Vaccine delivery by penetratin: mechanism of antigen presentation by dendritic cells

Cell-penetrating peptides (CPP) or membrane-translocating peptides such as penetratin from Antennapedia homeodomain or TAT from human immunodeficiency virus are useful vectors for the delivery of protein antigens or their cytotoxic (Tc) or helper (Th) T cell epitopes to antigen-presenting cells. Mice immunized with CPP containing immunogens elicit antigen-specific Tc and/or Th responses and could be protected from tumor challenges. In the present paper, we investigate the mechanism of class I and class II antigen presentation of ovalbumin covalently linked to penetratin (AntpOVA) by bone marrow-derived dendritic cells with the use of biochemical inhibitors of various pathways of antigen processing and presentation. Results from our study suggested that uptake of AntpOVA is via a combination of energy-independent (membrane fusion) and energy-dependent pathways (endocytosis). Once internalized by either mechanism, multiple tap-dependent or independent antigen presentation pathways are accessed while not completely dependent on proteasomal processing but involving proteolytic trimming in the ER and Golgi compartments. Our study provides an understanding on the mechanism of antigen presentation mediated by CPP and leads to greater insights into future development of vaccine formulations

Whole protein and defined CD8 + and CD4 + peptides linked to penetratin targets both MHC class i and II antigen presentation pathways

Cytoplasmic delivery and cross-presentation of proteins and peptides is necessary for processing and presentation of antigens for the generation of cytotoxic T cells. We previously described the use of the 16 amino acid peptide penetratin from the Drosophila Antennapedia homeodomain (penetratin, Antp) to transport cytotoxic T lymphocyte epitopes derived from ovalbumin (OVA) or the Mucin-1 tumor-associated antigen into cells. We have now shown that penetratin covalently conjugated to OVA protein and linked in tandem to CD4+ and/or CD8+ T-cell epitopes from OVA-stimulated T cells in vitro (B3Z T-cell hybridoma and OT-I and OT-II T cells). The induction of these responses was directly mediated by the penetratin peptide as linking a nonspecific 16-mer peptide to OVA or mixing did not induce CD8+ or CD4+ T-cell responses in vitro. Furthermore, interferon (IFN)-γ-secreting CD4+ and CD8+ T cells were induced which suppressed B16.OVA tumor growth in C57BL/6 mice. Tumor protection was mediated by a CD8+ T-cell-dependent mechanism and did not require CD4+ help to protect mice 7 days after a boost immunization. Alternatively, 40 days after a boost immunization, the presence of CD4+ help enhanced antigen-specific IFN-γ-secreting CD8+ T cells and tumor protection in mice challenged with B16.OVA. Long-term CD8 responses were equally enhanced by antigen-specific and universal CD4 help. In addition, immunization with AntpOVA significantly delayed growth of B16.OVA tumors in mice in a tumor therapy model

Penetratin tandemly linked to a CTL peptide induces anti-tumour T-cell responses via a cross-presentation pathway

Recently there has been increasing evidence to suggest that membrane translocating peptides enter cells by a receptor-dependent pathway. There have been some studies on the mechanism of major histocompatibility complex (MHC) class I presentation of membrane translocating peptides incorporating cytotoxic T lymphocyte epitopes. However, these have been on different cell lines and only a limited number of inhibitors of the antigen presentation pathway were used. Herein, we demonstrate a comprehensive study utilizing a full spectrum of inhibitors to various pathways of MHC class I to elucidate the mechanism of the membrane translocating peptide, penetratin from Antennapedia (Int). It is clear that Int, RQIKIWFQNRRMKWKK when tandemly linked to a cytotoxic T lymphocyte peptide of ovalbumin, SIINFEKL (IntSIIN) is endocytosed via phagocytosis or macropinocytosis by dendritic cells in an ATP-dependent manner and is processed by a proteasome- and tapasin-independent pathway for presentation and loading to MHC class I molecules. In addition, the majority of antigen is taken up by negatively charged receptors. IntSIIN activates T cells in vitro and in vivo and protects mice against challenge with an ovalbumin-expressing tumour