Development of prophylactic and therapeutic vaccines optimised for cellular processing and presentation to t lymphocytes and targeted to professional antigen presenting cells (protarvac)

Leistungen

Our strategy aims at enhancing the step of intracellular peptide transport into the endoplasmic reticulum by the TAP transporters to increase presentation of peptides to cytotoxic T cells. While the selectivity of the TAP pumps has been known for some time, a possible benefit of modulating TAP affinity for enhancing peptide presentation has so far neither been proposed nor tested. We have analyzed the effect of defined aminoterminal extensions on the efficiency of TAP transport and of presentation to T cells of several epitopes. In these studies, we have defined several sequences of between 1 and 3 residues that, upon addition to the aminoterminus of a peptide, increase TAP transporter affinity dramatically for all tested peptides. Moreover, for several epitopes, we have demonstrated that such extensions lead to highly efficient and increased epitope presentation to T lymphocytes. For peptide epitoeps with low TAP affinities, standard 3-amino acid extensions can be used that universalle enhcance TAP affinity. For peptides with intermediate TAP afffinity, individually designed extenions by one or two residues are preferrable. The mentioned extensions can be added to single epitopes but also used as linkers between two epitopes in synthetic poly-epitopes. Sequences designed to link poly-epitopes are also designed to minimize cleavage by cytosolic proteases (proteasome) within or after the linker but to increase cleavage before the linker. Such linkers are therefore designed to ensure optimal release from poly-epitopes of individual peptides in a form providing efficient access to the ER.

Strategy for identification of epitope clusters useful as CTL-inducing vaccine components. Most CTL epitopes in HIV antigens are concentrated in clusters. We found a strong correlation between the CTL epitope clusters and the degree of hydrophobicity of protein regions. Therefore, the hydrophilicity/hydrophobicity profile might be very useful for selecting protein regions for the production of potent, recombinant CTL-inducing HIV-vaccines.

Our strategies is that: 1) to restore mechanisms subverting CTL responses in chronic infections and 2) to enhance cross-presentation of soluble antigens. Concerning the point 1, we found direct evidence that both HCV and host determinants play a critical role in establishing a status of chronic low-level liver inflammation. Indeed, we found that circulating HCV core protein (HCVcore) inhibited the signal transduction pathway instrumental for IL-2 production, in turn essential for the full effector cell differentiation, and provided direct evidence of the existence of virus-specific CCR7-CD8+ regulatory T cells. These cells infiltrate the liver in chronically HCV-infected patients, and control hepatic effector CD8+ T cell responses via the production of IL-10. Thus HCV-core antagonists or IL-10 inihibitors may represent important targets for restoring the antiviral responses in HCV infection. As regards the mechanisms of cross-priming CD8+ T cell responses by DC (point 2), we set up a valid cross-presentation assay of soluble antigens by DCs and demonstrated that efficient cross-presentation of soluble antigens requires that the latter are protected from the endosomal processing machinery. This evidence suggests it could be exploited for the design of innovative strategies eliciting protective CD8 immunity.

Leistungen

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