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Genetic modification of vaccines by linking the Ag to lysosomal or endosomal targeting signals has been used to route Ags into MHC class II processing compartments for improvement of CD4+ T cell responses. We report in this study that combining an N-terminal leader peptide with an MHC class I trafficking signal (MITD) attached to the C terminus of the Ag strongly improves the presentation of MHC class I and class II epitopes in human and murine dendritic cells (DCs). Such chimeric fusion proteins display a maturation state-dependent subcellular distribution pattern in immature and mature DCs, mimicking the dynamic trafficking properties of MHC molecules. T cell response analysis in vitro and in mice immunized with DCs transfected with Ag-encoding RNA showed that MITD fusion proteins have a profoundly higher stimulatory capacity than wild-type controls. This results in efficient expansion of Ag-specific CD8+ and CD4+ T cells and improved effector functions. We used CMVpp65 and NY-ESO-1 Ags to study preformed immune responses in CMV-seropositive individuals and cancer patients. We show that linking these Ags to the MITD trafficking signal allows simultaneous, polyepitopic expansion of CD8+ and CD4+ T cells, resulting in distinct CD8+ T cell specificities and a surprisingly broad and variable Ag-specific CD4+ repertoire in different individuals.
Kreiter et al. (Tue,) studied this question.