Production of a Versatile Pan-DENV NS5 Polyclonal Antibody and Structural Epitope Analysis of Immunogen Performance

Abstract

Background: Dengue is a major mosquito-borne viral disease worldwide, with approximately 400 million infections reported annually and no fully effective antiviral treatments available. The non-structural protein NS5 is a promising antiviral target due to its critical role in viral RNA replication and host interactions. Although commercial monoclonal and polyclonal anti-NS5 antibodies are available, none were found to combine broad reactivity against all four dengue virus serotypes with suitability for Western Blot (WB), Immunofluorescence (IF), and Immunoprecipitation (IP) assays. This study aimed to develop and functionally characterize a versatile polyclonal antibody targeting a conserved region of DENV NS5 for immunodetection applications.

Methods: Conserved recombinant regions from the methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains of DENV2 NS5 were expressed in Escherichia coli and used as immunogens in a murine model.

Results: Both recombinant proteins exhibited predicted antigenic potential, but only the anti-NS5-RdRp antibody efficiently recognized native NS5 in infected cells. The resulting antibody detected NS5 from all four dengue serotypes in WB, with lower nonspecific background than a widely used commercial antibody. In addition, it proved effective in IF and IP assays in DENV1- and DENV2-infected cells, enabling co-immuno-precipitation of NS3, a known NS5 interactor. Retrospective structural epitope analyses suggested that the RdRp immunogen contained surface-accessible conformational features more compatible with antibody recognition in the native protein context.

Conclusion: These findings identify the anti-NS5-RdRp antibody as a valuable tool for dengue research and illustrate how structural epitope analyses can provide insights into differences in immunogen performance