Final Report Summary - MOSQUITOBLOCK (Integrated biomolecular methods to control mosquito-borne diseases)
Overview of Results and their Exploitation/Dissemination:
Based on studies reported in ticks and findings that the identified concealed antigens also seem to be effective in controlling mites, an initial strategy was adopted to search the mosquito genome databases for homologues of these proteins. Unfortunately these searches did not identify proteins with significant enough sequence homology to be considered worth pursing with sufficient confidence as possible antigen candidates. In view of this a literature data-mining exercise was performed in order to identify potential antigens in mosquitoes that could be classes as “concealed antigens” by virtue of their location in the gut and induction following a blood meal. To identify the widest number of candidates and increase the significance of the work towards controlling a host of mosquito borne diseases it was decided to perform this exercise on the 3 major mosquito species (Anopheles, Culex and Aedes). The results of this analysis lead to the identification of number of potential candidates many of which were proteases responsible for the digestion of protein components found in blood. The results of this analysis lead to the identification of the following: Anopheles gambiae – Trysin-1, Chitinase, Trypsin-2, Zinc carboxypeptidase A1, Chymotrypsin 2 and Chymotrypsin 1; Aedes aegypti – Vitelline membrane protein, early trypsin and Chymotrypsin II-like protein precursor; Culex quinquefasciatus - gut esterase 1, sterol carrier protein 2 variant 1, and early trypsin precursor.
Due to its important role in the spread of malaria it was decided to initially amplify the potential candidates identified in Anopheles gambiae. In the initials stages RNA was extracted from mosquitoes, used to generate cDNA and attempts made to amplify full length coding sequences for these candidates by PCR using gene-specific primers. Numerous PCR conditions, including the addition of DMSO, varying the magnesium concentration and use of touchdown cycling were tried to produce gene fragments with little success except for Trysin-1, which maybe a consequence of the small size of the gene. As the primary goal of this project is to generate peptides that can be used for antibody production an alternative strategy was devised for the other larger candidates which involved scanning the encoded peptide sequence for regions of high antigenicity and their subsequent amplification from genomic DNA as oppose to cDNA. Using a touchdown PCR method resulted in successful amplifying of the selected exons from all the remaining Anopheles antigen candidates using genomic DNA. The fragments for all these candidates were sequenced to confirmed authenticity and then cloned into a His-tagged bacterial expression vector to allow the production of purified protein for immunological testing of their antigen potential.
In the case of candidates identified from Aedes and Culex due to time constraints and the problems incurred generating gene fragments for the Anopheles candidates, it was decided to adopt a synthetic gene synthesis approach to obtaining full length gene sequences. These sequences were designed and generate in a “cloning ready state” by incorporating appropriate restriction sites before and after the start and stop codons to facilitate insertion into the same His-tagged bacterial expression vector as used for the Anopheles candidates. The 3 antigen candidates for both Aedes and Culex have been successfully inserted into the bacterial expression system, optimized for protein expression and translated proteins purified using metal-affinity chromatography ready for the next stage of testing for their potential in the generation of effective vaccines.
The next stage of testing, which unfortunately has not been possible to commence in the project, will involve the use of the purified antigens in an EliSpot assay to assess their ability to lead to T-cell activation thus confirming their ability to provoke an immune response. Positive candidates will progress to the next stage which will involve the assessment of their ability to provoke a B-cell response i.e. lead to the production of antibodies. Finally, antibodies generated from the candidates that successfully provoke a B-cell response will be tested in in-vitro feeding assays (sugar solutions or blood will be spiked with known concentrations of the appropriate antibody prior to feeding) in order to determine their ability to have a detrimental effect on mosquito growth and survival.
Conclusion on the Project:
In conclusion, during this project we have successfully identified several potential concealed antigens candidates from the 3 major mosquito species known to be vectors of disease. These antigens have been cloned into a bacterial expression system that incorporates a His-tag for purification by IMAC and the FMN-binding domain of human P450 oxidoreductase to facilitate visualization during purification. In the case of Aedes and Culex, we have successfully purified 3 antigens for each which are now ready for immunogenicity and antigenicity testing to assess their ability to lead to antibody production and the subsequent ability of these antibodies to affect mosquito survival.
Socio-economic Impact of the Project:
There is currently no mosquito vaccine on the market worldwide however the success seen with vaccines against ticks indicates that such an achievement is possible with the project undertaken here being a significant step towards the possible generation such vaccines. This project will benefit European competitiveness and boost the vaccine production sector as vaccine companies will be interested in developing the anti-mosquito candidate vaccines identified in Europe, for combating a problem that is widespread across over 50% of the world. An anti-mosquito vaccine will have a significant socio-economic impact as its production will lead to the creation of a significant numbers of new jobs within the Biosciences sector and the generate millions of Euro in income for Europe.
Northumbria University and Mansoura University plan to work together long-term on vaccine development, as the development of a vaccines against arthropods is vital to not only developing countries such as Egypt but also for Europe, in the context of global warming. Furthermore, the two Institutions plan to develop more exchanges through teaching (MSc programmes and PhD student exchanges) and research programmes, using this proposal as a catalytic project to involve more research and teaching staff after the 3rd year of this Fellowship.
Finally, as Dr. Ahmed Rashed as gain valuable experience in molecular biology, bioinformatics and protein expression/purified, he is now in a position to establish a Molecular Biology facility at his home Institute that will provide opportunities to gain addition funding alongside that obtained through collaboration with Northumbria University allow his home Institute to raise its ranking and increase its reputation both of which will facilitate the recruitment of high quality research staff.
Based on studies reported in ticks and findings that the identified concealed antigens also seem to be effective in controlling mites, an initial strategy was adopted to search the mosquito genome databases for homologues of these proteins. Unfortunately these searches did not identify proteins with significant enough sequence homology to be considered worth pursing with sufficient confidence as possible antigen candidates. In view of this a literature data-mining exercise was performed in order to identify potential antigens in mosquitoes that could be classes as “concealed antigens” by virtue of their location in the gut and induction following a blood meal. To identify the widest number of candidates and increase the significance of the work towards controlling a host of mosquito borne diseases it was decided to perform this exercise on the 3 major mosquito species (Anopheles, Culex and Aedes). The results of this analysis lead to the identification of number of potential candidates many of which were proteases responsible for the digestion of protein components found in blood. The results of this analysis lead to the identification of the following: Anopheles gambiae – Trysin-1, Chitinase, Trypsin-2, Zinc carboxypeptidase A1, Chymotrypsin 2 and Chymotrypsin 1; Aedes aegypti – Vitelline membrane protein, early trypsin and Chymotrypsin II-like protein precursor; Culex quinquefasciatus - gut esterase 1, sterol carrier protein 2 variant 1, and early trypsin precursor.
Due to its important role in the spread of malaria it was decided to initially amplify the potential candidates identified in Anopheles gambiae. In the initials stages RNA was extracted from mosquitoes, used to generate cDNA and attempts made to amplify full length coding sequences for these candidates by PCR using gene-specific primers. Numerous PCR conditions, including the addition of DMSO, varying the magnesium concentration and use of touchdown cycling were tried to produce gene fragments with little success except for Trysin-1, which maybe a consequence of the small size of the gene. As the primary goal of this project is to generate peptides that can be used for antibody production an alternative strategy was devised for the other larger candidates which involved scanning the encoded peptide sequence for regions of high antigenicity and their subsequent amplification from genomic DNA as oppose to cDNA. Using a touchdown PCR method resulted in successful amplifying of the selected exons from all the remaining Anopheles antigen candidates using genomic DNA. The fragments for all these candidates were sequenced to confirmed authenticity and then cloned into a His-tagged bacterial expression vector to allow the production of purified protein for immunological testing of their antigen potential.
In the case of candidates identified from Aedes and Culex due to time constraints and the problems incurred generating gene fragments for the Anopheles candidates, it was decided to adopt a synthetic gene synthesis approach to obtaining full length gene sequences. These sequences were designed and generate in a “cloning ready state” by incorporating appropriate restriction sites before and after the start and stop codons to facilitate insertion into the same His-tagged bacterial expression vector as used for the Anopheles candidates. The 3 antigen candidates for both Aedes and Culex have been successfully inserted into the bacterial expression system, optimized for protein expression and translated proteins purified using metal-affinity chromatography ready for the next stage of testing for their potential in the generation of effective vaccines.
The next stage of testing, which unfortunately has not been possible to commence in the project, will involve the use of the purified antigens in an EliSpot assay to assess their ability to lead to T-cell activation thus confirming their ability to provoke an immune response. Positive candidates will progress to the next stage which will involve the assessment of their ability to provoke a B-cell response i.e. lead to the production of antibodies. Finally, antibodies generated from the candidates that successfully provoke a B-cell response will be tested in in-vitro feeding assays (sugar solutions or blood will be spiked with known concentrations of the appropriate antibody prior to feeding) in order to determine their ability to have a detrimental effect on mosquito growth and survival.
Conclusion on the Project:
In conclusion, during this project we have successfully identified several potential concealed antigens candidates from the 3 major mosquito species known to be vectors of disease. These antigens have been cloned into a bacterial expression system that incorporates a His-tag for purification by IMAC and the FMN-binding domain of human P450 oxidoreductase to facilitate visualization during purification. In the case of Aedes and Culex, we have successfully purified 3 antigens for each which are now ready for immunogenicity and antigenicity testing to assess their ability to lead to antibody production and the subsequent ability of these antibodies to affect mosquito survival.
Socio-economic Impact of the Project:
There is currently no mosquito vaccine on the market worldwide however the success seen with vaccines against ticks indicates that such an achievement is possible with the project undertaken here being a significant step towards the possible generation such vaccines. This project will benefit European competitiveness and boost the vaccine production sector as vaccine companies will be interested in developing the anti-mosquito candidate vaccines identified in Europe, for combating a problem that is widespread across over 50% of the world. An anti-mosquito vaccine will have a significant socio-economic impact as its production will lead to the creation of a significant numbers of new jobs within the Biosciences sector and the generate millions of Euro in income for Europe.
Northumbria University and Mansoura University plan to work together long-term on vaccine development, as the development of a vaccines against arthropods is vital to not only developing countries such as Egypt but also for Europe, in the context of global warming. Furthermore, the two Institutions plan to develop more exchanges through teaching (MSc programmes and PhD student exchanges) and research programmes, using this proposal as a catalytic project to involve more research and teaching staff after the 3rd year of this Fellowship.
Finally, as Dr. Ahmed Rashed as gain valuable experience in molecular biology, bioinformatics and protein expression/purified, he is now in a position to establish a Molecular Biology facility at his home Institute that will provide opportunities to gain addition funding alongside that obtained through collaboration with Northumbria University allow his home Institute to raise its ranking and increase its reputation both of which will facilitate the recruitment of high quality research staff.