Dendritic Cell-Based ELISpot Assay for Assessing T-Cell IFN-γ Responses in Human Peripheral Blood Mononuclear Cells to Dengue Envelope Proteins

Dengue envelope (E) protein is a dominant antigen for vaccine development and E-based vaccines have shown partial or full protection against live-virus challenge in non-human primates. Generally, T cell responses can be investigated with peptides. However, hundreds of over-lapping peptides need to be synthesized to cover the whole sequence of a protein, which brings the cost up to a much higher level than purchasing a protein. We have developed an enzyme-linked immunospot (ELISpot) assay that uses intact E proteins instead of peptides for assessing IFN-gamma (IFN-γ) responses. The assay relies on professional antigen presenting cells, dendritic cells, to process and present the E proteins to stimulate T cells.Peripheral blood mononuclear cells (PBMCs) from dengue-exposed and naive subjects were selected for the assay development. IFN-γ production ranged from 53 to 513 spot forming units (SFUs) and 0-45 SFUs per million PBMCs in dengue-exposed and naive subject groups, respectively. The assay allowed quantification of E-specific IFN-γ secreting memory T cells in subjects 9 years after exposure to a live-attenuated virus vaccine and live-virus challenge. Our results suggest that the dendritic cell-based IFN-γ assay is a useful tool for assessing immunological memory for clinical research.