Identification of cross-neutralizing B-cell epitopes on the dengue virus envelope glycoprotein and their application in synthetic peptide based vaccine design

Dengue virus (DENV) is the causative agent for dengue fever. Classified under the flavivirus genus of the flaviviridae family and transmitted by mosquitos; DENV affects an approximately 50-100 million people per year which makes it the second most important tropical infectious disease after malaria. There is currently no vaccine or treatment for dengue fever. A key element in protection from dengue fever appears to be antibody. Development of a vaccine targeted against all 4 serotypes of dengue virus has been hampered by the potential complications following secondary infection. Previous studies have shown that antibodies generated against the precursormembrane protein (prM) are highly cross-reactive among the dengue virus serotypes but do not neutralize infection and potentially promote antibody dependent enhancement (ADE) of disease. This occurs as these antibodies can increase viral uptake into certain cell types, resulting in an increase in the total amount of virus produced. A means of overcoming this issue may be to design peptide vaccines that will generate a specific targeted antibody response against antigenic sequences within the Envelope (E) protein of all four dengue serotypes. In order to discover novel neutralizing antibody epitopes, the present research involves a multi-step epitope mapping strategy using the neutralizing antibodies present in the sera of individuals who have successfully cleared a dengue fever infection. The samples of anti-dengue immunoglobulin G (IgG) purified from patient sera were used for epitope screening against an array of 70 overlapping synthetic peptides spanning the entire E protein of dengue virus serotype 2. A combination of Enzyme-Linked Immunosorbent Assay (ELISA) and epitope extraction techniques revealed 29 epitopes recognized by anti-dengue antibodies on the E protein of DENV-2, of which nine were identified by both methods. Eight epitopes were identified in ELISA only and 12 epitopes were recognized in epitope extraction only. These epitopes span all three domains of the soluble E protein and the ectodomain of the native E protein. We have also used a multi-step computational analysis and predicted six antigenic regions on the DENV-2 E protein. These antigenic regions anchor atleast six epitopes identified by both wet-lab methods. In addition, our computational approach revealed several potential epitope candidates on the E protein of all four serotypes of DENV. The selected peptides were attached to a published B-cell T-helper epitope in order to serve as a vaccine candidate and evaluated in mice for their vaccine potency. Our study revealed 5 novel synthetic vaccine constructs that elicited humoral immune responses and neutralized one or more DENV serotypes in vitro, and are cross-reactive towards soluble recombinant E protein. The findings of this research may suggest new directions for epitope mapping and development of a much-needed anti-dengue vaccine.