Sequential immunization with domain III of dengue envelope proteins induces immune responses against dengue virus
Objective 
To investigate whether a novel sequential immunization strategy was more superior to the traditional immunization strategy in eliciting immune responses by using domain Ⅲ of dengue envelope proteins (EDⅢs) as immunogens. 
 
 
Methods 
EDⅢ subunit proteins of four serotypes of dengue viruses (DENVs) were expressed in a baculovirus expression system. SDS-PAGE and Western blot were performed to analyze the purity and specificity of purified recombinant proteins, respectively. In order to evaluate the immunogenicity of EDⅢ-based immunization strategies, female BALB/c mice were subcutaneously immunized with PBS, tetravalent mixture of four EDⅢ recombinant proteins, or the four EDⅢ proteins sequentially for four times with two weeks interval between each immunization. Two-week after the final immunization, splenocytes were isolated and analyzed by ELISPOT assay to evaluate T cell responses and serum samples were collected for plaque reduction neutralization test (PRNT). 
 
 
Results 
Both immunization strategies of sequential EDⅢ proteins and tetravalent EDⅢ proteins could elicit stronger antigen-specific Th2 (IL-4) cell responses in immunized mice than PBS did and the former was superior to the latter. Only the sequential immunization strategy could induce Th2 cell responses in immunized mice against peptide segments of DENV2 EDⅢ. Tetravalent EDⅢ proteins performed better than the sequential immunization strategy in inducing higher levels of neutralizing antibodies against DENV-1, DENV-2 and DENV-3, while both immunization strategies failed to generate neutralizing antibodies against DENV-4. 
 
 
Conclusion 
Sequential immunization with DENV EDⅢ proteins induced stronger T cell responses, but weaker neutralizing antibody responses against DENV than tetravalent EDⅢ proteins did. 
 
 
Key words: 
Tetravalent dengue vaccine; Sequential immunization; Antibody response; T cell response