ELISPOT assay. The mice immunized with tetravalent DENV-VLP were euthanized 7 days after the 3rd immunization and the spleen cells were isolated and stimulated in vitro with inactivated virions of each DENV type. IFN-γ (A), TNF-α (B), and IL-10 (C) producing lymphocytes were enumerated by ELISPOT assay. The mean number of spot forming cells (SFCs)/2 × 105 (splenocytes) is shown as virions-stimulated with an SEM bar. *indicates statistical significance (*P < 0.05; **P < 0.01).