Human cytomegalovirus protein pp65: an efficient protein carrier system into human dendritic cells

Abstract

Protein-based immunogens are usually poor inducers of CD8 + T cells. To enhance the induction of CD8 + T cells, one approach is the use of protein immunogens coupled to protein transduction domains (PTDs). These are small cationic peptide sequences that significantly enhance the uptake of fused proteins into dendritic cells (DC) and then mediate their presentation in the context of major histocompatibility complex class I (MHC-I) and MHC-II molecules. One drawback of this system is the high concentrations of PTD-fusion proteins required. Here, we show that proteins fused to the human cytomegalovirus tegument protein pp65were bound with higher efficiency to DCs than those fused to the described PTDs TatPTD and Penetratin. Furthermore, the fusion of pp65 to proteins led to an enhanced uptake of these proteins by DCs. Once taken up, CD4 + and CD8 + memory T cells were strongly stimulated ex vivo demonstrating that pp65 was efficiently processed and presented in the context of both MHC-I and MHC-II. These data make pp65 a promising delivery system to induce cellular immune responses by fused protein vaccines.

An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity

“…Mechanisms of immune evasion inhibiting MHC-I-dependent IE-1 antigen processing and presentation might also explain the lower frequency of IE-1-specific IFN-γ-producing cells detected in our ELISpot assay [65]. In addition, reduced processing and presentation efficiency due to protein stability, size and nuclear localization might play a role in the reduced reactivity to IE-1, as opposed to pp65 [64, 66, 67]. This proposition is supported by the observation that higher concentrations of IE-1 were required in our ELISpot assay, in comparison to pp65, to trigger a significant response.…”

Validation of T-Track® CMV to assess the functionality of cytomegalovirus-reactive cell-mediated immunity in hemodialysis patients

“…Moreover, mechanisms of immune evasion involving CMV-encoded unique short (US) proteins and resulting in the inhibition of the MHC-I-dependent antigen presentation pathway appear to be responsible for impaired IE-1 antigen processing and presentation, and thus in the low frequency of IE-1-reactive CD8 + T cells [55–57]. On the other hand, differential antigen uptake, processing and presentation by APC, possibly influenced by pp65 and IE-1 intrinsic properties [54, 58, 59], might explain inter-individual differences in the frequency of CMV antigen-specific T cells. Accordingly, comparable CD8 + T cell response to IE-1 and pp65 has also been described in some CMV-seropositive healthy donors [47, 60].…”

Delivery of functional DNA and messenger RNA to mammalian phagocytic cells by recombinant yeast

“…50 Briefly, peripheral blood mononuclear cells were separated into monocytes and PBL by adherence. PBL were kept frozen in liquid nitrogen (N 2 ) until use, whereas monocytes were differentiated into iDCs as described above.…”