Development underway for anti-mosquito vaccine
The aim of the EU-funded project MOSQUITOBLOCK (Integrated biomolecular methods to control mosquito-borne diseases) was to develop a non-chemical method of controlling mosquitoes, thereby protecting the foodchain, environment and non-pest insects. Work began with literature data mining. This enabled scientists to identify potential antigens in mosquitoes that could be classed as ′concealed′ antigens due to their location in the gut and induction following a blood meal. This was carried out for three major mosquito species (Anopheles, Culex and Aedes) in order to identify the widest number of candidates. The potential candidate gene Trysin-1 was found in the mosquito Anopheles gambia and amplified by polymerase chain reaction (PCR) method, using gene-specific primers. An alternative method was devised for larger candidates, which involved scanning the encoded peptide for sequence for regions of high antigenicity and their subsequent amplification from genomic DNA. A touchdown PCR technique was used to successfully amplify the selected exons from all the remaining Anopheles antigen candidates using genomic DNA. The fragments from all these candidates were cloned into a His-tagged bacterial expression vector to produce purified protein for immunological testing of their antigen potential. A gene synthesis approach was used for candidates identified from Aedes and Culex mosquitoes in order to obtain full length gene sequences. The three antigen candidates were successfully inserted into the bacterial expression system, optimised for protein expression and translated proteins purified using metal affinity chromatography. The next stage of testing for an effective vaccine, which could not commence during the project, will involve the use of purified antigen in an Enzyme-linked immunospot (EliSpot) assay. This will determine their ability to lead to T-cell activation, thereby confirming their ability to cause an antigen response. MOSQUITOBLOCK therefore successfully identified several potential concealed antigen candidates from the three major mosquito species recognised as vectors of disease. These antigens were cloned into a bacterial expression system that incorporates His-tag to facilitate visualisation during purification. In the case of Aedes and Culex, researchers successfully purified three antigens for each. These are now ready for immunogenicity and antigenicity testing to determine their ability to lead to antigen production and the ability of these antigens to influence mosquito survival. The project will help European competitiveness by boosting the vaccine production sector through the development of an anti-mosquito candidate vaccine, which will combat a major threat to humanity.
Keywords
MOSQUTIOBLOCH, concealed antigens, Culex, Aedes, Trysin-1, Anopheles gambia, polymerase chain reaction, gene-specific primers, genomic DNA, exons, touchdown PCR, His-Tag, enzyme-linked immunospot assay, T-cell