It has recently been demonstrated that a recombinant replication-deficient human adenovirus 5 (Ad5) vector expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) fused to the p31 invariant (Ii) chain confers broad, long-lasting T-cell immunity that completely protects C57BL/6 mice against lethal peripheral challenge. The current study questioned whether the same strategy, i.e. linkage of GP to an Ii chain, could be applied to a naked DNA vaccine. Following gene-gun immunization with the linked construct (DNA-IiGP), GP-specific CD4(+) T cells could not be detected by flow cytometry. However, inclusion of the Ii chain augmented the priming of GP-specific CD8(+) T cells directed towards both immunodominant (GP(33-41)) and subdominant (GP(276-286) and GP(92-101)) epitopes, and vaccination with DNA-IiGP conferred significantly improved protection against systemic LCMV infection compared with the unlinked construct. In contrast, substantial protection against peripheral challenge was not observed. Additional experiments with T-cell subset-depleted or perforin-deficient mice revealed that virus control in vaccinated mice depends critically on cytotoxic CD8(+) T cells. Finally, priming with the naked DNA vaccine was shown to augment the immune response raised by subsequent immunization with the Ad5 vector. In conclusion, this study showed that the immunoenhancing effect of Ii chain linkage is not limited to the Ad5 vector, but is also relevant with a DNA platform. Furthermore, given the fact that the Ii chain enhances the presentation of more than one epitope, this suggests that Ii-chain-based DNA vaccines may be promising candidates for various heterologous prime-boost regimes.