Final Report Summary - MOSQUITOBLOCK (Integrated biomolecular methods to control mosquito-borne diseases) Mosquitoes are the most prominent of many species of blood sucking arthropods that infest man and other warm-blooded animals, causing more human suffering than any other organism with over one million people dying from mosquito-borne diseases every year. In light of this there is a need for effective control measures. The identification of concealed antigens and their use as vaccine candidates has been successful in controlling other arthropods e.g. ticks and mites and thus the objective of this project is to identify concealed antigens in various mosquito species to evaluate their potential as vaccine candidate to stop mosquito-borne diseases in Europe and worldwide.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: 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.For the proteins early trypsin, Vitelline membrane protein and sterol carrier protein 2 variant 1 there were clearly soluble proteins and these were purified first but the other appeared to be predominantly insoluble and so initial attempts were made to purify these using Urea and denaturing conditions. On further experimentation it was found that the other three proteins did exist in the soluble fraction but with small amounts so the culture volume was increased (4.5 Liter LB made for each one) to reach to the require amounts of each protein. The 3 antigen candidates for both Aedes and Culex have been successfully inserted into the bacterial expression system. For E. coli expression of the various constructs, BLR (De3/pLysS) strains transformed with the appropriate plasmid were grown at 37°C in LB containing 50 μg/mL ampicillin and 34 μg/mL chlor¬amphenicol to an A600 of 0.4–0.8. Recombinant protein expression was induced by adding isopropyl-β-d-thiogalactopyranoside (IPTG) to 1 mM and growing cultures at 30°C overnight. Cells were harvested by centrifugation at 5000× g for 20 min and resuspended in 50 mL binding buffer/1 litre LB (20 mM Tris, pH 8.0 500 mM NaCl). Cell suspensions were lysed by freeze-thawing and sonication using several short bursts at high power, and then centrifuged at 16,000× g for 20 min at 4°C. After centrifuge at 16,000× g for 20 min at 4°C in Eppendorf tubes, the supernatants were loaded onto Ni-agarose affinity column (NTA) previously equilibrated in 10 column volume binding buffer. Proteins were washed consecutively with 5 column volume binding buffer and 5 column volume binding buffer/40 mM imidazole and eluted with binding buffer/300 mM imidazole. Purified fractions were collected into Eppendorf tubes by hand. The supernatant containing FMN-tagged protein was exchanged into PBS 1x buffer (pH 7.4) 20% (V/V) glycerol, by using a PD-10 column previously equilibrated with the same buffer. Purified antigens were sent to the company for Antibody Production 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 ProjectIn conclusion, during this project we have successfully identified several potential concealed antigens candidates. For Aedes and Culex we have successfully purified 3 antigens for each which have been sent to Charles River Ltd in the UK for antibody production to allow immunogenicity and antigenicity testing to subsequently carried to assess their ability to affect mosquito survival. Socio-economic Impact of the ProjectThis 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.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.
