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We have previously reported that the CD4+ T lymphocyte response against nuclear human CMV IE1 protein depends in part on endogenous MHC class II presentation. To optimize presentation by HLA-DR of the nuclear IE1 protein and increase the response by CD4+ T cells, we have constructed two different adenovirus vectors containing mutant versions of IE1, containing a HLA-DR3 epitope, fused to GFP. The first construct consisted of a sequence of 46 aa encoded by exon 4, called GFP-IE1 (86-131). The second construct consisted of the whole IE1 mutated on exon 4 nuclear localization signals, identified in this study, and deleted of already known exon 2 nuclear localization signals (GFP-IE1M). Both of these IE1 vectors expressed proteins with cytoplasmic localization, as evidenced by GFP expression, as opposed to control GFP-IE1, which was nuclear. GFP-IE1 (86-131) induced IE1-specific CD4+ T cell clone response that was &gt;30-fold more potent than that against GFP-IE1 and GFP-IE1M. The CD4+ T cell response was due to endogenous presentation followed by exogenous presentation at later time points. Presentation was dependent on both proteasome and acidic compartments. GFP-IE1 (86-131) was rapidly degraded by the APC, which may account for better presentation. Our data show potentiation of the CD4+ T cell response to a specific epitope through shortening and relocation of an otherwise nuclear protein and suggest applications in vaccination.

Optimization of CD4+ T Lymphocyte Response to Human Cytomegalovirus Nuclear IE1 Protein through Modifications of Both Size and Cellular Localization