Final Report Summary - TRYPADVAC2 (Development of an "anti-disease" vaccine and diagnostic tests for African trypanosomosis)
The aim of the project was to contribute to the improvement of livestock productivity in the developing world through limitation of trypanosome-associated pathology and accurate diagnostics of trypanosome infections.
It was proposed to develop immunisation strategies against pathogenic factors of the parasites ('anti-disease' vaccine approach). In a previous INCO project under the Fifth Framework Programme (FP5), we had focussed on congopain, an immunosuppressive Cystein protease (CP) of Trypanosoma congolense. As the effects of immunisation with congopain appeared limited, association with other antigens was deemed necessary. The current proposals expanded the initial work to screening and characterisation of other pathogenic molecules, especially those responsible for anemia. A number of other trypanosome proteases, CPs, serine and metallo- proteases, required characterisation of their biological roles in the parasite and the host. Trypanosomes also contain protease inhibitors, some of which have immunomodulatory effects. They may thus be manipulated, directly or in association with their partner enzymes, to modulate the disease. Other, non-proteolytic, pathogenic factors had been identified. The Glycosyl phosphatidyl inositol (GPI) anchor of the T. Brucei Variant surface glycoprotein (VSG) had been shown to be a major inducer of TNF, a cytokine that mediates anemia. Finally, development in the field of proteomics and progress in the genome mapping of trypanosomes provided tools to study new pathogenic pathways.
In order to improve diagnosis of the disease, procedures for antibody detection were to be developed and / or validated. They are based on recombinant technology, which circumvent problems associated with the current use of parasite extracts. Recombinant and synthetic peptides from CPs and heat shock proteins, both previously identified as major antigens, as well as newly described molecules, were to be assessed for their diagnostic potential.
Work performed
The work performed and results achieved during the four years of the project can be summarised under the following six themes:
1. Molecular studies on trypanosome proteases and their inhibitors (WP1, WP2, WP3, WP4, WP6)
The previous INCO-DEV Trypadvac project in FP5 centred exclusively on congopain. It was shown that congopain indeed acts as a pathogenic factor of T. congolense during infection, but immunisation / challenge experiments remained inconclusive. The present Sixth Framework Programme (FP6) Trypadvac2 project had proposed to expand the anti-disease vaccine concept to other pathogenic factors, while still attempting to improve the protective potential of congopain.
Congopain-like cysteine proteases (clan A enzymes)
Knowledge of the three-dimensional structure of congopain is essential for the production of an effective anti-disease vaccine based on congopain. It is all the more important that not only does congopain dimerises at physiological pH (a unique feature of this class of proteases), but also that protective epitopes may be dimmer-associated.
Cathepsin B (clan A enzyme)
An exciting finding of the project has been the discovery of numerous cathepsin B-coding genes in the trypanosome genome.
Metacaspases (Clan CD enzymes)
Metacaspases are cysteine protease present in plants, fungi and protozoa, but not in mammals. As a consequence they have diagnostic and vaccine potential in the context of this project.
Trypanosomal serine oligopeptidases
The genes coding for T. congolense and T. vivax oligopeptidase B were identified by bioinformatics mining of the Sanger trypanosome genome database, and the ORF cloned by PCR.
Natural inhibitors of trypanosomal cysteine and serine proteases
The general objective within the project was to characterise the pathogenic roles and examine the 'protective potential' of natural peptidase inhibitors of parasite origin: Inhibitors of cysteine peptidases (ICP) or inhibitors of serine peptidases (ecotin, also known as ISP).
2. Proteomic studies - (WP6)
In this part of the project we endeavoured to identify, using proteomics tools, novel pathogenic factors from T. congolense and T. evansi to use as candidate antigens in an anti-disease strategy.
3. Immunological and pathophysiology studies on anaemia - WP5
This theme focussed on Glycosyl phosphatidyl inositol (GPI) molecules that are cell membrane anchor molecules, shown to have pro- as well as anti-inflammatory activities. Regarding trypanosomosis a number of infection-linked pathological features are a direct result of the proinflammatory activity of GPI molecules. Consequently, vaccination with GPI was attempted to prevent infection-associated complications such as the incidence of severe anemia as well as host fat and energy metabolism disturbances. Specifically, the objective was to assess the protective capacity of a GPI vaccination for cattle to provide protection against T. vivax and / or T. Congolense induced pathology.
4. Antibody and antigen detection tests based on previously identified molecules - WP7
In this part of the project, we aimed to develop serologic diagnostic tests based on antigens previously identified, namely BiP (immunoglobulin heavy chain binding protein) subfamily of heat shock proteins 70 (Hsp 70), congopain, evansain and oligopeptidase B. The bulk of the development work was carried out at CIRAD in Montpellier by partner 1 and while other partners were involved in providing field samples, expertise, monoclonal control antibodies, or antigens. The work has focussed mainly on BiP, an immunodominant antigen in trypanosome infections, and at a late stage of the project also on evansain.
5. Antibody detection tests based on newly identified antigens - WP8
To develop serodiagnostic tests for Trypanosoma congolense, T. vivax, T. brucei s.l. and T. Evansi several antigens and corresponding antisera were collected. Representative sera were selected from a collection of sera from natural and experimental infections of different mammal host species. Nineteen recombinant and three native antigens were provided by partners on the projects.
6. Assessment of the 'anti-disease' protective potential of a pathogenic factor cocktail vaccine (across work packages). Six antigens developed during the project under work packages WP1, WP2, WP3 and WP4 (CB1, CB12, C2, FL28, OpdB and ICP) were selected for testing in an immunisation / challenge field trail.
Deliverables
For all work packages, most of the objective were reached, and in some cases, exceeded. Concerning the anti-disease strategy, objectives were not reached in GPI-anchor vaccination experiments in cattle, the reason being no protection was observed in mice, there was hence no need to embark on costly cattle experiments. Some delays were incurred in the crystallisation of congopain, but results are pending. Likewise, progresses were made in the obtention of null mutants for cathepsin L (congopain), cathepsin B, oligopeptidase B, and ISP 1 and 2, but the analysis of phenotypes in vitro and in vitro is ongoing.
On the diagnostic tests front, a large number of antigens were tested, and some tests validated. Species-specific indirect-ELISA tests could not be achieved, due to the close-proximity of the antigenic fabric of the three main species of parasites. The preliminary results regarding the use of cathepsin B as a T. congolense-specific test however are encouraging for this particular theme.
Antigen- (capture-) ELISA, that has occupied scientists in the field for the past 20 years, still remains elusive, in our case due to problems encountered to obtain monoclonal antibodies in a timely manner. Finally, the ultimate withdrawal of DiaMed in year 3, the private partner whose role was to adapt test to rapid format, implies that this objective could not be reached.
Conclusions
In the course of this project, significant progresses have been made towards the development of an anti-disease vaccine and diagnostic tests for trypanosomosis. For both themes, the results at the end of the project may not appear sensational. One must keep in mind though that we are dealing here with a particularly shrewd parasite, with highly evolved mechanisms to evade host immune response, making the development of a vaccine, and as a corollary development of serologic tests, a particularly daunting task.
We have seen that animals immunised with a cocktail of pathogenic factors did generally better than controls, validating the concept of an anti-disease vaccine, but at a level that would not warrant pursuing commercially the venture in the present form. Likewise, some antigens with a diagnostic potential were characterised, and some tests validated, but sensitivity often remained low, and optimisation work is required. On upstream research level, positive outcomes are patent. A large number of trypanosomal pathogenic factors were characterised, through cloning, expression and enzymatic characterisation, and for some crystallisation studies, and gene-silencing, a very important emerging technology. New tools were developed regarding in vitro culture and cyclical transformation of trypanosomes, analysis of parasite proteome and secretome, and molecular adjuvants.
The project has generating close to twenty papers in peer-reviewed, with another ten submitted or in preparation. It should also not be understated that the project served as the basis of twenty Master's theses and eight PhDs, and further strengthened North-South collaborations through important movement of personnel for technology transfer and capacity building.
A vaccine against trypanosomosis has been a venture of many decades, and the road may be still long. Our results, as disappointing as they may look, clearly bring some stones to the edifice, and prove worthwhile the efforts of the consortium and the support of the European Commission.
It was proposed to develop immunisation strategies against pathogenic factors of the parasites ('anti-disease' vaccine approach). In a previous INCO project under the Fifth Framework Programme (FP5), we had focussed on congopain, an immunosuppressive Cystein protease (CP) of Trypanosoma congolense. As the effects of immunisation with congopain appeared limited, association with other antigens was deemed necessary. The current proposals expanded the initial work to screening and characterisation of other pathogenic molecules, especially those responsible for anemia. A number of other trypanosome proteases, CPs, serine and metallo- proteases, required characterisation of their biological roles in the parasite and the host. Trypanosomes also contain protease inhibitors, some of which have immunomodulatory effects. They may thus be manipulated, directly or in association with their partner enzymes, to modulate the disease. Other, non-proteolytic, pathogenic factors had been identified. The Glycosyl phosphatidyl inositol (GPI) anchor of the T. Brucei Variant surface glycoprotein (VSG) had been shown to be a major inducer of TNF, a cytokine that mediates anemia. Finally, development in the field of proteomics and progress in the genome mapping of trypanosomes provided tools to study new pathogenic pathways.
In order to improve diagnosis of the disease, procedures for antibody detection were to be developed and / or validated. They are based on recombinant technology, which circumvent problems associated with the current use of parasite extracts. Recombinant and synthetic peptides from CPs and heat shock proteins, both previously identified as major antigens, as well as newly described molecules, were to be assessed for their diagnostic potential.
Work performed
The work performed and results achieved during the four years of the project can be summarised under the following six themes:
1. Molecular studies on trypanosome proteases and their inhibitors (WP1, WP2, WP3, WP4, WP6)
The previous INCO-DEV Trypadvac project in FP5 centred exclusively on congopain. It was shown that congopain indeed acts as a pathogenic factor of T. congolense during infection, but immunisation / challenge experiments remained inconclusive. The present Sixth Framework Programme (FP6) Trypadvac2 project had proposed to expand the anti-disease vaccine concept to other pathogenic factors, while still attempting to improve the protective potential of congopain.
Congopain-like cysteine proteases (clan A enzymes)
Knowledge of the three-dimensional structure of congopain is essential for the production of an effective anti-disease vaccine based on congopain. It is all the more important that not only does congopain dimerises at physiological pH (a unique feature of this class of proteases), but also that protective epitopes may be dimmer-associated.
Cathepsin B (clan A enzyme)
An exciting finding of the project has been the discovery of numerous cathepsin B-coding genes in the trypanosome genome.
Metacaspases (Clan CD enzymes)
Metacaspases are cysteine protease present in plants, fungi and protozoa, but not in mammals. As a consequence they have diagnostic and vaccine potential in the context of this project.
Trypanosomal serine oligopeptidases
The genes coding for T. congolense and T. vivax oligopeptidase B were identified by bioinformatics mining of the Sanger trypanosome genome database, and the ORF cloned by PCR.
Natural inhibitors of trypanosomal cysteine and serine proteases
The general objective within the project was to characterise the pathogenic roles and examine the 'protective potential' of natural peptidase inhibitors of parasite origin: Inhibitors of cysteine peptidases (ICP) or inhibitors of serine peptidases (ecotin, also known as ISP).
2. Proteomic studies - (WP6)
In this part of the project we endeavoured to identify, using proteomics tools, novel pathogenic factors from T. congolense and T. evansi to use as candidate antigens in an anti-disease strategy.
3. Immunological and pathophysiology studies on anaemia - WP5
This theme focussed on Glycosyl phosphatidyl inositol (GPI) molecules that are cell membrane anchor molecules, shown to have pro- as well as anti-inflammatory activities. Regarding trypanosomosis a number of infection-linked pathological features are a direct result of the proinflammatory activity of GPI molecules. Consequently, vaccination with GPI was attempted to prevent infection-associated complications such as the incidence of severe anemia as well as host fat and energy metabolism disturbances. Specifically, the objective was to assess the protective capacity of a GPI vaccination for cattle to provide protection against T. vivax and / or T. Congolense induced pathology.
4. Antibody and antigen detection tests based on previously identified molecules - WP7
In this part of the project, we aimed to develop serologic diagnostic tests based on antigens previously identified, namely BiP (immunoglobulin heavy chain binding protein) subfamily of heat shock proteins 70 (Hsp 70), congopain, evansain and oligopeptidase B. The bulk of the development work was carried out at CIRAD in Montpellier by partner 1 and while other partners were involved in providing field samples, expertise, monoclonal control antibodies, or antigens. The work has focussed mainly on BiP, an immunodominant antigen in trypanosome infections, and at a late stage of the project also on evansain.
5. Antibody detection tests based on newly identified antigens - WP8
To develop serodiagnostic tests for Trypanosoma congolense, T. vivax, T. brucei s.l. and T. Evansi several antigens and corresponding antisera were collected. Representative sera were selected from a collection of sera from natural and experimental infections of different mammal host species. Nineteen recombinant and three native antigens were provided by partners on the projects.
6. Assessment of the 'anti-disease' protective potential of a pathogenic factor cocktail vaccine (across work packages). Six antigens developed during the project under work packages WP1, WP2, WP3 and WP4 (CB1, CB12, C2, FL28, OpdB and ICP) were selected for testing in an immunisation / challenge field trail.
Deliverables
For all work packages, most of the objective were reached, and in some cases, exceeded. Concerning the anti-disease strategy, objectives were not reached in GPI-anchor vaccination experiments in cattle, the reason being no protection was observed in mice, there was hence no need to embark on costly cattle experiments. Some delays were incurred in the crystallisation of congopain, but results are pending. Likewise, progresses were made in the obtention of null mutants for cathepsin L (congopain), cathepsin B, oligopeptidase B, and ISP 1 and 2, but the analysis of phenotypes in vitro and in vitro is ongoing.
On the diagnostic tests front, a large number of antigens were tested, and some tests validated. Species-specific indirect-ELISA tests could not be achieved, due to the close-proximity of the antigenic fabric of the three main species of parasites. The preliminary results regarding the use of cathepsin B as a T. congolense-specific test however are encouraging for this particular theme.
Antigen- (capture-) ELISA, that has occupied scientists in the field for the past 20 years, still remains elusive, in our case due to problems encountered to obtain monoclonal antibodies in a timely manner. Finally, the ultimate withdrawal of DiaMed in year 3, the private partner whose role was to adapt test to rapid format, implies that this objective could not be reached.
Conclusions
In the course of this project, significant progresses have been made towards the development of an anti-disease vaccine and diagnostic tests for trypanosomosis. For both themes, the results at the end of the project may not appear sensational. One must keep in mind though that we are dealing here with a particularly shrewd parasite, with highly evolved mechanisms to evade host immune response, making the development of a vaccine, and as a corollary development of serologic tests, a particularly daunting task.
We have seen that animals immunised with a cocktail of pathogenic factors did generally better than controls, validating the concept of an anti-disease vaccine, but at a level that would not warrant pursuing commercially the venture in the present form. Likewise, some antigens with a diagnostic potential were characterised, and some tests validated, but sensitivity often remained low, and optimisation work is required. On upstream research level, positive outcomes are patent. A large number of trypanosomal pathogenic factors were characterised, through cloning, expression and enzymatic characterisation, and for some crystallisation studies, and gene-silencing, a very important emerging technology. New tools were developed regarding in vitro culture and cyclical transformation of trypanosomes, analysis of parasite proteome and secretome, and molecular adjuvants.
The project has generating close to twenty papers in peer-reviewed, with another ten submitted or in preparation. It should also not be understated that the project served as the basis of twenty Master's theses and eight PhDs, and further strengthened North-South collaborations through important movement of personnel for technology transfer and capacity building.
A vaccine against trypanosomosis has been a venture of many decades, and the road may be still long. Our results, as disappointing as they may look, clearly bring some stones to the edifice, and prove worthwhile the efforts of the consortium and the support of the European Commission.
