Efficient delivery of Antennapedia homeodomain fused to CTL epitope with liposomes into dendritic cells results in the activation of CD8+ T cells.

The in vivo induction of a CTL response using Antennapedia homeodomain (AntpHD) fused to a poorly immunogenic CTL epitope requires that the Ag is given in presence of SDS, an unacceptable adjuvant for human use. In the present report, we developed a hybrid CTL epitope delivery system consisting of AntpHD peptide vector formulated in liposomes as an alternative approach to bypass the need for SDS. It is proposed that liposomes will prevent degradation of the Ag in vivo and will deliver AntpHD recombinant peptide to the cytosol of APCs. We show in this work that dendritic cells incubated with AntpHD-fused peptide in liposomes can present MHC class I-restricted peptide and induce CTL response with a minimal amount of Ag. Intracellular processing studies have shown that encapsulated AntpHD recombinant peptide is endocytized before entering the cytosol, where it is processed by the proteasome complex. The processed liposomal peptides are then transported to the endoplasmic reticulum. The increase of the CTL response induced by AntpHD-fused peptide in liposomes correlates with this active transport to the class I-processing pathway. In vivo studies demonstrated that positively charged liposomes increase the immunogenicity of AntpHD-Cw3 when injected s.c. in mice in comparison to SDS. Moreover, addition of CpG oligodeoxynucleotide immunostimulatory sequences further increase the CD8+ T cell response. This strategy combining lipid-based carriers with AntpHD peptide to target poorly immunogenic Ags into the MHC class I processing pathway represents a novel approach for CTL vaccines that may have important applications for development of cancer vaccines.

Enhanced immune response to T-independent antigen by using CpG oligodeoxynucleotides encapsulated in liposomes.

Immunostimulatory CpG oligodeoxynucleotides (ODN) have been tested as immunoadjuvants for various vaccines including T-cell independent (TI) antigens. Findings from previous reports suggest that close physical association of CpG ODN to the antigen could enhance its adjuvant effect. As an alternative to chemical conjugation of CpG ODN to the antigen, the current study is aimed at determining the benefit of using liposomes as a carrier for CpG ODN to improve the immune response to biotinylated liposomes (Bx-liposomes), a model of a TI antigen. Liposomes with suboptimal concentration of hapten (1% biotin) were not immunogenic. However, when CpG ODN encapsulated in Bx-liposomes were used to immunize mice, a hapten-specific response was obtained as indicated by antibody-mediated elimination of re-administered Bx-liposomes. CpG ODN co-administered with empty Bx-liposomes could not achieve the same effect, indicating the requirement for encapsulation of the adjuvant. Using both intravenous (i.v.) and subcutaneous (s.c.) immunization methods, it was found that IgM levels, but not IgG levels were elevated. Immunization in nude mice confirmed that the immune response obtained was TI. The use of non-CpG ODN and an ODN with alternatively flanked CpG motifs showed no adjuvant effect. Incorporation of poly(ethylene)glycol (PEG)-modified lipid in liposomes enhanced the immune response even further. In conclusion, our data shows that liposomes are a useful delivery vehicle for CpG ODN as an immune adjuvant for TI antigens.

Characterization of hybrid CTL epitope delivery systems consisting of the Antennapedia homeodomain peptide vector formulated in liposomes.

Peptide carriers, such the homeodomain of Antennapedia molecule (AntpHD), which spontaneously cross cellular membranes, have been exploited to deliver antigenic peptide Cw3 to the major histocompatibility complex (MHC) class-I presentation pathway and to prime cytotoxic T cells (CTL). However, the in vivo use of AntpHD recombinant peptide has been limited because CTLs can only be primed in the presence of sodium dodecyl sulfate (SDS) as adjuvant. In this report, we have exploited liposomes to protect the AntpHD-Cw3 from serum degradation and to facilitate the delivery of the recombinant peptide into the MHC class-I pathway of antigen-presenting cells. We have demonstrated that AntpHD recombinant peptide spontaneously associates with liposomes and this association is stable in vitro. However, exchange studies assessing the transfer of the peptide to model membranes or cells in vitro indicates that approximately 50% of the liposome-associated peptide is readily exchangeable. This is consistent with trypsin-protection assays, which have shown that approximately 40% of the liposome-associated peptide is protected from hydrolysis. Importantly, macrophages and dendritic cells are able to internalize AntpHD recombinant peptide associated with liposomes resulting in efficient delivery of the CTL peptide into the cytosol. These studies have demonstrated that dendritic cells treated with AntpHD-Cw3 in liposomes sensitize CTL clones to lyse syngeneic target cells expressing Cw3 epitope. This strategy, which combines liposomes and a peptide vector, provides a new approach for introducing molecules into the MHC class-I antigen presentation pathway of dendritic cells.

Introduction of exogenous antigens into the MHC class I processing and presentation pathway by Drosophila antennapedia homeodomain primes cytotoxic T cells in vivo.

The homeodomain of the Antennapedia molecule (AntpHD) spontaneously crosses cellular membranes and can be used to deliver up to 50 additional amino acids to the cytoplasm. We exploited this approach to deliver antigenic peptides to the MHC class I processing and presentation pathway. AntpHD-based fusion peptides expressing the 170-179 HLA-Cw3 CTL epitope (pCw3) were produced in bacteria. Incubation of these fusion peptides with H-2d target cells resulted in efficient delivery to the cytosol as indicated by protease resistance and confocal microscopy. Moreover, this introduction of an exogenous Ag resulted in sensitization of the cell to lysis by a CTL clone specific for the 170-179 HLA-Cw3-derived peptide. Sensitivity of the Ag processing to brefeldin A but not to chloroquine is consistent with the delivery of AntpHD fusion peptides to the conventional class I-associated processing pathway. Immunization of DBA/2 (H-2d) mice with AntpHD pCw3 fusion peptide in the presence of SDS primed H-2Kd-restricted HLA-Cw3-specific CTL. Similar results were obtained with AntpHD fusion peptides expressing the 147-156 influenza nucleoprotein peptide. The strategy outlined in this paper provides a new approach for introducing molecules into the MHC class I Ag-presenting pathway. This approach has clear relevance to the design of synthetic peptide-based vaccines.