Enhancement of immune responses to an HIV env DNA vaccine by a C-terminal segment of listeriolysin O

An effective vaccine against AIDS should induce both cellular and humoral immune responses. Here we report that immunization of mice with a DNA plasmid encoding a chimeric protein consisting of HIV89.6 Env gp140 and the listeriolysin O (LLO) C-terminal segment ( 59 amino acids) significantly enhanced both humoral and cellular immune responses against the HIV89.6 Env protein. Plasmid DNA expression vectors with genes codon-optimized for mammalian expression were synthesized for HIV89.6 gp140 as well as for chimeric protein gp140 - LLO, in which the coding sequence for the C-terminal 59 amino acids of LLO were fused in frame to the 39 end of the codon-optimized gene for gp140. All plasmid vectors produced high levels of protein expression, and the gp140 - LLO chimeric protein was cleaved and secreted as efficiently as gp140. Analysis of humoral immune responses by ELISA showed that the chimeric gp140 - LLO construct induced higher antibody responses than the gp140 construct in immunized mice, more notably in the IgG2a antibody subtype. Intracellular cytokine staining and flow cytometry analysis showed that the gp140 - LLO construct induced significantly higher levels of cytotoxic T lymphocyte immune responses against the HIV 89.6 Env protein than those observed with the gp140 construct. Our results thus demonstrate that the C-terminal segment of LLO can be effectively employed to enhance both cellular and humoral immune responses against the HIV89.6 Env antigen in the context of a DNA vaccine.