CLONING, EXPRESSION AND IMMUNOLOGICAL SCREENING OF DENGUE VIRUS ENVELOPE PROTEIN CONSTRUCTS

Abstract

Dengue virus (DENV) infects humans by causing mild fever and sometimes, advances to severe haemorrhagic fever characterized by capillary leakage. The mosquito vector Aedes aegypti is found predominantly in the tropical and subtropical regions and places more than half of the world’s population at risk of contracting the virus. The main strategy for controlling infection from spreading is vector control using insecticides and vaccination. Currently, DengvaxiaTM is the only licensed vaccine and is administered to individuals with a history of viral exposure, as recommended by the World Health Organization (WHO). DengvaxiaTM elicits cross-reactive antibodies in seronegative individuals, sometimes leading to the development of severe dengue illness, including Dengue Haemorrhagic fever (DHF). Therefore, with the mosquito vector developing resistance towards available insecticides and the absence of a fully effective vaccine, there is a need to develop not only a protective vaccine but also one that does not establish cross-reactive antibodies that could result in severe disease. Recent studies suggest the presence of protective epitopes on the envelope protein eliciting neutralizing antibodies which prevent viral infection of host cells. In this study, the protective envelope epitopes were identified, and constructs designed for antigenicity testing based on structural modelling of DENV-2 and Insect specific flavivirus (ISF) envelope proteins. The DENV-2 protective epitopes were introduced on the ISF envelope backbone replacing the corresponding residues following modelling, resulting in the DENV-ISF chimera. As controls, both ISF and DENV envelopes proteins were successfully cloned and expressed in HEK 293F mammalian cells and tested alongside the DENV-ISF chimera. To check for proper design of the recombinant chimera, differences in DENV-2 and ISF domain II (DII) and correct folding of the expressed recombinant DENV-2 antigen, a monoclonal antibody targeting DENV domain vi II (mAbDENVDII) implicated to produce cross-reactive antibodies was used to probe the recombinant proteins. The results showed that DENV-2 recombinant envelope protein (containing DII) had a significantly high response across different antibody dilutions, in comparison to DENV-ISF (p=0.0026) and ISF (p=1.4e-5). The antigenicity of the recombinant proteins was subsequently tested with DENV immune pooled sera, and the findings showed that DENV-2 had a significant high response in comparison to DENV-ISF (p=0.016) and ISF (p=0.005). To further examine antibody specific to DENV binding capacity on the antigens, a depletion assay and a whole virus ELISA assay on the antibody depleted sera was performed. The results showed that across the first four dilutions, DENV-2 and DENV-ISF had reduced responses although not statistically significant when compared to ISF (p=0.767 and p=1 respectively). A neutralization assay with one round of envelope specific depleted sera showed no significant differences in response among the recombinant proteins. These results indicate that more depletion rounds may be needed to bind DENV specific antibodies on the chimeric construct before performing neutralization assays to test ISF as a delivery platform for the protective DENV epitopes. The lack of response of ISF and DENV-ISF to the mAbDENVDII show that there is the potential use of ISF as a delivery platform for epitopes, especially DENV envelope protective epitopes.