertoire of an immunised dromedary (or llama) in phage display vectors, and to select the antigen-specific Nbs from these ‘immune’ Nb libraries (Lauwereys et al., EMBO J. 1998 17(13):3512-20). The optimised protocol is fast and straightforward. In addition, the method is far superior to the selection of antigen binders from conventional antibodies. Moreover, Nbs are economically produced by bacterial, fungal or plant expression systems to a high yield and possess excellent biophysical properties (e.g. specificity, solubility, stability, long shelf-life). As nanobodies are produced as recombinant molecules, it is also relatively easy to produce larger nanobody complexes, generating for example bivalent or bivalent-Fc coupled nanobodies. In addition, in order to reduce possible immunogenicity of nanobodies when injected into non-camelids, they can be customized by altering specific amino acid sequences. For example, for use in man, nanobodies are ‘humanized’ to reduce the risk of the induction of anti-nanobody antibodies in the circulation. While NANOTRYP is not expected to generate anything new, beyond the current state-of-the-art insights at the level of Nb generation, it does provide the opportunity to transfer the knowledge of how to generate anti-parasite Nbs to members of the participating African research communities. Given the relative ease with which Nbs can be generated, this transfer of knowledge can later result in a whole range of new applications, originating from the views and needs of these partners.
Over the last decade, VIB has been involved in the development of new approaches to HAT and livestock trypanosomiasis diagnosis. Indeed, RT-PCR methods as well as PNA-FISH techniques for differential diagnosis of T.rhodesiense and T.gambiense (Radwanska et al. Am J Trop Med Hyg. 2002 67(6):684-90, Radwanska et al. J Clin Microbiol. 2002 40(11):4295-7., Radwanka et al. Am J Trop Med Hyg. 2002 67(3):289-95.) were pioneered in collaboration with VIB, and were based on the earlier discovery by VIB of a species-specific trypanosome resistance genes (i.e. SRA, De greef et al. Ann Soc Belg Med Trop. 1992;72 Suppl 1:13-21 , Xong et al. Cell. 1998 Dec 11;95(6):839-46.). However, considering the burden that these molecular biology techniques pose at the level of equipment infrastructure and maintenance, as well as the high costs involved in routine PCR screening under field conditions, these innovations were not explored beyond the ‘proof-of-principle’ in screening for HAT. In addition, molecular biology approaches for the preventive screening of HAT causing parasites in the African livestock herd are considered to be unaffordable on a large and systematic scale. Hence, in an alternative approach, VIB has generated fluorescent anti-trypanosome Nbs that greatly facilitate parasite detection in blood of experimentally infected animals. This technique was shown to be successful for T.b.brucei T.b.rhodesiense T.b.gambiense T.evansi and T.vivax trypanosomes. However, as parasite levels in human infections are often 1000-times lower than those levels found in experimental mouse infections, the minimal detection limit of fluorescent parasite visualisation techniques remains a problem in field conditions. Hence, the project proposed here will focus on Nb-based antigen capturing diagnostic methods that can drastically increase parasite detection limits.
Before the initiation of NANOTRYP, all Nb work done with respect to the diagnosis of trypanosomiasis had been executed at a laboratory scale, involving well-characterized, cloned parasite stabilates. In order to field-validate the diagnostic tools, two partners with key positions in assessing HAT and livestock trypanosomiasis respectively participated in NANOTRYP.
The first partner is the Foundation for Innovative New Diagnostics (FIND). FIND is an independent Swiss Foundation dedicated to the development of diagnostic tests for poverty related infectious diseases of public health importance. Prior to its participation in NANOTRYP, FIND has used other diagnostic programmes (such as their tuberculosis programme) as a model to develop a rigorous and systematic approach to the needs-driven development, evaluation, demonstration and access of diagnostic technologies. This is done in partnership with academic and research institutes, ministries of health in endemic countries, and with biotechnology companies in the private sector, in the framework of contractual agreements that ensure eventual access to affordable tools in the public sector of developing countries. A critical obstacle to the development of improved assays for HAT is access to patients, quality diagnostic and clinical data, carefully collected and stored reference materials, and to sustained field programs with the capacity for long-term follow-up. The partnership with WHO, ministries of health and national research institutes in endemic countries has enabled the establishment of screening sites for collection of biological materials, and a specimen bank owned by the WHO. Clinical trial sites were also being strengthened through a memorandum of understanding between FIND and the WHO. The establishment of a specimen bank has guaranteed more efficient use of limited resources, limited the need for field trials, promotes product comparisons and facilitated quality control. This as tol be used as a first step in the evaluation of diagnostic tests developed through the project. One of the key requirements in the HAT diagnostics programme at FIND is the development of an access strategy for diagnostics that ensures that these tools reach the population at risk in a sustainable and affordable manner. The strategy that FIND has developed includes advocacy at the level of the African Union, ministries of health, finance and agriculture of endemic countries, and affected communities. The second partner of NANOTRYP crucial for Nb diagnostic development is the University Edwando Mondlane in Mozambique (UEM)). UEM selected to validate Nb trypanosomiasis diagnostic tools in on livestock infections. At UEM, regular field diagnosis screenings for trypanosomiasis are performed, covering most of the trypanosome affected area of Mozambique. Hence, within the context of this project, Nb-tools were assessed in a completely uncontrolled environment.
Besides problems with accurate HAT and AAT diagnostics, problems with anti-trypanosome treatments hamper the eradication of trypanosomiasis. To date, anti-trypanosome treatment is hampered by (i) a limited choice of effective compounds, and (ii) severe treatment-related side effects due to the limited specificity of the available drugs. Starting with existing anti-trypanosome nanobodies, and including at a later stage newly generated nanobodies, NANOTRYP aimed at generating chemical chimeras in which nanobodies are used to specifically target drug compounds towards the membrane of the trypanosome. This part of the project was to rely on the state-of-the-art expertise of the Spanish partner FIBOA. This partner engaged in efforts to use an encapsulated Nb-targeted delivery system for existing drugs, mainly aiming at improving the balance between efficacy and toxicity, and as such enhancing the drug quality of existing compounds. Indeed, site-specific delivery is a recognised strategy for improving the therapeutic efficiency and safety of drugs, and nanobodies have already proven to be successful in this context (Stijlemans et al. Biol Chem. 2004 279(2):1256-61). The primary purpose of drug delivery systems is to deliver the necessary amount of drug to the target site efficiently and precisely. In addition, a fundamental aspect of these delivery systems is that they should work without modifying the desired biological features of the drug. Considerable efforts have focused on developing new strategies for controlled drug release systems based on biodegradable polymers such as cyclodextrins, which are mainly used as complexing agents to increase the aqueous solubility of poorly water-soluble drugs and to increase bioavailability and stability of drugs (review in Brewster and Loftsson. Adv Drug Deliv Rev. 2007 Jul 30;59(7):645-66). Indeed, cyclodextrins allow the formation of non-covalent complexes with ‘guest’ drug molecules, altering the physicochemical properties of the drug, and improving their water solubility. Hence, at FIBAO the aim will be to couple the advantages of nanobody-mediated drug delivery to the benefits of cyclodexrtin-controlled drug delivery methods. Important here is that VIB has already proven that coupling an anti-trypanosome nanobody to the human ApoL1 lytic domain increase lytic activity of the ApoL1 protein a 100 fold. However, as ApoL1 is the only human serum defence molecule that acts against a wide range of trypanosomes, modification of this molecule combined with wide spread use could result in a disastrous outcome in terms of ApoL1 resistance build-up. Hence, as an alternative strategy, NANOTRYP focused on encapsulation of exsisting drugs and peptides with unique anti-trypanosome properties.
As indicated above, the Nb-ApoL1 anti-trypanosome treatment approach provided the basis for the drug-targeting strategy of NANOTRYP. However, previous work only used targeting strategies in an experimental mouse model only. In order to evaluate the possibility of Nb-based drug treatment in the field, this project incorporated research on the treatment of trypanosome infected non-human primates. Human African Trypanosomiasis is a disease in which the crossing of the trypanosome into the central nervous system is the cause of several complications at the level of diagnosis and treatment. To date, little is known about the immunobiological mechanisms that are involved in this aspect of the disease, partly because of the fact that experimental rodent infection models do not offer the right settings to mimic HAT in this aspect. For example, basic information such as timing of parasite entry into the brain in HAT is still not known and is not possible to assess in rodent models. Hence, in order to address the question of parasite diagnostics as well as anti-trypanosomiasis treatment, experimental research models need to extend beyond the conventional laboratory rodent models to mimic more realistic HAT-associated problems. At the Institute of Primate Research in Kenya (IPR), trypanosome research is conducted using a vervet monkey (Chlorocebus aethiops syn. Cercopithecus aethiops) model that can be used to mimic several aspects of HAT. Including IPR as a research partner in this proposal allowed for the testing of Nb-based diagnostics in function of disease progression, and was to allow the testing of Nb-based drug therapy during these different disease stages as well.
In contrast to HAT, livestock trypanosomiasis is not hallmarked by neurological complications, but by severe infection-associated immunopathology (anaemia) and very low levels of circulating parasites. The participation of UEM in NANOTRYP provided experimental as well as field models for livestock trypanosomiasis, allowing the testing of Nb-based diagnostics in realistic conditions.
Finally, the NANOTRYP project also provided the build-up of research capacity in the participating African research institutes, as well as at the organization of a trypanosomiasis awareness campaign at all places where field trials would be performed.
First, the NANOTRYP project included the use of nanobodies in the T. brucei rhodesiense research model currently being studied at IPR. However, NANOTRYP also supported a sustainable research project in the field of T.b.gambiense trypanosomiasis, as very little is known to-date about the disease kinetics of this slow-progressing HAT variant. The project allowed IPR to engage in a longer-term fundamental research program and as such contributed to the goal of building sustainable research capacity in trypanosomiasis affected countries, through the support of the local research community. This aspect of research strengthening benefited from the research training of IPR members at both VIB and FIBAO.
Second, while HAT has a tremendous detrimental direct effect on the human population of sub-Sahara Africa, animal trypanosomiasis causes an equally severe problem as it renders vast areas of up to 60% of Africa’s grazing land unsuitable for livestock production. Hence, the UEM partner from Mozambique, where several livestock infective trypanosome species are present, participated in the development of new Nb-based strategies to fight this economic problem. Also, training of UEM researchers at VIB and FIBAO served the purpose of strengthening the local research community. In addition, UEM engaged in raising the community awareness about trypanosomiasis, with special focus on combining field work and education with respect to disease transmission, control and treatment.
In addition to the training at VIB and FIBAO of staff from IPR and UEM, 2 scientific workshops aimed specifically at transferring the Nb-knowledge to a wider public within the African research community.
Third, with the participation of FIND, NANOTRYP created a direct link to an established HAT prevention and treatment programme. Indeed, the FIND/HAT programme, executed jointly with WHO and with funding from amongst others the Bill & Melinda Gates Foundation, has links with industry, academic and research institutes in developed and endemic countries. While the proposed project fitted perfectly within the FIND diagnostics development pipeline, it also served to further expand the scope of FIND activities within the awareness programme to include the important component of livestock reservoirs.
Training in diagnosis and management of HAT by FIND was crucial for accelerate control of HAT in sub-Sahara Africa. Advocacy with ministries of health, agriculture and finance will also in the future encourage allocation of national resources to HAT control and ownership by affected countries. Companies developing diagnostic tests for HAT are being lobbied by FIND to manufacture them at prices that are affordable by the public sectors of endemic countries. These are crucial elements of a successful global access plan. In addition, the role that livestock play in disease epidemiology is now being highlighted, to ensure that livestock disease control is made an integral component of HAT control programs. Advocacy highlights the importance of HAT and livestock trypanosomiasis at international, national, and community levels, and a global access strategy. Here, NANOTRYP did not only focus on the development and implementation of Nanobody-based tools developed within the programme, but through the awareness campaigning tasks took into account the bigger picture of both HAT and AAT problems, including all available information on existing control strategies as well.
Project Results:
Overall Strategy
At the start of NANOTRYP, the main goals of the project were defined as:
1. the development of new, easy-to-use, inexpensive and reliable Nb-based diagnostic tools,
2. the build-up of a knowledge exchange and capacity building program that would enhance the access of Nb-technology by members of the African (trypanosomiasis) research community, and increase efforts in disease awareness programme.
In order to attain these goals, NANOTRYP is subdivided into 9 individual work packages, with one work package (WP1) dedicated to the nanobody generation itself, and one work package (WP9) dedicated to project management.
The generation of Nb libraries against all 4 major African trypanosome species was needed for the execution of all the other WPs. By the time NANOTRYP was initiated, the Nb generation technique was proven to be very effective and already the generated a Nbs against a cloned T. brucei parasite had been published (Stijlemans et al. J Biol Chem. 2004 Jan 9;279(2):1256-61). However, taking the use of Nbs to a next level, we aimed within NANOTRYP to design a rapid diagnostic test for the 4 major African trypanosome species. Prior to the start of the project, the VIB partner had 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, NANOTRYP first needed to develop new Nbs, with a detection range broad enough to detect all human infective T.brucei species present in the field as well as animal trypanosomes. In terms of sensitivity, the requirement for a useful Nb-based RTD would be a detection limit of 100 parasites per ml. From the start it was clear to all NANOTRYP participants that any antigen detection test would always be inferior with respect to sensitivity, when compared to an antibody detection test. However, at the level of specificity and it usefulness as test-of-cure, the antigen detection test is far more advantageous.
In order be successful in the development of a field applicable RTD for trypanosomiasis, NANOTRYP relied on the pre-existing contacts between the FIND, Switzerland and SD, Korea. Initially, the aim was to involve a commercial partner in the format transformation of a laboratory scale Nb-based diagnostic tool such as an ELISA, to a dipstick-like device for field use. SD has proven in the past to hold crucial knowledge in the field of conventional antibody-based dipstick development. In this domain they already collaborated with FIND. In the context of NANOTRYP, SD attempted to transfor their antibody technology into Nanobody technology.
When NANOTRYP was initiated, the project also included to use of Nbs for trypanosome-specific drug targeting of existing drugs, aiming at improving their therapeutic index and hence reducing toxicity associated with treatment. Only one Work Package was dedicated to this task, and the work was limited to a single well established rodent laboratory model for experimental tryopanosomiasis, using pentamidine as trypanocide. This proposal was building on our previously published data (Baral et al. Nat Med. 2006 12(5):580-4)
When dealing with trypanosomiasis, one crucial aspect of the research is the understanding of the host-pathogen interaction. To date, most academic knowledge of trypanosomiasis is based on either in vitro work, or experimental mouse models. While the first omits the relevant host interaction altogether, the second studies a host-parasite interaction that often does not reflect the relevant issue for the natural host. One example of this is the study of anemia in rodent trypanosomiasis models, while anemia is not a severe disease associated problem in HAT. The second is the encyphalicit complications of HAT, for which there is no good experimental rodent model. Hence, NANOTRYP included the International Primate Research centre (IPR, Kenya) as a partner is order to all the development of relevant models for HAT, which in a later stage would be crucial for diagnostic testing during the late stage of the disease. NANOTRYP did not include experimental testing of Nb-drug targets in non-human primates. During all experiments associated to NANOTRYP, animals were treated with a well established anti-trypanosome treatment according to protocols approved both in the home institution as well as by the institutions of the EU partners. Along the same research line, also infections in livestock anomals were included in the NANOTRYP research programme. For this purpose, UEM provided the bulk of the material, i.e. samples for both experimentally and naturally infected larger animals.
Finally, at the start of NANOTRYP, a list of deliverables was made, that allowed the follow-up of progress of the project. These mainly included the libraries of Nbs against the 4 main pathogenic species of trypanosomes (D1.1-D1.4) a Nb-based RTD (dipstick format) for these trypanosomes (D2.1-D2.4) The production of a commercial RTD for HAT, including the validation evaluation and implementation (D4.1-D4.4) a chimeric Nb-drug compound as well as a Nb-peptide construct with trypanocidal activity, validated in an experimental mouse model (D5.1 5.2) testing of Nb immunogenicity in non-human primates (D6.1) and the set-up of models for diagnostic testing in these animals for both T. rhodesiense and T. gambiense (D6.2- 6.4) optimized diagnostic method for animal trypanosomiasis (D7.1) initiating activities of awareness campaigning, training and education which included writing of an evaluation report of HAT awareness campaigning (D8.1) writing of an evaluation report of AAT awareness campaigning (D8.2) writing of a paper on the current status of trypanosomiasis diagnosis and new EC developments (D8.3) the organization of a Mid-term EC symposium on trypanosomiasis (D8.4) and the delivery of a number of joint master and PhD degrees (D8.5). Finally, three management deliverables were proposed that included the project management plans and quality assurance, the Consortium Agreement signature, and the Annual Project Reports including Financial summary report (D9.1-9.3).
Important to mention is that during the execution of NANOTRYP, a number of alterations were implemented as a result of discussion with the EU midterm evaluation panel.
• First, it was decided to drop the initial deliverable D3.1 which was a magnetic Nb-based antigen capturing tool. Based on the results obtained during the first year of NANOTRYP, this approach was considered too complicated and error-prone for later filed implementation.
• Secondly, it was proposed to limit the work on Nb-drug delivery to a single rodent model for T. brucei infections, and not to attempt to develop a strategy for larger animals or non-human primates. This resulted in term in a refocus of D6.2-6.4 in which initially a Nb-drug treatment modality was foreseen. The initial argumentation of the Mid-term review panel (that included a DNDi member) was the fact that a Nb-based compound would not suite the Target Product Profile for HAT which is an oral drug. However, two reasons were provided by the NANOTRYP consortium to persuade the panel to allow the continuation of the proof-of-concept work, being: (1) a large HAT reservoir, in particular in East Africa where the most acute form of HAT occurs is found in livestock animals, for which oral treatment is NOT the way the go, and (2) in case of late stage HAT with T. rhodesiense, oral treatment is often impossible as victims are too weak to eat, drink of swallow medication. Hence also in the this case the TPP as defined by DNDi is not ideal.
• Third, D8.5 initially only focussed on the delivery of one joined VIB/FIBAO PhD, and the Mid-term review panel asked to broaden this effort. Hence this deliverable was expanded resulting in the delivery of 4 PhDs and 7 Master thesis graduations towards the end of the NANOTRYP programme.
Results obtained in individual Work Packages
WP1: Nanobody generation
Original Objectives: The objective of WP1 is the generation of extended batteries of nanobodies directed against surface exposed epitopes of T.brucei T.congolense T.vivax and T.evansi parasites. In a first approach, Nbs will be generated against all 4 major African trypanosome species. In an additional approach, cross-reactive nanobodies, recognizing more than 1 species will be identified.
Results: NANOTRYP generated multiple libraries of binders against all parasites targeted. For T. brucei 6 libraries were made, for T. congolense and T. evansi 3 libraries were made and for T. vivax, two libraries were made, all based on material obtained from alpaca vaccinations. In addition, after the knowledge of Nb generation was transferred from VIB to both FIBAO and IPR, both partners initiated the construction of a library based on lymphocyte mRNA derived from camels. FIBOA used material that originated from a naturally infected camel identified on the Canary Islands (Spain), while IPR experimentally infected and treated a camel, allowing obtaining an anti-T. congolense response in a natural host.
T. brucei was the first parasite to be targeted by the NANOTRYP project. Having obtained a number of Nanobodies capable of binding intact parasites, it was shown that simple immunofluorescent microscopy using these Nbs made it much easier and more reliable to correctly score the infection status of suspected samples. In a similar approach, NANOTRYP showed that specific Nbs can be used in FACS to score suspected samples for infection. This was done for various clones of human infective T. b. gambiense and T. b. rhodesiense parasites.
Figure 1: T. b. gambiense brucei (A,C) and T. b. brucei (B) were stained with ALEXA488 fluorescent tagged Nb63(A), N69(B) and Nb72(A). NbBruc2.2 was subsequently used in FACS on T. brucei MITat 1.1 MITat 1.4 and MITat 1.5 infected blood samples (red = negative control).
Finally, after the screening of 6 libraries constructed using different T. brucei variants, two promising Nanobodies were selected that showed good cross-reactive binding on all samples tested. These Nbs (Figure 2 below) were transferred to WP2. Both Nb863 and Nb971 tagted conserved proteins. Screening on various purified VSG molecules (Variant Surface Glycoprotein) showed that they were not targeting a conserved region of these components of the trypanosome coat.
Figure 2: After screening of 6 libraries constructed using different T. brucei variants, two promising Nanobodies were selected that showed good cross-reactive binding on all samples tested
For T. congolense, 3 libraries were constructed and very stringent parameters were used to select for a Nb that would be suitable for binding all T. congolense parasites available to us in a laboratory setting. Several Nbs were found to be useful for fluorescent parasite detection as shown in Figure 3. However, finally only a single Nb was withheld that met all our criteria (Nb474) for sandwich Ag-capturing development. This Nb was subsequently transferred to WP2.
Figure 3: Assessment of T. congolense Tc13 specificity of 3 Nbs of the Tc2 library in an on live T. congolense parasites omitting any cold-step incubation.
T. vivax library construction was performed having only a single laboratory stock as our target. From this library 11 Nbs were obtained of which most could be used in fluorescent detection of parasites in infected blood samples. All these Nbs were transferred to WP2 aiming at developing a sandwich Ag capturing test.
For T. evansi a first library was constructed using a vaccination protocol in which a mix soluble protein extracts, obtained from 5 different T. evansi stocks, was used. With this strategy, 4 Nbs were obtained that were capable of binding a 6th T. evansi parasite that was not part of the initial vaccination protocol. Perfomring various ELISAs in which the detection limit for the 4 different Nbs was assessed, only Nb392 was withheld for further use in WP2. At the same time, Nb392 was also produced as a GFP fusion construct for the use in fluorescent microscopy and FACS. Following the generation of Nb392, we engaged in the construction of a new anti-T. evansi library with a slightly different approach. Rather than using a mix of antigens for every vaccination boost, we purified T. evansi soluble protein extracts and subsequently use one specific extract fo a single vaccination/boost only, hence exposing the alpaca to 6 different ‘parasites’ during the subsequent weeks of the protocol. Interestingly, this strategy resulted towards the end of the NANOTRYP proposal in 16 different Nb families that will be be used in future strategies.
Figure 4. Left panel: Solid-phase ELISA coating serum and lysates from T.evansi STIB 816, T.evansi 0101399B, T.evansi 150399B, T.evansi 120399C and T.vivax (negative control). Right panel: Solid-phage ELISA coating two-fold serial dilution of antigen (T.evansi STIB816) and subsequent recognition by Nb392.
One of the goals of NANOTRYP WP1 was to generate a Nb-based Ag capturing system. While for a long period of time it seemed that this goal would not be attained, in the end ant anti-T. evansi Nb392 appeared to target a very conserved protein that is part of the flagella of the parasite. Hence this Nb was tested in fluorescence microscopy for binding to various parasites. Figure 5 shows identical staining patters on T. brucei, T. congolense, T. vivax and T. evansi. Future identification of the exact target may hence contribute to the development of a truly cross-recognizing Ag capturing device for the diagnosis of trypanosomiasis.
Figure 5: Binding of Nb392 to fixed permeabilzed T. evansi parasites (left panel) and T. congolense parasites (right), indicting the recognition of a highly conserved flagellar protein. Staining on T. brucei and T. vivax (both not shown here) gave a similar result.
Conclusion: WP1 was mostly executed according to the initial planning. During the progress made, vaccination protocols were altered mainly with respect to the challenge strategy during the subsequent vaccination boosts. Form both the T. congolense work and the T. evansi work most valuable lessons were learned that will guide future work. Using mixed prtein extracts derived from several non related stabilates improves the change of obtaining Nbs with good binding characteristics against consrevd targets. However, in the case of this project, only one truly cross-reactive Nb was obtained, suggesting that immunodominance of a limited number of compounds results in a biased outcome. Obtaining only one Nb does not allow the construction of a sandwich system (the aim of WP2), but it allowed to identify the target which in the future now can be used for a much more targeted immunization strategy. This in turn will greatly enhance the chance of finding a second binder that will allow the construction of a sandwich system. Using different trypanosome stocks for vaccination and selection of binders, as was done for T. congolense, resulted in a single Nb that could be used in a sandwich system. Indeed as Nb474 appears to recognize a target with repeated epitopes it was successfully transferred to WP2. In a third approach (used for T. evansi) successive alpaca vaccinations were performed with each time a different trypanosome variant. This resulted in the selection of 16 families of cross-recognizing Nbs. Whether or not all of those are ssuitable for transfer to WP2 is still under investigation. In contrast to the success with T. congolense and T. evansi, the generation of applicable anti-T. vivax Nbs was less successful. The main issue encountered here is the fact that in general, T. vivax cannot be propagated in laboratory rodents, making it impossible to have access to protein extracts of various isolates that have been properly characterized. Hence, using different isolates for vaccination and Nb selction was not an option here. In addition, it appears that the surface organization of T. vivax must be quite radically different from other trypanosome as we were able to use T. vivax as a ‘negative’ control for specificity during the selection of binders against all other parasites. The molecular reason for this is has not been elucidated at this stage.
For the generation of anti-T. brucei nanobodies (including binders for the human infective T. b. gambiense and T. b. rhodesiense), the most successful approach was the generation of a Nb library starting form material obtain from an experimentally infected camel (using T. evansi for infection – subsequently drug cured before any infection-associated pathology signs became apparent). This strategy resulted in two Nb-pairs that were successfully transferred to WP2.
A final remark for WP1 that is important to reiterate on is the fact that even when Nbs that are not suitable for the development of a sandwich Ag-capturing tool, they can still be very efficiently used in immunofluorescent detection of trypanosomes. This was shown in NANOTRYP both for the collection an anti-T. vivax Nbs, as well as the cross-recognizing anti-T. evansi Nb.
WP2: Development of a Nb-based lateral flow assay
Original Objective: The objective of WP2 is to use the batteries of species-specific nanobodies from WP1, and select couples of Nbs to make specific dip-stick tools for the detection of the 4 major African trypanosome species
Results: WP2 started with the transformation of an existing VSG-specific Nb-sandwich system (that was used as a proof of principle to put the NANOTRYP consortium together) into a dot-blot system. A dot-plot is the laboratory format that closes resembles an RTD for field use. As this initial system was however unable to detect human infective T. b. gambiense and T. b. rhodesiense parasites, this WP swiftly moved on to indentify Nbs that were capable detecting Both HAT parasites, and subsequently tested the Nbs identified in ELISA (see above WP1 Figure 2) in a dot-blot setting.
Figure 6: Sandwich Dot-blot and ELISA system using parasite lysate as antigen. Left panel (A): T. b. rhodesiense lysate captured with various His-tagged Nbs (as indicated) and detected with a mix of both Nb61 and Nb64 biotin-tagged Nbs. Left panel (B): idem. For T.b.gambiense. Right panel: ELISA with T. b. gambiense lysate using His-tagged Nbs for coating and biotin-tagged Nbs for detection
After having obtained convincing data showing that several Nb-sandwich systems were capable of capturing parasite antigen in infected samples, an MTA was drafted with Standard Diagnostics (SD, Korea), i.e. the commercial partner identified by FIND (see WP4) that would engage in actual RTD development. At SD, Nbs were processed according to internal protocols in order to make them suitable tools for antigen capture and detection. In short, 16 different Nb-capturing/Ag-detection combinations were tested (Figure 7)
The test format designed followed the conventional later flow Ag capturing design that has been successfully commercialized by SD for a whole range of diseases, using conventional antibodies rather than Nanobodies.
Results obtained are visualized below:
Figure 7: Various combinations of Nbs were used for coating and detection, with results boxed in yellow providing the information that the combination Nb971/971-gold provided sensitive and specific Ag recognition. This is the same Nb pair that was indentified in WP1 – see Figure 2 – as the best Nb combination in ELISA.
Unfortunately, despite the initial hopeful results, SD realized that the Nb-gold conjugate used for detection was unstable and that production using a standard protocol yielded unacceptable batch-to-batch variations. After several rounds of discussion and two visits by the VIB partner to SD, it was decided that two technical issues hampered further development, i.e. the fact that (i) both capturing and detecting Nbs in this system bind the same epitope, resulting in saturation of the Ag in the first step of the assay, leading to subsequent lowering of capturing capacity, and (ii) that Nb-gold conjugation needs to be optimized, most lileky using a protocol that is not part of the standard production methods at SD. The last trail runs at SD were performed in April 2013, towards the end of the 6 month extension period. In the near future, we will initiate alternative protocols, involving most likely a new partner, in order to solve the technical problems encountered at SD.
Later flow test development for T. congolense, T. evansi and T. vivax were not initiated, but in an alternative way forward, NANOTRYP did develop a highly sensitive Nb-based ELISA system for the detection of T. congolense infections as part of WP2 and also WP7. Using Nb474 for both capturing and detection, we were able to construct a system that could be used for both diagnosis of active cases, and could be used as test-of-cure.
Figure 8: VIB tested the Nb474 ELISA system on a large range of well characterized T. congolense laboratory strains as well as in an experimental infection setting performed at the Onderstepoort Veterinary Institute in South Africa. Here, T. vivax and T. brucei infections were both used as negative controls
Following the results obtained in Figure 7, the Nb474 system was extensively tested together with the UEM and IPR partners (see WP7).
Conclusion: As outlined above, NANOTRYP did not deliver a final format for a Nb-based diagnostic lateral flow test for trypanosomiasis. The preliminary results obtained by Standard Diagnostics (SD, Korea) presented in Figure 6 were hopeful (in particular as they confirmed the VIB ELISA results of Figure 2) but technical problems with the colloid-gold detection system used in conventional RTDs could not be resolved in a timely manner. One of the main consequence of this ‘failure’ is the fact that a large portion of WP4 – the testing in a field setting of the developed device – was not executed. Looking back on the project, the fact that SD was unable to transform the Nb-based ELISA into a dipstick was impossible to anticipate, as from the start we did have successful preliminary data from a prototype VSG specific dipstick developed by VisionBiotech, South Africa. Taken that both SD and VisionBiotech are now owned by the same industrial group, moving back to South Africa with the project was not an option. One lesson learned during NANOTRYP is the fact that the engagement of SD into the project happened far too late into the project. On one hand, more than a year of three-way negotiations between FIND, SD and VIB were needed to come to a proper MTA. Secondly, SD initiated the RTD development only after a suitable Nb pair for HAT had been identified. From an SD perspective, this is understandable as optimizing the technology in advance with an irrelevant Nb-pair would have been a lost investment if later-on we would have been unable to deliver a suitable Nb-pair. From The NANOTRYP side however, it would have provided more time to solve issues with the Nb-gold coupling, a technique that once resolved should be pretty standard for all Nbs. Finally, only when the full SD RTD development strategy was revealed (this happened after the failure to produce a prototype), it became clear that the successful ELISA sandwich system did not fully meet the conceptual requirements for a lateral flow design. Indeed, while the detection of a multimeric target with repeated epitopes allows in ELISA for the use of a Nb homo-sandwich setup, resulting in high sensitivity and specificity, a lateral flow design needs to be constructed around the use of two Nbs targeting different epitopes of the same target, i.e. the design of a Nb hetero-sandwich system is a must. On the positive site, the combined WP1 and WP2 packages showed that it is possible to develop a Nb-based sandwich system that can be used successfully trypanosomiasis detection. In the immediate future, we will now concentrate on different ways to solve the Nb-detection system in a RTD context, and secondly, we have now designed a new Nb selection strategy to fish out hetero-sandwich pairs right from the start of the procedure. While NANOTRYP has come to an end, the WP2 package has provided us with extremely valuable information for future efforts. The NANOTRYP consortium remains fully convinced that development of new diagnostic tools are crucial for the future fight against trypanosomiasis, and that an Ag-detecting tool in the end is the way to go, as only such assay can be used for (i) active case diagnosis, (ii) test-of cure after treatment or drug failure detection, and (iii) diagnosis of re-infection after successful treatment.
WP3: Development of a Nb-based magnetic bead assay
Original Objectives: The objective of WP3 is to develop an alternative trypanosome detection method as back-up assay for the dipsticks developed in WP2. Here the same Nbs from WP1 will be coupled to magnetic beads, allowing the development of an alternative magnetic-bead detection method for all 4 major African trypanosome species.
Results: During the first reporting period of NANOTRYP, various Nbs were used to set up a magnetic bead capturing assay. The assay is based on the principle of using Nb-coated paramagnetic beads for incubation with target blood samples, containing parasite antigens. Subsequently, beads are collected using a Pickpen® device, washed and Ag-binding is measured using a colorimetric substrate conversion reaction similar to those used in ELISA. After optimizing washing conditions as presented below, the assay sensitivity was brought to 100 parasites/sample. While this sensitivity is good enough for diagnostic screening tool, the entire procedure of development turned out to be too laborious to properly be performed under realistic field conditions.
Figure 9: Pickpen® capturing of T. brucei AnTat 1.1 using a variety of incubation and washing conditions and a combination of the pan-reactive Nb33 and Nb1 from Library 1. Virtually identical results were obtained with Nb7 and Nb8 from the same library.
Conclusion: Taken the technical difficulties of performing this assay under optimal conditions, in particular the requirement for large volumes of washing buffer, prompted the consortium to halt this WP. This decision was approved by the Mid-term reviewing panel that assessed the NANOTRYP program in September 2010.
WP4: Diagnostic field evaluation
Original Objectives: To evaluate T. brucei specific dip-stick and magnetic bead assays, demonstrate their use, and develop a strategy for their access to the population at risk
Result: The first task of WP4 was to indentify a commercial partner willing to transform a laboratory scale Nb-diagnostic tool into a field applicable format. Based on the experience of FIND in other projects, Standard Diagnostics (SD, Korea) was contacted. After three-way negotiations involving VIB, FIND and SD, and MTA was signed between the VIB and SD and the most promising Nb pairs obtained in WP1 and WP2 were transferred (see above, results WP2). However, due to the technical problems encountered during lateral flow development (see above, results WP2), the subsequent tasks allocated to WP4, i.e. the validation, evaluation, demonstration and implementation were not executed.
Conclusion: Unfortunately, the main tasks of WP4 with respect to bringing a Nb-based diagnostic tool to the field were not fulfilled due to the lack of a proper functioning test prototype. The efforts to develop such prototype will continue in the future.
WP5: Nanobody-drug coupling
Original Objectives: The aim of WP5 is to develop a Nb-based targeting strategy for site-specific drug delivery, in order to improve current anti-parasite therapies. Two different approaches will be employed: i) coupling existing anti-trypanosome drugs to Nbs; and ii) coupling trypanolytic peptides to Nbs to target the parasite.
Results: During the NANOTRYP project, a nanobody based drug delivery system consisting in a cyclodextrin molecule linked to the six histidines tail present in the carboxiterminal end of nanobody NbAn33 was generated. Next step was the occlusion a trypanocidal drug into Nb33-cyclodextrin complex without affecting its trypanosome binding capacity. For this purpose, two trypanocidal drugs were synthesized and occluded into Nb::CD conjugate: nitrofurazone and tionitrofurazone. Both are hydrophobic nitroaromatic compounds. By drug occlusion assays into Nanobody:cyclodextrin conjugate we determined that the occlusion rate of both Nifurtimox and Nitrofurazone was 40%. Tionitrofurazone occlusion rate was lower but improved after increasing the ionic strength. We have analyzed the trypanocidal activities of nitrofurazone and tionitrofurazone complexes (figure 10) and both complexes kill trypanosomes in vitro.
Figure 10: Growth curves of bloodstream AnTat 1.1 in the absence (circles) and the presence of Nb:CD:Nitrofurazone and Nb:CD:Tionitrofurazone complexes (squares) and Nb:CD conjugate (triangles).
However, two were the major limitations of the Nb::CD trypanocidal drug delivery system: i) Its specify for the occlusion of hydrophobic drugs. ii) Its limited loading capacity. Only one drug molecule per Nb::CD conjugated and according to the occlusion rate for nitrofurazone and tionitrofurazone about 60 % of Nb::CD molecules are not loaded with drug. In order to overcome these limitations we decided to alter the the original plan. Two different polymeric nanoparticles coated by a Nb that specifically target T. brucei cell surface (NbAn33) and loaded with the trypanocidal drug pentamidine have been synthesized. The natural polymers chitosan and PLGA have used to generate noparticles of chitosan and PLGA termed NbAn33-pentamidine-chNPs and NbAn33-pentamidine-PLGANPs respectively. Both drug carries displayed a potent in vitro trypanocidal activity, as the half-maximal inhibitory concentration (IC50) of pentamidine-loaded, nanoparticles was 14 and 7 fold lower 7 respectively than free pentamidine (P<0.0001) (figure 11).
Figure 11: Trypanocidal activity of pentamidine formulations. A) Gray column: IC50 value for free pentamidine. Red column: IC50 value for pentamidine-chNPs. Blue column: IC50 value for NbAn33-pentamidine-chNPs. Errors bars indicate SD from 3-9 independent experiments. Fold reductions are indicated in the graph. B) Green: nanobody-PLGA-pentamidine nanoparticles; Purpure: PLGA-pentamidine nanoparticles; Blue: pentamidine.
We next tested the trypanocidal activity of the Nb::pentamidine-chitosanNP. The minimal full curative dose of pentamidine in a mouse model of acute infection of T. brucei was previously established as four doses of 2.5 mg∙kg-1 administrated daily by i.p. injection in four consecutive days, starting upon detection of parasites in blood (day 3 after infection) (Fig. 11b). When mice were treated with a 10-fold lower dose of pentamidine (4 x 0.25 mg∙kg-1) the parasites disappeared from the peripheral blood after the third dose. However, the infection relapsed and the mice began to die at day 22 after infection, curing only 20% of the treated animals (Fig. 12). Having established the suboptimal pentamidine curative dose, we treated mice with equal dose of pentamidine loaded into NbAn33-NPs. Clearance of parasites from this group was complete after the first dose and the treatment successfully cured 100% of the animals (Fig. 12). Infected mice were treated with the same pentamidine dose loaded into NPs non-coated by the nanobody. In this group, parasites disappeared from the blood after the first dose, however 40% of the treated mice succumbed to the infection (Fig. 12). Next, a dose 100 times lower than the minimal curative dose of pentamidine was tested. At that low concentration (4 x 0.025 mg∙kg-1), free pentamidine did not cure mice from trypanosome infection. Remarkably, treatment with 4 doses of NbAn33-pentamidine-chNPs at 0.025 mg∙kg- cured all treated mice. However, the same low dose of pentamidine loaded into chNPs non-coated by the NbAn33, , did not cure mice, with 100% of the treated animals dying from the infection.
Figure 12: Therapeutic effect in T. b. brucei acute infection mouse model. Survival (Kaplan-Meier plot) of female C57BL/6J mice infected with T. b. brucei AnT1.1 (inoculum 1x104 parasites). The treatment started once the parasites were detected in blood, at 3 day after infection and consisted in a daily dose in four consecutive days. Treatment with pentamidine, pentamidine-chNPs (pentamidine-loaded PEGlycated chitosan nanoparticles), NbAn33-pentamidine-chNPs (nanobody-coated pentamidine-loaded PEGlycated chitosan nanoparticles), NbAn33-chNPs empty (nanobody-coated non pentamidine-loaded PEGlycated chitosan nanoparticles) and vehicle (physiological serum).
All know mechanisms associated to pentamidine resistance are dependent on drug uptake associated to surface membrane proteins (transporters and aquaglycerolporins). A tripanocidal drug vehicle consisting in pentamidine loaded nanoparticles coupled to a nanobody that bind to the surface of the parasite, like Nb33, sould overcome drug resistance associated to pentamidine uptake. To test this hipotheis we generated a T. brucei Antat 1.1 cell line resistant to 25 ng/ml pentamidine by slowly increasing drug concentration added to the culture medium. We named this cell line T. brucei R25. We next compare the IC50 of penamidine (Fig. 13), pentamidine loaded nanoparticles and pentamidines loaded NPs coated with Nb33 in our resistant cell line. As observed in wild type cells, the IC50 obtained with both Nb::NPs was significantly lower compared to pentamidine alone (12.5 nM, 5.9nM and 87.6 nM respectively).
Figure 13: IC50 reduction of the described formulations.
The second proposed approach was to develop a Nb-based anti-trypanosomiasis treatment through coupling trypanolytic antimicrobial peptides (AMPs) to specific nanobodies that target trypanosomes. In the initial proposal one of the strategies proposed was to couple lytic peptides to Nbs by chemical methods. However conjugation with nanobodies may affect the lytic capacity of the peptides at the surface membrane. To circumvent this problem a protease cleavage site was added to the end of each synthetic peptide. Thus, all synthetic lytic peptides employed carried at their carboxy-terminal end a tail of five residues containing the Cathepsin B cleavage sequence. Cathepsin B is a lysosomal protease that is also present in T. brucei. If the Nb-lytic peptide conjugate do not kill the parasite by cell membrane disruption when they are on the cell surface, the complex of surface protein-Nbs-(Cathepsin B cleavage site)-lytic peptides will be internalised by the very active endocytic pathway. Upon delivery to the lysosome, the lytic peptides/nanobodies/receptor complexes will be processed by Cathepsin B. Released lytic peptides will then be free to insert into and disrupt the lysosomal membrane causing cell death.
First, the trypanocidal activity of two AMPs, temporin A and temporin 1Lc were assayed. Both Nb::Temporin bioconjugates were able to reduce the IC5O of the AMP about 4-fold lower than temporines (figure 14).
Figure 14. Effect of AMPs on T. brucei bloodstream forms viability. Curve dose response (viability percent versus Temporin 1Lc concentration).
Conclusion: WP5 was mostly executed according to the initial planning. During the progress made, two different polymeric nanoparticles coated by a Nb that specifically target T. brucei cell surface (NbAn33) and loaded with the trypanocidal drug pentamidine have been synthesized. The natural polymers chitosan and PLGA have been used to generate noparticles of chitosan and PLGA termed NbAn33-pentamidine-chNPs and NbAn33-pentamidine-PLGANPs respectively. Both drug carries displayed a potent in vitro trypanocidal activity, as the half-maximal inhibitory concentration (IC50) of pentamidine-loaded, nanoparticles was 14 and 7 fold lower respectively than free pentamidine (P<0.0001). Furthermore, the curative dose of pentamidine-loaded nanobody-nanoparticles was 100-fold lower than pentamidine alone in a murine model of acute African trypanosomiasis. Crucially, pentamidine-loaded nanoparticles displayed undiminished in vitro and in vivo activity against a trypanosome cell line resistant to pentamidine.
We have also generated two anti-trypanosome nanobodies carrying each one an antimicrobial peptide (temporin A and temporin 1Lc) covalently attached via Cathepsin B cleavage sites. Both Nb::peptide conjugates were active in vitro against T. brucei bloodstream forms.
WP6: Nb-tools for diagnosis and treatment of T.b.rhodesiense and T.b.gambiense infections
Original Objective: The objective of WP6 is to take the result from WP2, WP3, and WP5 to more field-related trypanosomiasis models. Here, a monkey model for T. brucei (rhodesiense), in which blood-brain-barrier complications may hamper drug efficacy, will be assessed. WP2 and WP3 will be applied in the sense that both a T.brucei specific dipstick assay and a trypanosome magnetic bead detection assay will be assessed in this infection model. WP5 will be evaluated as new treatment methods for experimental trypanosomiasis, in a model that is much closer to the human situation than the usual mouse model. In this WP, Nbs could also be subjected to host-specific mutagenesis to reduce possible immunogenicity that will be checked for at the start of this WP..
Results:
Determination of Immunogenicity of Nb in mammalian hosts
For chemotherapy of HAT, it is envisaged that the Nb-drug conjugates will need to be repeatedly (but with short intervals) injected into infected subjects. Thus, their immunogenicity requires evaluation, under conditions in which several subsequent daily injections would be administered. The current study evaluated the immunogenicity of NbA33, a recently generated nanobody for use in HAT studies under NANOTRYP project. The study was carried out in vervet monkeys (Chlorocebus aethiops) which were to be used as the ultimate animal model for treatment and diagnostic tests during the project.
Three groups of experimental animals were used divided and inoculated with various antigens thus;
• Group 1: Inoculated with NbAn33 at 50ug intramuscularly (IM) for 3 consecutive days
• Group 2: Negative control, phosphate buffer saline (PBS) administered at 1ml for 3 days
• Group 3: Positive control, infected with Leishmania donovani promastigotes
The monkeys were sedated and sampled for blood at weekly interviews for 5 weeks. This regime was meant to simulate the treatment protocol for diminazene aceturate as carried out during experimental infection treatments in this lab animal model. Peripheral mononuclear lymphocytes (PBMCs) were harvested and blast assays carried out while serum was analysed for anti-Nb stimulation.
The mean ODs of the monkeys treated with NbAn33 at all the sampled days were not significantly different from those inoculated with PBS buffer. However monkeys inoculated with L. donovani had a consistent elevation of OD values to significantly higher between 7 and 35 days post-inoculation in comparison to either PBS or NbAn33 groups (Fig 15a and Fig 15b).
Figure 15a: Mean OD values in vervets testing for immunogenicity against NbAn33
Figure 15b: Blast assays in vervets testing for immunogenicity against NbAn33
Conclusion:
The study showed that NbAn33 have undetectable immunogenicity in mammalian hosts and therefore it may be safe as a vehicle for targeted delivery of HAT drugs.
Diagnosis and treatment of early and late stage Tb rhodesiense infection in vervet monkey model
Vervet monkeys were experimentally infected with T. b. rhodesiense IPR 001 isolate. The animals were monitored for disease through ear prick (Ndungu 1994; Sayer & Schimdt, 1983), sampled at weekly intervals under sedation for blood (for serum separation and haematology) and cerebrospinal fluid (CSF). At 28 days after infection the animals were treated with diminazene aceturate (DA). It is assumed that treatment at this stage clears trypanosomes from the haemolymphatic system and allows those in the CSF to thrive, become neuroinvasive and precipitate late stage disease (meningoencephalitis). The animals were monitored post DA-treatment for late stage disease clinically and by immunological assays. The experiment was stopped by humane euthanasia of the experimental animals approximately 51-56 days when relapse parasitaemia alongside salient clinical signs of paresis and paralysis were observed in the animals. Non-infected control animals subjected to similar experimental procedures were also included in the study. All the experimental procedures were approved by the Institutional Ethical Review Committee (IRC) of IPR, including an oversight International Advisory Board (IAB) with a membership drawn from Europe and USA. Two MSc students were enrolled for this task.
Analyses for immunospecific IgM and IgG was carried out using enzyme linked immunosorbent assays (ELISA), cytokines IL-6 and IFN-γ were assayed using flow cytometry as well as ELISA whereas albumin and total protein were quantified using colorimetric protein assays.
The four experimentally infected animals had a typical clinical presentation of the disease with patent parasitaemia 4 days after infection which peaked at 9 days after which the parasitaemia fluctuated with typical waves of parasitaemia. Late stage disease was established by demonstration of typical meningoencephalitic lesions including perivascular lesions (Figure 16)
Figure 16a: perivascular cuffing in the brainstem of a vervet monkey infected with Tb rhodesiense
Figure 16b: perivascular cuffing in the cerebellum of a vervet monkey infected with Tb rhodesiense
Detailed immunological analysis further showed a significant elevation of IFN-gamma and IL-6 in serum of experimentally infected animals as depicted in the Figure 17. These molecules were not detectable in the CSF during early stage disease.
Figure 17a: Serum IFN-γ spike 7dpi in early stage HAT
Figure 17b: Serum IL-6 spike 21dpi in early stage HAT
Characterisation, diagnosis and treatment of Tb gambiense in a NHP model
The T.b. gambiense is responsible for over 90% of total cases of HAT (Malvy et al., 2011). The infection is characterized by low parasitaemia with no specific clinical symptoms especially during the early stage when trypanosomes are confined to the hemolymphatic system (Inojosa, 2006). This has limited the use of standard diagnostic techniques, with an estimated 20 to 30% of patients being undiagnosed (Robays et al., 2004). The serological test—card agglutination test for trypanosomiasis (CATT)—for T. b. gambiense has varying sensitivities and cannot decisively differentiate between active and cured cases (Lejon, 2010). The parasitological detection techniques have limited sensitivity hence it is possible that some of these unconfirmed CATT-seropositive individuals are actually infected (Garcia et al., 2000). Vast improvements have been made by the development of a rapid diagnostic test (www.finddiagnostics.org accessed 6 September 2013). In order to study the pathogenesis of this chronic form of the disease, several attempts have been made at characterising a laboratory animal model, with limited success. Two attempts have been made in this project to characterise a nonhuman primate model of Tb gambiense, in two different species (vervets and Sykes monkey).
Alongside the immunological characterisation, molecular detection of the parasite DNA in various tissues and fluids was made. The loop mediated isothermal amplification (LAMP) technique was first described by Notomi et al. (2000). It amplifies DNA using an enzyme with strand displacement activity under isothermal conditions using four to six primers. LAMP has been used extensively in HAT studies. In the diagnosis of T. brucei using the PFR A protein (Kuboki et al., 2003), T. gambiense using ITS2 gene (Thekisoe et al., 2007), Trypanozoon subgenus using RIME (Njiru et al., 2008) and T. rhodesiense using the SRA gene (Njiru et al., 2008). LAMP has several advantages over other detection formats. These include: rapidity, high sensitivity, specificity, several product detection formats and amplification at isothermal conditions.
DNA was isolated from the blood, urine, cerebrospinal fluid and saliva samples of Sykes monkeys obtained at regular intervals during the study. T .b. gambiense primers described by Njiru et al (2011). The LAMP reaction was performed for 80 mins in a LoopAmp Turbidimeter® using purified trypanosome DNA from the T. b. gambiense IL 3253
Body Compartment Duration of infection when test was Positive
PCR LAMP
Blood/serum 7 – 123 dpi 7 – 168 dpi
CSF - 168 – 175 dpi
Urine - 28 – 105 dpi
Saliva 14 – 35 dpi 14 – 112 dpi
Parasitaemia was detectable between 7 and 98 dpi
CSF parasites were not detected by microscopy for the entire duration of the study
The overall detection rate of parasite DNA in all the samples was low. Whole blood has a higher detection rate (14%) as compared to the other samples- urine (8%), CSF (6%) and saliva (2%). The parasites could not be detected in the later stages of the disease in any of the samples. This may imply the infection in the animals had resolved. A similar study in the vervet model reported higher detection rates in all the four sample groups.
Conclusion:
The following conclusions can be made from the studies carried out under WP6:
1) NbAn33 has undetectable immunogenicity in mammalian hosts and therefore it may be safe as a vehicle for targeted delivery of HAT drugs
2) The clinical disease, biochemistry and immunological analysis of Tb rhodesiense nonhuman primate model was characterised.
3) Partial characterisation of the Tb gambiense model has been accomplished leading to the detection of parasites DNA in urine and saliva opening possibilities of non-invasive samples for diagnosis
WP7: Treatment testing in cattle and goat
Original objective: The objective of WP7 is to take the result from WP2 and WP3 to larger animal models, and in particular assess the use of Nb-based tools for the diagnosis infected goats, focussing on T. brucei, T.congolense T.vivax and T.evansi while using the tools from WP5, treatment for T. brucei infections will be assessed as well.
Results:
Collection of sera and field isolates
Eleven field missions were carried out aiming at the collection of serum samples and Trypanosoma isolates from naturally infected animals. Eight field missions were done in Matutuíne District, Maputo Province (100 Km South of the City of Maputo) and three in the districts of Nicoadala and Inhassunge, Zambézia Province (1600 Km North of the City of Maputo). The field work was executed at dip-tanks or corridors. From each animal, blood was collected from the coccygeal vein, using two 7 ml vacuum tubes (with and without anti-coagulant [EDTA]). All the samples were parasitologicaly analysed in the field, using the micro-haematocrit centrifugation technique, “Buffy-coat”. All the parasitological positive samples were inoculated into mice or in goats in the case of T. vivax, pure or mixed infections. All the samples were PCR analysed (18S PCR –RFLP) in the Biotechnology Center-UEM for molecular species specific identification. The serum was prepared from the tubes without anti-coagulant and stored and labelled according to the parasitological and molecular results. All serum samples from negative animals in both assays were discarded.
During the field missions 1048 bovines were screened and a panel of 124 reference sera (103 T. congolense positive; 7 T.vivax; 14 T. congolense and T. vivax mixed infections) was established. Furthermore, two hundred cattle serum samples were collected from a Trypanosoma free area of Maputo Province (Boane District), to constitute a panel of bovine negative sera. From the field diagnosed positive animals a collection of 45 isolates (43 T. congolense and 2 T. vivax) was added to the cryobank.
Experimental Infections
All experimental infections were performed in fly proof conditions. Five goats and 50 bovines were infected with T. congolense and three goats with T. vivax . Before each experimental infection 20 ml of blood were collected from jugular vein into plain tubes to prepare the pre-infection serum sample (approximately 10 ml). For the experimental infection, stabilates from a specific isolate were removed from the cryobank, thawed in water at 38oC and and injected I.P. in two Balbc mice. The infected mouse blood was diluted in PSG, to a concentration of 104 trypanosomes/ml, and subsequently I.V. inoculated in an experimental goat or bovine. For the T. vivax infections the process of isolate amplification after retrieval from the cryobank was done, using goats. During the course of the experimental infection the parasitaemia and the PCV was determined by micro-haematocrit centrifugation technique and followed daily until patency. After patency the above mentioned parameters were checked weekly. A serum sample (5 ml) was prepared weekly and each animal was followed until ninety days post-infection. All the serum samples were appropriately labelled and stored at -80oC.
The biological material collected up to now combined with the present availability of nanobodies will allow for the evaluation of any nanobody- based diagnostic assay. As part of the NANOTRYP effort to evaluate the potential of a Nb-based diagnostic tool, Steven Odongo (VIB) visited UEM and tested a Nb ELISA that had been optimized previously at the VIB. The results obtained show that the Nb474/474 combination ELISA developed in WP1/2 can be used to detect trypanosomiasis infections. The specificity of the assay is sufficient to discriminate T. congolense trypanosomiasis form other common bovine infections such as theileria and babesia. T. vivax Trypanosomose was not detected with the assay, making the Nb474/474 a specific detection tool for T. congolense. While testing the ELISA, the storage requirements for test samples were addressed as well. Storage for up to 2 weeks at 4°C of infected samples did not hamper the correct identification of infections. However, samples that were stored for up to 3 weeks at 4°C started to show an unacceptable level of false negative results. Under field conditions this is not be considered to be an issue as samples would most likely be tested within 24 hours after isolation.
In addition to the UEM sample collection, the Nb474/474 ELISA was also tested on a sample collected obtained from IPR, Makerere University (Uganda), Onderstepoort Veterinary Institute (South Africa) as well as a collection that was obtained through a more recent collaboration with GALVmed. In the latter collection, samples pre- and post-treatment were included, showing that within 48h after treatment, a negative ELISA score was obtained.
Conclusion: Combined, in all cases similar results were obtained showing that the Nb approach proposed in NANOTRYP is a successful, and offers the potential to be developed as a test-of-cure. However, as indicated in WP4, for the next step, i.e. development of an field applicable RTD, the Nb474 target has to be identified and a second detecting Nb has to be generated in order to allow the use of a hetero-sandwich system.
Figure 18: Cattle sera characterized by buffy coat technique (BCT) were cross-examined with T. congolense Nanobody®_ELISA (Nb_ELISA). A,B: Local cattle reared in trypanosome endemic regions of Mozambique (right) or Kenya (left) were bled and whole blood examined for trypanosomes by BCT followed by cross-examination of their sera for T. congolense by Nb_ELISA. C: Performance of Nb_ELISA evaluated on sera of cattle experimentally infected with T. congolense (IL1180). High OD450nm are consistent with sera where T. congolense infection is involved indicating the test’s ability to detect T. congolense infection. More so the sensitivity of Nb ELISA on experimentally infected cattle was 87%. D: Assessment of Nb_ELISA for possible cross-reactivity with Theileria, Babesia, or Anaplasma. Nb_ELISA was performed on hemolyzed whole blood confirmed positive for the named protozoan parasites. Negative (-ve) were whole blood from naive cattle. The Nb_ELISA was scored as positive 2/3 trypanosome samples, 5/44 theileria samples, and negative on babesia and anaplasma samples. Therefore, apparent cross reactivity observed with theileria could be attributed to mixed infection with T. congolense that was missed by BCT. These positive samples will be cross-checked by PCR.
WP8: Awareness campaigning, education and training
Original Objectives: The aim of WP8 is to ensure that the project results are disseminated to the affected community. This WP includes training of students in HAT/AAT endemic countries, training of PhD students as well as generating education material for local communities. Finally, also an EU mini-symposium envisaged the communication of FP7 research efforts to the wider public.
Results:
In Uganda, FIND awareness campaigns were carried out by Gulu University in the T.b. gambiense region in the north of the country. The specific objectives of the campaigns included a) understanding the local community’s perception of the disease, b) appraising the community’s level of understanding of the symptoms of HAT and nagana, transmission, diagnosis, treatment and control, and c) sensitizing leaders and communities living in these areas so that they seek early diagnosis and treatment for both human and livestock diseases, in order to remove reservoirs of infection. Familiarization and interpersonal meetings were held with key district and selected sub-county officials, to brief them on the purpose of the project and get their understanding on the severity of the problem. Community mobilization campaigns at the district, sub-county and at community levels focussed on the importance of early diagnosis of HAT and Nagana, the animal-human infection relationship, understanding the ecology of tsetse flies and their role in transmission of the diseases, local community perception of sleeping sickness and Nagana, and community coping strategies and adaptations. Advocacy materials that were developed and distributed included a) posters on trypanosomiasis and how it is spread, b) posters on actions in control of trypanosomiasis, c) Question and Answer booklet (Frequently Asked Questions) for community mobilization, d) leaflets in the various local languages in the endemic areas highlighting how trypanosomiasis is spread and control methods and T shirts, e) radio talk shows in the two dialects zones of Adjumani and Nwoya – Amuru, and f) training workshop for village Health Teams.
The Ministry of Health (MOH) of Uganda and the National Livestock Resources Research Institute (NaLIRRI) focused on the T.b. rhodesiense region in the south east and central Uganda. In addition to using advocacy materials that were similar to those used in northern Uganda, the MOH held advocacy workshops in primary schools and distributed a Comic Book of HAT that was developed by the WHO. They also held workshops with senior government officers and parliamentarians, resulting in a significant increase in budgetary allocation for African Trypanosomiasis control by the government, within the Annual budget. A similar outcome was also reported by Tanzania.
In Tanzania, awareness campaigning was carried out by both the Ministry of Health and Social Welfare (MOHSW) and the Ministry of Livestock and Fisheries Development (MOLFD). Various government institutions involved in tsetse and trypanosomiasis research and control came together and established the Network for Mapping African Trypanosomiasis in Tanzania (NetMATT). Institutions that became members of the network included a) Ministry of Livestock Development and Fisheries (Coordinator), b) National Institute of Medical Research (NIMR), c) Tsetse and Trypanosomiasis Research Institute (TTRI), d) Ministry of HeaIth and Social Welfare (MOHSW), e) Institute of Resource Assessment (IRA) - University of Dar es Salaam (UDSM), f) Tanzanian National Parks Authority (TANAPA), g) Tropical Pesticide Research Institute (TPRI), and h) Sokoine University of Agriculture (SUA). The activities that NeTMATT undertook included a) developing tools to guide surveillance, intervention and the targeting of resources, b) consolidating mapping data to support the FIND/PATTEC HAT advocacy programme, c) using reports and arising publications as tools for advocacy, to guide further research and leverage funding, d) strengthening data collection and mapping of African trypanosomiasis in Tanzania through collaboration between various stakeholders (including government ministries, research institutions and universities), e) facilitating sharing of data between institutions, and f) improving the quality of trypanosomiasis data analysis by enhancing the amount and quality of mapping and spatial analysis that is undertaken using the data. The outcome of the activities relating to characterization of health centres was series of Geographic Information System (GIS) maps showing the health centres where HAT has been reported during the past 10 years, and the changes in numbers of cases by year.
A key observation was a dramatic fall in the number of cases of HAT, indicating that elimination of the disease in this country is imminent.
While livestock trypanosomiasis is widespread in Nigeria, cases of HAT have in recent years only been reported in Delta State, which is in the south of the country. Nigeria has intensified surveillance and control of both diseases in the entire country, with the aim of eliminating them. During the reporting period, FIND supported Nigeria to intensify awareness campaigning, and to actively screen communities in Delta State for HAT.
In addition to the efforts coordinated by FIND, UEM dedicated a significant effort to awareness campaigning in the Matutíne District in the South of Mozambique which borders the North-East region of Kwazulu-Natal in South Africa. These two geographical areas are relatively isolated from the main belt of trypanosomiasis in East and Southern África and represent the south most distribution of the genus Glossina in África. Glossina brevipalpis and G. austeni are the only species present and Trypanosoma congolense, T. vivax and T. brucei brucei and T. simiae are the species associated with livestock trypanosomiasis in this region. T. congolense is undoubtedly the most important species, with prevalences higher than 30% reported in some locations. Livestock production, mainly represented by cattle and goats, is considered an important activity for the local rural families and trypanosomiasis is generally perceived as a major constraint for the improvement of herd production and productivity. The campaign launched at Matutuíne District, targeted especially school children and cattle owners and had as main objective to increase the awareness towards trypanosomiasis by creating an environment conducive to dialogue, simple but technically accurate messages, and making use of adequate material.
Launch of awareness campaign
The campaign was launched in the presence of the Permanent Secretary of the District, the Heads of the five District Administrative Areas, the head of District Services for Economical Activities , the Provincial Director of Education, the State Veterinary at District level and Technicians, including the livestock technicians from the five District Administrative Areas, the representative of the Provincial Director of Agriculture, The representative of the Director The Biotechnology Center – UEM (CB-UEM), two researchers from CB-UEM and representatives from the main local Communities.
All the activities were filmed and photographed by the “Centro de Comunicação e Imagem – UEM”. Moreover, a full text on the NANOTRYP promoted campaign appeared in the Mozambican main daily newspaper “Notícias”, in the UEM newsletter and the campaign was further advertised by a half hour interview in Mozambican main TV channel “TVM 1”.
Dissemination materials
Two types of posters, both printed in Portuguese and in the local language Xi-Ronga, 250 of each type were put up in all the schools shown in the Table below, and at the headquarters of the five District Administrative Areas. Six thousand leaflets in Portuguese and one thousand in Xi-Ronga, containing the main messages on tsetse and trypanosomiasis, were also made and delivered to all students, teachers and livestock owners targeted by the campaign.
Schools in which awareness campaigns were conducted
School name Number of students District Administrative Area
EPC Bela-Vista 980 Bela-Vista
EPC Mudada 272
EPC Salamanga 463
EPC Tinonganine 205
Instituto Agro-industrial de Salamanga 200
EPC Nsime 346 Catembe-Nsime
EPC Zitundo 139 Zitundo
EPC Gala 33
EPC Catuane 211 Catuane
EPCNdlala 225
EP1 Mhala 93 Machangulo
EP1 Ngomene 123
EPC Ndelane 206
EPC Machangulo 246
EP1 Mabuluco 41
EPC Nhonguane 117
Main activities of the campaign
Seminar with the teachers
One hundred and twelve teachers participated in the campaign. A twenty five minutes seminar with the teachers was held in all the schools. A power point presentation containing exactly the same content of the leaflets and the posters was made. Just before the seminar a pre-exposure questionnaire was administered to the students. It is expected that at school level the teachers will assume an active role in conveying the message to the students.
Seminars with the cattle owners
A seminar with the presentation of a power point containing exactly the same content of the leaflets and the posters was conducted. Just as in the schools the seminar was preceded by the administration of a pre-exposure questionnaire.
Pre-exposure questionnaire
Immediately before the campaign, a questionnaire on the content of the dissemination material was administered to all members of the target audience (students, teachers and cattle owners). The questionnaire has nine questions with different marks. The difference between the marks and the total of correct answers will be analysed by the appropriate statistical methods and used to measure campaign efficacy.
Post-exposure questionnaire
The same questionnaire prepared for the pre-exposure phase was administered sixty days after the launching of the campaign in each school and dip-tank.
Dip-tanks
Nine dip-tanks, including Felipe, Mudada, Tinonganine, Salamanga, Gala, Zitundo, Catuane, Ndala e Machangulo, were involved in the campaign with the participation of approximately one hundred cattle owners.
Besides efforts to educate local comities and bring awareness of the trypanosomiasis problem to the different stakeholders, NANOTRYP also dedicated a considerable effort to education both at the level of PhD and Master training, as well as research training from both IPR and UEM researchers at the European VIB and FIBAO partner laboratories.
Training of IPR researcher at VIB
Christopher Kinyanjui Kariuki (IPR) visited VIB for two consecutive training periods of three months each (Jan-March 2011 and Oct.-Dec. 2011). During this period, Mr Kariuki developed a set of Nanobodies that can be used as tools for the work ongoing at IPR, in which the T. brucei. rhodesiense IPR001 parasite stock is being used. A detailed progress report of the work performed was included in the previous progress report / description of WP1.
This work formed part of Mr Kariuki’s MSc training at Jomo Kenyatta University of Agriculture & Technology (JKUAT), Nairobi. Mr Kariuki returned home to initiate the nanobody generation work leading to immunization of camels for generation and extraction of mRNA. However the next stages of nanobody generation still require heavy investment which IPR may not have, and therefore the collaboration with VUB to access the VIB Nanobody generation Centre is likely to remain alive beyond the life of this project.
Training of UEM and IPR researcher at FIBAO
Mr. Mwadime and Mr. Mucache were successfully training in laboratory techniques that included Immunofluorescence of the Tb brucei parasite Antat 1.1 with the Nb (Nanobody) and fluorochromes e.g. Rodamine, In vitro culture of the Tb brucei. Maintenance of culture parasites and working with in vitro based systems, IC 50 and Alamar Blue Assays involving, the Nb, Nb tagged to cyclodextrin, Pentamidine, Pentamidine + Nicotinamide and Pentamidine delivered in a variety of Nanocapsules such as Chitosan and PLGA, Endocytosis measurement of nanoparticles loaded with fluorochromes by FACS analysis, and finally Protein molecular weight determination methods including SDS and use of a Nanodrop™, General lab techniques such as microscopy and parasitemia.
Training of UEM researcher at VIB
Hermogenes Mucache (UEM) visited VIB for a training period of three months (May-Aug. 2010). During this period, Hermogenes participated in the development of a set of NANOBODIES that can be used as tools for the detection of T. evansi. A detailed progress report of the work performed was included in the previous reporting document / description of WP1. At the end of the last reporting period, a second student form UEM joinded VIB, i.e. Paula Alberto Macucule. She joined the laboratory at VIB to get acquainted with basic techniques in library panning and Nb selection.
Joined PhD training program VIB/FIBAO
A joint PhD was initiated at the start of the NANOTRYP project, and results obtained are included in the report on WP1 and WP2. The PhD (of Florencia Le Greca) covers the work on T. vivax. The PhD has passed through a first round of defence at VIB and will be finalised at the end of August 2013.
Addition training efforts
During the mid-term evaluation, a comment was made that pointed to the fact that the exchange of a single PhD student between two of the partner laboratories cannot be considered a ‘PhD training Program’. However, it should be mentioned here that the NANOTRYP budget only incorporated a budget for 1 student. Nevertheless, in the last reporting period we have extended the PhD training to include several more students.
• Juan Diego Unciti Broceta (FIBAO) spend a 2 month training period at VIB (May-June 2011), focussing on the generation of a Nb library against the non-variant OpdB T. brucei antigen. After having received this training, Juandi returned to FIBOA and initiated the generation of a specific library for T. evansi.
• Steven Odongo (Uganda) joined the NANOTRYP project at the start of 2011 and has been focussing at the development of a Nanobody sandwich system for T. congolense. This project includes (i) the generation, selection and production of Nbs, and (ii) the use of Nbs in the development of an ELISA based diagnostic tools, that could later be transformed into a dip-stick (lateral flow) format
• Fyei Obishakin (Nigeria) joined the NANOTRYP project in late 2010, and focuses on the development of a Nanobody-based diagnostic tool for T. evansi. His PhD has a dual focus that includes the assessment of immunopathology of T. evansi infections as well as the development of diagnostic tools for the infection, and hence about 50% of his project is directly associated with NANOTRYP.
Directly related to NANOTRYP is the fact that other research teams working on other parasitic diseases have gained interest in Nanobody technology and have asked for training for PhD students. Within a European context it is worth mentioning that the COST BM0802 action (Cooperation for Science and Technology) has been used to exchange both a PhD and Master student between th VIB and the University of Alcala, Spain (Prof Antonio Jiménez Ruiz), with the aim of developing Nbs as molecular tools for studying the fundamental biology of Leishmania. In turn, this collaboration has lead to two additional collaborations (within the COST action) with the University Medical Centre of Leiden, The Netherlands (Dr. Shahid Khan), and the University of Tubingen, Germany (Prof. Michael Duszenko).
Conclusion:
Besides the obvious achievements of the NANOTRYP education efforts, the key achievements of the awareness campaigns included:
• Broad coverage - the awareness campaigns were carried out using different forums that varied from country to country, including among others, presentations of projects at international, regional and national conferences, community workshops, news articles in the local press, TV interviews and documentaries, road shows, banners and brochures. These approaches ensured that the campaigns impacted all the levels that were targeted.
• The awareness and ownership of HAT problem at local, national, regional and international levels was enhanced. This is reflected by a decision by the WHO to identify HAT as one of the diseases targeted for elimination by the year 2020. Meanwhile in early 2012, a large group of funding agencies and industry partners passed the London Declaration, committing to support efforts to eliminate the disease.
• Strategies for intensified control and elimination of tsetse and trypanosomiasis were better harmonized at the national, regional and international levels. As a result, the prevalence of the disease is currently being reported with higher accuracy than before. Indeed Uganda has now initiated a national project to intensified surveillance and control of T.b. gambiense HAT.
• The ability of health workers to suspect HAT was improved at all levels including public and private clinics and traditional healers in endemic areas. This has resulted in accelerated control of the disease, and could be contributing to the dramatic fall on the number of cases of HAT reported in recent years.
• Many endemic countries have now allocated annual budgets for trypanosomiasis control, while others have increased the annual budgetary allocation.
• Characterization of health facilities in HAT endemic regions has strengthened the countries’ ability to re-allocate resources to priority areas based on the evidence provided. The information generated is also providing guidance on targeting of diagnostics to facilities based on the available capacity at those facilities.
• The capacity of health personnel to utilize available diagnostic tools for HAT was strengthened through training, contributing to better detection of cases and proper management of the disease.
• Education on the importance of accurate diagnosis created an environment for sustainable introduction of novel diagnostic tools in endemic countries. This will be critical as efforts to eliminate HAT are intensified.
• A number of countries are now able to apply early warning systems to predict disease outbreaks, and therefore take appropriate measures to prevent escalation of the disease.
Potential Impact:
An important component of the NANOTRYP project was an awareness campaign on the importance of both human and animal African trypanosomiasis. This activity was mainly executed by FIND and UEM. In addition to this, NANOTRYP also included a large education component for academic trainees. Together, VIB, FIBAO, IPR and UEM organised a multitude of research exchanges and provided the possibility for the 4 PhD programmes to be executed.
With respect to awareness campaigning itself, FIND took the lead, and at the international level, funding agencies were lobbied to commit resources for research and control of the disease. Within the endemic countries, governments were urged to allocate resources for control of the disease within their national budgets. Medical and veterinary personnel were reminded to include the disease in their differential diagnosis, while communities were made more aware of the importance of these diseases.
The focus of the campaigns was communities that are at risk of infection, or who keep livestock in areas infested with tsetse flies, medical personnel working in health centres located in endemic regions, and policy makers in governments of endemic countries. Implementation of advocacy activities was guided by a Strategic Plan on Advocacy (SPA) for African Trypanosomiasis developed by FIND, the Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC) office of the African Union, and endemic countries. Phase one of the Strategic Plan covered the period January 2008 to December 2011. The selected countries appointed contact persons, who worked with PATTEC, and developed country-specific work plans and budgets. The work plans were guided by a country’s capacity to implement activities against specific outputs that were agreed on and included in the SPA.
The key achievements and impacts of the awareness campaigns included:
• Broad coverage - the awareness campaigns were carried out using different forums that varied from country to country, including among others, presentations of projects at international, regional and national conferences, community workshops, news articles in the local press, TV interviews and documentaries, road shows, banners and brochures. These approaches ensured that the campaigns impacted all the levels that were targeted.
• The awareness and ownership of HAT problem at local, national, regional and international levels was enhanced. This is reflected by a decision by the WHO to identify HAT as one of the diseases targeted for elimination by the year 2020. Meanwhile in early 2012, a large group of funding agencies and industry partners passed the London Declaration, committing to support efforts to eliminate the disease.
• Strategies for intensified control and elimination of tsetse and trypanosomiasis were better harmonized at the national, regional and international levels. As a result, the prevalence of the disease is currently being reported with higher accuracy than before. Indeed Uganda has now initiated a national project to intensified surveillance and control of T.b. gambiense HAT.
• The ability of health workers to suspect HAT was improved at all levels including public and private clinics and traditional healers in endemic areas. This has resulted in accelerated control of the disease, and could be contributing to the dramatic fall on the number of cases of HAT reported in recent years.
• Many endemic countries have now allocated annual budgets for trypanosomiasis control, while others have increased the annual budgetary allocation.
• Characterization of health facilities in HAT endemic regions has strengthened the countries’ ability to re-allocate resources to priority areas based on the evidence provided. The information generated is also providing guidance on targeting of diagnostics to facilities based on the available capacity at those facilities.
• The capacity of health personnel to utilize available diagnostic tools for HAT was strengthened through training, contributing to better detection of cases and proper management of the disease.
• Education on the importance of accurate diagnosis created an environment for sustainable introduction of novel diagnostic tools in endemic countries. This will be critical as efforts to eliminate HAT are intensified.
• A number of countries are now able to apply early warning systems to predict disease outbreaks, and therefore take appropriate measures to prevent escalation of the disease.
Awareness campaigns supported with NANOTRYP funds focused on Uganda, Tanzania and Nigeria in collaboration with the governments of those countries and PATTEC. This was done alongside a larger program on advocacy in all countries reporting cases of HAT in Africa, and funded by the BMGF and the DFID. A detailed account of the achievements is reported in the following section.
In Uganda, awareness campaigns were carried out by Gulu University in the T.b. gambiense region in the north of the country. The specific objectives of the campaigns included a) understanding the local community’s perception of the disease, b) appraising the community’s level of understanding of the symptoms of HAT and nagana, transmission, diagnosis, treatment and control, and c) sensitizing leaders and communities living in these areas so that they seek early diagnosis and treatment for both human and livestock diseases, in order to remove reservoirs of infection. At the onset, familiarization and interpersonal meetings were held with key district and selected sub-county officials, to brief them on the purpose of the project and get their understanding on the severity of the problem. Community mobilization and campaigns at the district, sub-county and at community levels focussed on the importance of early diagnosis of sleeping sickness and Nagana in humans and animals, respectively, the animal-human infection relationship, understanding the ecology of tsetse flies and their role in transmission of the diseases, local community perception of sleeping sickness and Nagana, and community coping strategies and adaptations. Advocacy materials that were developed and distributed included a) posters on trypanosomiasis and how it is spread, b) posters on actions in control of trypanosomiasis, c) Question and Answer booklet (Frequently Asked Questions) for community mobilization, d) leaflets in the various local languages in the endemic areas highlighting how trypanosomiasis is spread and control methods and T shirts, e) radio talk shows (30 minutes each) in the two dialects zones of Adjumani and Nwoya – Amuru, and f) training workshops for village health teams on awareness of trypanosomiasis.
The Ministry of Health (MOH) of Uganda and the National Livestock Resources Research Institute (NaLIRRI) focused on the T.b. rhodesiense region in the south east and central Uganda. However, in addition to using advocacy materials that were similar to those used in northern Uganda, the MOH carried advocacy workshops in primary schools and distributed a Comic Book of sleeping sickness that was developed by the WHO. They also held workshops with senior government officers and parliamentarians, resulting in a significant increase in budgetary allocation for African Trypanosomiasis control by the government, within the Annual budget. A similar outcome was also reported by Tanzania. The latest data from these activities indicates that the T.b. gambiense focus may be shrinking northwards, reducing the risk of merging with the T.b. rhodesiense belt. This is important because the two forms of the disease are not distinguishable clinically, yet treatment is different. A merger of the two would therefore pose a challenge in management of the disease.
In Tanzania, awareness campaigning was carried out by both the Ministry of Health and Social Welfare (MOHSW) and the Ministry of Livestock and Fisheries Development (MOLFD). Various government institutions involved in tsetse and trypanosomiasis research and control came together and established the Network for Mapping African Trypanosomiasis in Tanzania (NetMATT). Institutions that became members of the network included a) Ministry of Livestock Development and Fisheries (Coordinator), b) National Institute of Medical Research (NIMR), c) Tsetse and Trypanosomiasis Research Institute (TTRI), d) Ministry of HeaIth and Social Welfare (MOHSW), e) Institute of Resource Assessment (IRA) - University of Dar es Salaam (UDSM), f) Tanzanian National Parks Authority (TANAPA), g) Tropical Pesticide Research Institute (TPRI), and h) Sokoine University of Agriculture (SUA). The activities that NeTMATT undertook included a) developing tools to guide surveillance, intervention and the targeting of resources, b) consolidating mapping data to support the FIND/PATTEC HAT advocacy programme, c) using reports and arising publications as tools for advocacy, to guide further research and leverage funding, d) strengthening data collection and mapping of African trypanosomiasis in Tanzania through collaboration between various stakeholders (including government ministries, research institutions and universities), e) facilitating sharing of data between institutions, and f) improving the quality of trypanosomiasis data analysis by enhancing the amount and quality of mapping and spatial analysis that is undertaken using the data. The outcome of the activities relating to characterization of health centres was a series of GIS maps showing the health centres where HAT has been reported during the past 10 years, and the changes in numbers of cases by year.
While livestock trypanosomiasis is widespread in Nigeria, cases of HAT have in recent years only been reported in Delta State, which is in the south of the country. Nigeria has intensified surveillance and control of both diseases in the entire country, with the aim of eliminating them. Nigeria was supported to intensify awareness campaigning, and to actively screen communities in Delta State for HAT. Active screening was carried out using the CATT test (the screening test in current use), and case confirmation by microscopy. While a significant percentage of people were found positive with CATT, none of them was confirmed by parasitology. This also indicates that the prevalence of HAT in Nigeria is very low, and elimination of the disease in this country is also imminent.
The second NANOTRYP partner that provided a particular valuable contribution to the impact of the programme with respect to affected population was UEM – The University Eduardo Mondlane (Mozambique). From the Eduardo Mondlane University standpoint to evaluate the social impact of the Nanotryp project, aspects associated to the enhancement of the Mozambican Research Group on African Animal Trypanosomiasis, the exposure of Mozambican students and researchers to world class scientific seminars and the involvement of communities affected by animal trypanosomiasis in awareness campaigns should be considered.
Boosting Trypanosomiasis Reasearch in Mozambique
The Nanotryp project contributed directly to the increase in the research activities in the area of animal trypanosomiasis in Mozambique. Young researchers participating in the project had the opportunity of training both at national level and in the laboratories of participating institutions in Belgium and in Spain. In Mozambique the researchers were actively involved in intensive field activities combined with the establishment and validation of modern techniques like PCR and PCR-RFLP for Trypanosoma species identification. At the laboratories of European participating institutions these researchers had the opportunity to familiarize themselves with the theory and practice of nanobody technology as well as with advance techniques applied to cellular immunology.
It is worth mentioning that, apart from the technical and scientific experience acquired by the Mozambican researchers, in the course of this project a substantial amount of very valuable biological material was collected which will enhance dramatically the probability of future participation of the Mozambican Research Group on African Animal Trypanosomiasis in specialized research networks.
Last but not least, the project has contributed to the boost of researcher’s individual careers and through that, they manage to get hold permanent positions at the university. It is of crucial social importance to train and retain a generation of competent young African scientists in order to achieve a substantial improvement on the continental scientific output, which is directly linked with the possibility of further job creation at African institutions of science and technology.
Nanotryp Scientific Workshop
In the context of the Nanotryp project, a scientific workshop was held in February 2011, at the Veterinary Faculty of Eduardo Mondlane University. The workshop was a unique opportunity to expose students and researchers to world class scientific seminars, addressing a broad spectrum of topics, including Nanobody principles and applications, advances in the pharmacology of typanocidal compounds, immunology and diagnostics. It is worth mentioning the inclusion in the workshop program of practical classes.
This scientific exercise was extremely important not only by the relevance of its contents, but mainly by setting a model of academic excellence in a place where this kind of contribution are greatly required.
Awareness Campaign (see also WP8)
The trypanosomiasis and tse tse fly awareness campaign carried out in the context of WP8 of the Nanotryp project was arguably the activity with greater social impact. The involvement in the campaign of more than 3000 students, approximately 100 primary school teachers and 100 local cattle owners generated a tremendous social movement. In African Animal Trypanosomiasis the activities related with disease control are normally assumed by the government through the veterinary services, generally in collaboration with one or more international agencies. The participation of affected communities has been largely neglected and normally the small holders have lost completely the sense of ownership regarding the main control interventions.
A particular feature of this campaign was to define as main target children from primary schools. In the Mozambican rural areas, particularly in the South, children play a major role in cattle husbandry. Generally, the children are the responsible to take the cattle to the pastures and they are also the ones who bring the animals to the dip-tanks or crush-pen for any type of veterinary intervention.
During the campaign, making use of simple, but technically accurate messages, emphasis was put on the basic concepts of disease ethiology and epidemiology as well as on the rational management of trypanocidal drugs. It is expected a much more proactive involvement of individuals and communities on all aspects of disease management and control.
The third major partner that provided a direct impact for the trypanosomiasis affected community was IPR, the International Primate Research centre, Kenya. Here the main contribution of NANOTRYP has been in the following fields:
1. Technology Transfer and Training
The biggest single gain with a long lasting effect is the training that this project accorded the Kenyan partners. Four MSc students at Kenyan universities were successfully trained by carrying out an aspect during the life of the studies that comprised the tasks on this project thus;
i. Mr Chris Kinyanjui, Jomo Kenyatta University of Agriculture & Technology (2009, JKUAT). Mr Kinyanjui was trained at VUB on nanobody generation using Tb rhodesiense IPR 001 isolate. This training formed his MSc research project. Mr Kinyanjui was mid-way through the program offered a scholarship to pursue molecular biology studies at MSc level in Belgium, which was an offshot of his engagement with VUB. He is due to return home on completion of his studies to resume his position at IPR.
ii. Mr Alex Gaithuma – MSc, (2009,JKUAT), developing the non-human primate model of Tb rhodesiense
iii. Ms Dawn Maranga – MSc (2013, JKUAT) characterizing the immunological profile of the Tb rhodesiense nonhuman primate model. Ms Maranga was later offered a research fellowship following the departure of the first research fellow. This allowed the HAT research project to proceed to its logical conclusion.
iv. Ms Beatrice Gachie – MSc (on-going, JKUAT) characterizing the diagnostic value of non-invasive procedures in development of nonhuman primate model of Tb gambiense
v. Mr Victor Mwadime trained at FIBAO on Nb::drug coupling. He is currently undertaking his MSc studies (University of Nairobi) and is expected to carry out his research project on the basis of this training.
2. Nonhuman primate models of Tb rhodesiense and Tb gambiense
The NHPs model of Tb rhodesiense has been fully established and has taken its place in the IPR HAT research programs. Developed as part of the Nanotryp project, this model attracted two other research grants for preclinical testing of diagnostic products. It is anticipated that this laboratory animal model will be even more useful as a testing tool for treatment and diagnostic tests especially as the clamour for eradication of the disease gains momentum.
The development of a NHP model of Tb gambiense was also supported by Nanotryp. Two series of infections were carried out in vervet as well as Sykes monkeys with encouraging results. Some diagnostic tests based on non-invasive samples such as saliva and urine indicated that these tests are possible considerations for monitoring infection. This would be a vast improvement over the existing tests which require patients to undergo a painful lumbar puncture over a period of two years before cure can be ascertained. Even more important is that this test was able to demonstrate relapse before detection of parasites in blood.
IPR is the biggest Primate Centre in Africa and continues to attract research scientists from all over the world. It is hoped that this development in the field of HAT will provide the necessary leverage needed to attract firms and investigators interested in elucidating more information to plug the existing knowledge gaps.
Following the training at VUB and FIBAO, IPR proceeded to carry out immunization of camels under field settings in Kenya. The camel farm was identified and terms of engagement entered into. The farm is found within camel trypanosomiasis zone (0.121639N 36.630885E). The immunization and subsequent processing of samples for nanobody preparation was a demonstration of the practicality of the training. However the process stopped short of completion due to inability to transfer the end product in Kenya (RNA) to the University of Brussels laboratory where the Nb production would have gone to completion. This implies that the technology transfer has been successful except for the heavy investment to process the materials to full generation of Nb. The reliance on the facility at Brussels is likely to be so in the foreseeable future. The understanding with the farm owners was such that long-term engagement may be addressed as and when needs arise.
The initial aim of this project was to transfer this Nb technology to other tropical diseases of importance under the Kenyan research priority. This has been achieved by initiating the diagnostic potential of Nb against Leishmaniasis a neglected disease. It is anticipated that the import/export hitches between Kenya and Belgium will be sorted to allow the full processing of this initiative.
3. Field evaluation of T. congolense nanobody
IPR provided a platform for testing of nanobodies against the T, congolense which was carried out in a field setting where livestock trypanosomiasis is still a major constraint to animal production. The interaction with farmers was a very fulfilling experience when carrying out sensitization control strategies for application post research. The presence in the field was taken positively and seen that the government still cares about the prevalent problem affecting farmers. It is anticipated that the test results will have a bearing on the way livestock trypanosomiasis is diagnosed in the future. Kenya stands to benefit greatly considering T. congolense is the main cause of cattle, sheep and goats morbidity affecting agricultural productivity in about 60% of arid and semi-arid land surface where livestock is the main source of economic activity.
4. Infrastructure Development: Equipment
Through Nanotryp, IPR benefited from procurement of a haemtology analyser which replaced an old and frequently breaking down equipment. The equipment is centrally placed and therefore available to all users in IPR. This way the maintenance has been transferred from the Nanotryp project to the users through levy of user charge. All data on Nanotryp project was generated through this machine.
The procurement of a fluorescent microscope allowed IPR to carry out varied staining tests on trypanosomes as well as other parasites of important research programs at IPR. The Acridine Orange (AO) staining is gaining ground as the stain to use in order to visualize trypanosomes and other protozoan parasites much faster and easily than traditional laboratory stains. The application of this technology is likely to only be better utilized at IPR in the coming years for training and research purposes. Already tests in Leishmania and malaria parasites have been carried out with encouraging results.
In addition to helping to sustain the scientific infrastructure, the Nanotryp project paid to IPR 12.5% of all grant money received, according to Institutional Policy. The funding went into construction of a new building for group housing animals in experiments as part of the animal welfare improvements (Figure 20). All animals in the experiments were taken through environment enrichment protocols. These infrastructure developments will have a huge beneficial effect on the future of IPR well beyond the life of Nanotryp Project, particularly because IPR has initiated the process of international accreditation by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC International). The new building has been put up with advice from reputable international firm (Arrowmight Inc) which has provided consultancy services in the renovation of several NHP centres in Europe. The dimensions meet the current EU guidelines and exceed the American rules (The Guide).
Figure 20: New building for group housing animals in experiments as part of the animal welfare improvements
In the future, animals will be housed in compartible pairs as the minimum housing requirements. The animal house is built to use as much natural light as possible hence the translucent sunroof sheets and open sides to allow natural air circulation since the animals will be housed within their natural/home setting.
Besides efforts to educate local comities and bring awareness of the trypanosomiasis problem to the different stakeholders, NANOTRYP also dedicated a considerable effort to education both at the level of PhD and Master training, as well as research training from both IPR and UEM researchers at the European VIB and FIBAO partner laboratories.
Training of IPR researcher at VIB
Christopher Kinyanjui Kariuki (IPR) visited VIB for two consecutive training periods of three months each (Jan-March 2011 and Oct.-Dec. 2011). During this period, Mr Kariuki developed a set of Nanobodies that can be used as tools for the work ongoing at IPR, in which the T. brucei. rhodesiense IPR001 parasite stock is being used. A detailed progress report of the work performed was included in the previous progress report / description of WP1.
This work formed part of Mr Kariuki’s MSc training at Jomo Kenyatta University of Agriculture & Technology (JKUAT), Nairobi. Mr Kariuki returned home to initiate the nanobody generation work leading to immunization of camels for generation and extraction of mRNA. However the next stages of nanobody generation still require heavy investment which IPR may not have, and therefore the collaboration with VUB to access the VIB Nanobody generation Centre is likely to remain alive beyond the life of this project.
Training of UEM and IPR researcher at FIBAO
Mr Mwadime and Mr Mucache were successfully training in laboratory techniques that included Immunofluorescence of the Tb brucei parasite Antat 1.1 with the Nb (Nanobody) and fluorochromes e.g. Rodamine, In vitro culture of the Tb brucei. Maintenance of culture parasites and working with in vitro based systems, IC 50 and Alamar Blue Assays involving, the Nb, Nb tagged to cyclodextrin, Pentamidine, Pentamidine + Nicotinamide and Pentamidine delivered in a variety of Nanocapsules such as Chitosan and PLGA, Endocytosis measurement of nanoparticles loaded with fluorochromes by FACS analysis, and finally Protein molecular weight determination methods including SDS and use of a Nanodrop™, General lab techniques such as microscopy and parasitemia.
Training of UEM researcher at VIB
Hermogenes Mucache (UEM) visited VIB for a training period of three months (May-Aug. 2010). During this period, Hermogenes participated in the development of a set of NANOBODIES that can be used as tools for the detection of T. evansi. A detailed progress report of the work performed was included in the previous reporting document / description of WP1. At the end of the last reporting period, a second student form UEM joinded VIB, i.e. Paula Alberto Macucule. She joined the laboratory at VIB to get acquainted with basic techniques in library panning and Nb selection.
Joined PhD training programme VIB/FIBAO
A joint PhD was initiated at the start of the NANOTRYP project, and results obtained are included in the report on WP1 and WP2. The PhD (of Florencia Le Greca) covers the work on T. vivax. The PhD has passed through a first round of defence at VIB and will be finalised at the end of August 2013.
Addition training efforts
During the mid-term evaluation, a comment was made that pointed to the fact that the exchange of a single PhD student between two of the partner laboratories cannot be considered a ‘PhD training Program’. However, it should be mentioned here that the NANOTRYP budget only incorporated a budget for 1 student. Nevertheless, in the last reporting period we have extended the PhD training to include several more students.
• Juan Diego Unciti Broceta (FIBAO) spend a 2 month training period at VIB (May-June 2011), focussing on the generation of a Nb library against the non-variant OpdB T. brucei antigen. After having received this training, Juandi returned to FIBOA and initiated the generation of a specific library for T. evansi.
• Steven Odongo (Uganda) joined the NANOTRYP project at the start of 2011 and has been focussing at the development of a Nanobody sandwich system for T. congolense. This project includes (i) the generation, selection and production of Nbs, and (ii) the use of Nbs in the development of an ELISA based diagnostic tools, that could later be transformed into a dip-stick (lateral flow) format
• Fyei Obishakin (Nigeria) joined the NANOTRYP project in late 2010, and focuses on the development of a Nanobody-based diagnostic tool for T. evansi. His PhD has a dual focus that includes the assessment of immunopathology of T. evansi infections as well as the development of diagnostic tools for the infection, and hence about 50% of his project is directly associated with NANOTRYP.
Directly related to NANOTRYP is the fact that other research teams working on other parasitic diseases have gained interest in Nanobody technology and have asked for training for PhD students. Within a European context it is worth mentioning that the COST BM0802 action (Cooperation for Science and Technology) has been used to exchange both a PhD and Master student between th VIB and the University of Alcala, Spain (Prof Antonio Jiménez Ruiz), with the aim of developing Nbs as molecular tools for studying the fundamental biology of Leishmania. In turn, this collaboration has lead to two additional collaborations (within the COST action) with the University Medical Centre of Leiden, The Netherlands (Dr. Shahid Khan), and the University of Tubingen, Germany (Prof. Michael Duszenko).
List of Websites:
www.nanotryp.eu
P1 Flanders Institute for Biotechnology VIB - Stefan Magez - stemagez@vub.ac.be
P2 ARTTIC in Brussels, SPRL ARTTIC - Paul Crompton - pdc@arttic.be
P4 Institute of Primate Research IPR - Maina N’Gotho - ngothojm@yahoo.co.uk
P5 University Eduardo Mondlane UEM - Luis Neves - nidzi@tvcabo.co.mz
P6 Foundation for Innovative New Diagnostics FIND - Joseph Ndung'u - joseph.ndungu@finddiagnostics.org
P7 Fundacion para la Investigation Biosanitaria de Andalucia Oriental - Alejandro Otero FIBAO - Jose Garcia Salcado - jagarciasalcedo@yahoo.es