Our previous studies have shown that targeting DNA vaccine-encoded major histocompatibility complex class I epitopes to the proteasome enhanced CD8⁺T-cell induction and protection against lymphocytic choriomeningitis virus (LCMV) challenge. Here, we expand these studies to evaluate CD4⁺T-cell responses induced by DNA immunization and describe a system for targeting proteins and minigenes to lysosomes. Full-length proteins can be targeted to the lysosomal compartment by covalent attachment to the 20-amino-acid C-terminal tail of lysosomal integral membrane protein-II (LIMP-II). Using minigenes encoding defined T-helper epitopes from lymphocytic choriomeningitis virus, we show that the CD4⁺T-cell response induced by the NP_309–328epitope of LCMV was greatly enhanced by addition of the LIMP-II tail. However, the immunological consequence of lysosomal targeting is not invariably positive; the CD4⁺T-cell response induced by the GP_61–80epitope was almost abolished when attached to the LIMP-II tail. We identify the mechanism which underlies this marked difference in outcome. The GP_61–80epitope is highly susceptible to cleavage by cathepsin D, an aspartic endopeptidase found almost exclusively in lysosomes. We show, using mass spectrometry, that the GP_61–80peptide is cleaved between residues F₇₄and K₇₅and that this destroys its ability to stimulate virus-specific CD4⁺T cells. Thus, the immunological result of lysosomal targeting varies, depending upon the primary sequence of the encoded antigen. We analyze the effects of CD4⁺T-cell priming on the virus-specific antibody and CD8⁺T-cell responses which are mounted after virus infection and show that neither response appears to be accelerated or enhanced. Finally, we evaluate the protective benefits of CD4⁺T-cell vaccination in the LCMV model system; in contrast to DNA vaccine-induced CD8⁺T cells, which can confer solid protection against LCMV challenge, DNA vaccine-mediated priming of CD4⁺T cells does not appear to enhance the vaccinee's ability to combat viral challenge.

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