Title
RECOMBINANT DENGUE 2 VIRUS NS3 PROTEIN CONSERVES STRUCTURAL ANTIGENIC AND IMMUNOLOGICAL PROPERTIES RELEVANT FOR DENGUE VACCINE DESIGN
Authors
Abstract
Introduction: The NS3 protein is a multifunctional nonstructural protein of flaviviruses implicated in the polyprotein processing. The predominance of cytotoxic T cell lymphocytes epitopes on the NS3 protein suggests a protective role of this protein in limiting virus replication. In this work, we studied the antigenicity and immunogenicity of a recombinant NS3 protein of the Dengue virus 2.
Materials and Methods: The full-length NS3 gene was cloned and expressed as a His tagged fusion protein in Escherichia coli. The NS3 protein was purified by two chromatography steps. The immunodetection was carried out by Western Blot using polyclonal antibodies. The antigenic characterization of DENV2-NS3 protein was made by ELISA, Western Blot and immunoflourescence. Mice were immunized with the recombinant NS3 protein in order to determine the capacity to induce anti- recombinant NS3 antibodies.
Results: The recombinant NS3 protein was recognized by anti-protease NS3 polyclonal antibody and anti-DENV2 HMAF by Western Blot. This purified protein was able to stimulate the secretion of high levels of gamma interferon and low levels of interleukin-10 and tumor necrosis factor-a in mice splenocytes, suggesting a predominantly Th-1-type T cell response. Immunized BALB/c mice with the purified NS3 protein showed a strong induction of anti-NS3 IgG antibodies, essentially IgG2b, as determined by ELISA. Immunized mice sera with recombinant NS3 protein showed specific recognition of native dengue protein by Western blotting and immunofluorescence techniques.
Conclusions: The successfully purified recombinant protein was able to preserve the structural and antigenic determinants of the native dengue protein. The antigenicity shown by the recombinant NS3 protein suggests its possible inclusion into future DENV vaccine preparations.
Materials and Methods: The full-length NS3 gene was cloned and expressed as a His tagged fusion protein in Escherichia coli. The NS3 protein was purified by two chromatography steps. The immunodetection was carried out by Western Blot using polyclonal antibodies. The antigenic characterization of DENV2-NS3 protein was made by ELISA, Western Blot and immunoflourescence. Mice were immunized with the recombinant NS3 protein in order to determine the capacity to induce anti- recombinant NS3 antibodies.
Results: The recombinant NS3 protein was recognized by anti-protease NS3 polyclonal antibody and anti-DENV2 HMAF by Western Blot. This purified protein was able to stimulate the secretion of high levels of gamma interferon and low levels of interleukin-10 and tumor necrosis factor-a in mice splenocytes, suggesting a predominantly Th-1-type T cell response. Immunized BALB/c mice with the purified NS3 protein showed a strong induction of anti-NS3 IgG antibodies, essentially IgG2b, as determined by ELISA. Immunized mice sera with recombinant NS3 protein showed specific recognition of native dengue protein by Western blotting and immunofluorescence techniques.
Conclusions: The successfully purified recombinant protein was able to preserve the structural and antigenic determinants of the native dengue protein. The antigenicity shown by the recombinant NS3 protein suggests its possible inclusion into future DENV vaccine preparations.