Presentation of lipopeptide by dendritic cells induces anti-tumor responses via an endocytosis-independent pathway in vivo.

Cross-presentation by DCs is the major mechanism by which exogenous antigens activate CTLs. However, the mechanisms of entry and presentation of vaccine peptides by DCs remain unclear. In this study, we determined that the mechanisms of antigen presentation differed between nonlipidated and monopalmitoylated peptide antigens. We found that a nonlipidated long peptide could be taken up by DCs and that the peptide could be colocalized with early endosomes. The uptake of nonlipidated peptides by DCs was inhibited at low temperatures or by the depolymerization of actin filaments or microtubules. In contrast, lipidated peptides were internalized by DCs at low temperatures, and internalization was not inhibited when actin filaments or microtubules were depolymerized. Moreover, lipidated peptide, but not nonlipidated peptide, was internalized by nonphagocytic Jurkat cells. The endosomal/lysosomal and proteasomal degradation pathways were necessary for nonlipidated presentation leading to the activation of CD8(+) T cells, but the proteasomal degradation pathway alone was sufficient to process lipidated peptides for MHC class I presentation. We further found that lipidated peptides could enhance peptide-specific T cell responses in vitro and in vivo and induced stronger antitumor responses than nonlipidated peptides. Taken together, our results demonstrate that DCs present lipidated peptides through an endocytosis-independent pathway to promote strong anti-tumor effects in vivo.

Enhanced antigen presentation and CTL activity by transduction of mature rather than immature dendritic cells with octaarginine-modified liposomes.

To improve uptake and cross-presentation of exogenous antigens (Ag) by dendritic cells (DCs), octaarginine-modified liposomes (R8-Lip) were used as a novel strategy for protein-Ag transduction. Immature DCs endocytose macromolecules efficiently. While mature DCs lose their ability to capture Ag, but have an increased capacity for T-cell activation. Thus Ag-transduction has been performed mostly in immature DCs. In the present study, R8-Lip were efficiently taken up by both immature and mature DCs. DCs transduced after maturation were highly efficient at cross-presentation of Ag and induced higher cytotoxic T-lymphocytes (CTL) activity than were DCs transduced before maturation. The mechanism of Ag presentation involved the escape of R8-Lip from endosomes to cytosol, which require the acidic environment. The Ag released was then processed by a proteasome-dependent pathway. This novel transduction approach is clinically applicable, easy to perform, and has more practical advantages than current protein transduction methods.

Immunoprotection against murine bladder carcinoma by octaarginine-modified liposomes incorporating cell wall of Mycobacterium bovis bacillus Calmette-Guérin.

OBJECTIVE: To develop a prototype of a non-live bacterial agent that consists of a cell wall (CW) preparation from heat-killed bacillus Calmette-Guérin (BCG-CW) incorporated into octaarginine-modified cationized liposomes as a vector (R8-liposome-BCG-CW), and to evaluate its immunoprotective potentiation in mice, as although BCG is an established effective immunotherapy for nonmuscle-invasive bladder cancer, more active and less toxic treatments are needed. MATERIALS AND METHODS: The cellular interaction of R8-liposome-BCG-CW co-cultured with mouse bladder cancer cell line (MBT-2) was examined by confocal laser scanning microscopy. MBT-2 cells (7 x 10(5)) were subcutaneously inoculated with 1 mg BCG, 0.1 mg or 1 mg BCG-CW, 0.1 mg or 1 mg R8-liposome-BCG-CW in female C3H/HeN mice. The MBT-2 cells pretreated with BCG or R8-liposome-BCG-CW were re-challenged at 6 weeks. The sizes of the primary and re-challenged tumours were evaluated at 4 and 10 weeks, respectively. RESULTS: Confocal laser scanning microscopy showed the enhanced incorporation of R8-liposome-BCG-CW into MBT-2 cells after 1 h of co-incubation. 0.1 mg R8-liposome-BCG-CW completely inhibited the growth of MBT-2 tumours while 0.1 mg BCG-CW alone did not (P = 0.002). Mice vaccinated with a mixture of MBT-2 cells and R8-liposome-BCG-CW inhibited the growth of re-challenged tumour of MBT-2 cells pretreated with BCG or R8-liposome-BCG-CW but did not inhibit that of MBT-2 cells with no pretreatment at 10 weeks, with mean (sd) tumours sizes of 54 (60) mm(2) (P < 0.001) or 69 (43) mm(2) (P = 0.003) compared with 309 (125) mm(2), respectively. CONCLUSION: The immunotherapeutic potential of BCG-CW was enhanced by improving cellular association using the R8-liposomes delivery system. Development of this non-live bacterial agent may contribute to providing a more active and less toxic tool as a substitute for live BCG as immunotherapy against nonmuscle-invasive bladder cancer in the future.

An improved SUMO fusion protein system for effective production of native proteins.

Expression of recombinant proteins as fusions with SUMO (small ubiquitin-related modifier) protein has significantly increased the yield of difficult-to-express proteins in Escherichia coli. The benefit of this technique is further enhanced by the availability of naturally occurring SUMO proteases, which remove SUMO from the fusion protein. Here we have improved the exiting SUMO fusion protein approach for effective production of native proteins. First, a sticky-end PCR strategy was applied to design a new SUMO fusion protein vector that allows directional cloning of any target gene using two universal cloning sites (Sfo1 at the 5'-end and XhoI at the 3'-end). No restriction digestion is required for the target gene PCR product, even the insert target gene contains a SfoI or XhoI restriction site. This vector produces a fusion protein (denoted as His(6)-Smt3-X) in which the protein of interest (X) is fused to a hexahistidine (His(6))-tagged Smt3. Smt3 is the yeast SUMO protein. His(6)-Smt3-X was purified by Ni(2+) resin. Removal of His(6)-Smt3 was performed on the Ni(2+) resin by an engineered SUMO protease, His(6)-Ulp1(403-621)-His(6). Because of its dual His(6) tags, His(6)-Ulp1(403-621)-His(6) exhibits a high affinity for Ni(2) resin and associates with Ni(2+) resin after cleavage reaction. One can carry out both fusion protein purification and SUMO protease cleavage using one Ni(2+)-resin column. The eluant contains only the native target protein. Such a one-column protocol is useful in developing a better high-throughput platform. Finally, this new system was shown to be effective for cloning, expression, and rapid purification of several difficult-to-produce authentic proteins.

New packaging method of mycobacterial cell wall using octaarginine-modified liposomes: enhanced uptake by and immunostimulatory activity of dendritic cells.

Despite the potential of mycobacterial cell wall (CW) components to serve as immunotherapeutic agents, this application is hampered by the molecules' unfavorable physicochemical properties, such as its high molecular weight, poor solubility and negatively charged nature. Here we describe a new mycobacterial CW delivery system that uses an efficient and simple packaging method. This is achieved by incorporating mycobacterial CW into liposomes and attaching arginine octamers (R8) to the liposome surface. R8-modified liposomes improve the uptake of mycobacterial CW by dendritic cells (DC) and enhance its immunostimulatory activity. High R8 surface density promoted high levels of mycobacterial CW uptake by DC compared to low density R8-modified liposomes. Maturation markers (CD80, CD86, MHC Class II molecules) showed significantly enhanced expression on DC pulsed with high density R8-modified liposomes containing mycobacterial CW. Moreover, R8-modified liposomes with mycobacterial CW incorporated induced production of IL-12 p40 by DC, at levels similar to those produced by lipopolysaccharide-pulsed DC. We assert that R8-modified liposomes with mycobacterial CW incorporated should have tremendous potential as immune-potentiating agents.

Virosome and ISCOM vaccines against Newcastle disease: preparation, characterization and immunogenicity.

The purpose of this study was to prepare and characterize virosomes and ISCOMs containing envelope proteins of Newcastle disease virus (NDV) and to evaluate their immunogenicity in target animals (chickens). Virosomes were prepared by solubilization of virus with either Triton X-100 or octyl glucoside (OG) followed by detergent removal. Biochemical analysis revealed that these virosomes contained both the haemagglutinin-neuraminidase protein (HN) and the fusion protein (F), with preserved biological activity. Acidic environment triggered the fusion between virosomes and chicken erythrocyte ghosts. Formation of ISCOMs was achieved by solubilizing phospholipids, cholesterol, envelope protein antigen and Quil A in Triton X-100. The ISCOM particles were formed by removal of the detergent. In each formulation the relative HN content correlated with the capability to agglutinate red blood cells. The immunogenicity of these lipid-based subunit vaccines was determined in chickens after subcutaneous immunization. The relative HN content of the subunit vaccines correlated with the haemagglutination-inhibition (HI) antibody titres. Virosomes prepared with Triton X-100 and ISCOMs offered high clinical protection (> 80%) upon challenge with virulent NDV. Virosomes prepared with OG yielded lower clinical protection despite high HI antibody titres. Virosomes with reduced antigen density showed poor immunogenicity and protection. In conclusion, ND virosomes and ISCOMs were found to be immunogenic and provided good protection.