Bioscience Reports, Vol. 22, No. 2, April 2002 ( 2002) MINI REVIEW Liposomal Delivery of CTL Epitopes to Dendritic Cells Ghania Chikh1 and Marie-Paule Schutze-Redelmeier1,2,3 Receiûed Noûember 17, 2001 The induction of strong and long lasting T-cell response, CD4C or CD8+, is a major requirement in the development of efﬁcient vaccines. An important aspect involves delivery of antigens to dendritic cells (DCs) as antigen presenting cells (APCs) for the induction of potent antigen-speciﬁc CD8+ T lymphocyte (CTLs) responses. Protein or peptide-based vaccines become an attractive alternative to the use of live cell vaccines to stimulate CTL responses for the treatment of viral diseases or malignancies. However, vaccination with proteins or synthetic peptides representing discrete CTL epitopes have failed in most instances due to the inability for exogenous antigens to be properly presented to T cells via major histocompatibility complex (MHC) class I molecules. Modern vaccines, based on either synthetic or natural molecules, will be designed in order to target appropriately professional APCs and to co-deliver signals able to facilitate activation of DCs. In this review, we describe the recent ﬁndings in the development of lipid-based formulations containing a combination of these attributes able to deliver tumor- or viral-associated antigens to the cytosol of DCs. We present in ûitro and pre-clinical studies reporting speciﬁc immunity to viral, parasitic infection and tumor growth. KEY WORDS: Liposomes; dendritic cells; cytotoxic T lymphocyte; vaccine. INTRODUCTION The goal of current vaccines is to induce an immune response that can protect the host against challenging diseases such as cancer, complex viruses or parasites. This protection depends mainly on activated tumor-, viral- or parasitic-speciﬁc CD8+ cytotoxic T lymphocytes (CTLs). CTLs kill neoplastic or infected cells through recognition of antigenic peptides presented by MHC class I molecules on the surface of the target cells. MHC class I-restricted presentation to cytotoxic T lymphocytes (CTLs) is generally limited to antigens actively synthesized by target cells, due to the intracellular sites of peptide generation occurring in the cytosol and loading onto MHC class I molecules in the endoplasmic reticulum (ER) (Germain, 1994). Exogenous antigens are not able to access the sites of peptide processing since the 1 Systemic Therapy Program, Advanced Therapeutics, British Columbia Cancer Research Center, Dept of Advanced Therapeutics, 601 West 10th Avenue, Vancouver, B.C., V5Z 1L3, Canada 2 Dept of Pathology, University of British Columbia, Vancouver, Canada 3 To whom correspondence should be addressed. Fax: 1 (604) 877 6011; e-mail: email@example.com 339 0144-8463 02 0400-0339 0 2002 Plenum Publishing Corporation 340 Chikh and Schutze-Redelmeir cytosol and ER do not exchange material with the lumen of endocytic compartments. This prevents neighbor cells, that may have phagocytosed viral infected or tumor cells but are not infected or transformed themselves, from CTL lysis (Moore et al., 1988). Antigen-expressing cells cannot directly initiate immune responses and the induction of cellular responses requires ingestion of the antigens by professional APCs. This process is called ‘‘cross priming,’’ APCs such DCs are involved in crosspriming and can present exogenous internalized antigens in the context of MHC class I molecules and stimulate naıve T cells (Albert et al., 1988). CTL responses ¨ have been induced to replicating vaccines such as retroviral vectors expressing antigens; however despite some relative success these vaccinal vectors are immunogenic and contain safety risks. Exogenous proteins and peptides represent the safest means of vaccination. However, in terms of generating an immune response to tumor, viral or parasitic associated peptides, researchers have identiﬁed numerous obstacles. Peptide vaccination depends on the loading of empty MHC molecules on APCs in ûiûo. However, single administration of peptide without a means of targeting activation APCs can potentially lead to loading of MHC class I molecules on nonprofessional APCs, which could result in tolerance (Toes et al., 1998, Diehl et al., 1999, Grossmann et al., 2001). One example was the vaccination using CTL epitopes derived from adenovirus type 5 early region (Ad5E1) oncogenes which speciﬁcally tolerized T cells causing enhanced tumor outgrowth (Toes et al., 1998). Neither modiﬁcation of the Ad5E1 peptide vaccine through incorporation of the peptide into liposomes or by ligation of the peptides to lipid tails was able to restore immune response. Only Ad5E1-derived peptides presented on DCs evoked a strong tumorprotective CTL response. These ﬁndings have important implications for the development of peptide-based immune intervention strategies and emphasize the superior nature of antigen-pulsed DCs over other peptide-based vaccination protocols as well as the crucial importance of the mode of peptide-antigen delivery in setting the balance between T cell stimulation and tolerization. Although DC pulsed with peptides appear to be good candidates for a clinical use in human tolerization (Mayordomo et al., 1995, Dallal and Lotze, 2000), ex ûiûo approaches suffer from two problems, one related to generation of high numbers of DCs from a clinically practicable manner, and the other one resulting from multiple rounds of immunization at fairly short intervals that can at times lead to the emergence of non-cytolytic CD4C T cells exhibiting the characteristic phenotype of Th2 cells. There are many attempts being made to simplify immunization protocols, and targeting speciﬁcally the DC systems in ûiûo has not been achieved yet and consequently represents an active area of research. DENDRITIC CELLS Dendritic cells are highly specialized APCs, capable of inducing naıve T-cell ¨ response. DCs in the periphery capture and process antigens, express lymphocyte co-stimulatory molecules such as B7–1, B7–2, CD40, migrate to lymphoid organs and secrete cytokines to initiate immune responses (Banchereau and Steinman, 1998). When pulsed with proteins, DCs are powerful stimulators of immunity when administered as a vaccine, even for weak antigens (Mayordomo et al., 1995; Dallal Dendritic Cells and Liposomes 341 and Lotze, 2000). These cells express 50-fold higher levels of MHC molecules than macrophages, providing more peptide MHC ligand for T-cell receptor engagement (Banchereau and Steinman, 1998). In addition to their ability to process exogenous antigens for class II presentation, these cells are also able to process antigens for MHC class I presentation. During migration from peripheral tissues to lymph nodes, DCs mature, resulting in a down-regulation of endocytic activity while the capacity to present MHC peptide complexes to T lymphocytes is maximized. They represent a heterogenous population of cells present at low number in most peripheral tissues, where they ingest antigens by phagocytosis, macropinocytosis and receptor-mediated endocytosis. DCs also express several receptors that determine their maturation. Maturation is induced either by pathogens, through components of the bacterial wall (e.g., lipopolysaccharide) or viral products (e.g., double strand RNA), or by inﬂammatory stimuli such as cytokines (e.g. IL–1 or TNF-α ). Helper T lymphocytes trigger complete DC maturation through CD40 CD40L-dependent and -independent interactions (Aiba 1998; Piemonti et al., 1999; Terheyden et al., 2000; Bauer et al., 2001). The existence of transport from endosomes to the cytosol in APCs was initially reported by Kovacsovics-Bankowski and Rock, 1995, who showed that upon phagocytosis of antigen-coated latex beads, MHC class I presentation occurred in macrophages and that the cells become sensitive to a toxin with a cytosolic target. Induction of macropinocytosis in macrophages also showed cross presentation and transport of internalized antigen to the cytosol (Norbury et al., 1997). More recently Rodriguez et al., 1999, showed that DCs have developed a membrane transport pathway linking the lumen of endocytic compartment and the cytosol. DCs play a crucial role in the initiation of primary immune response and represent therefore the best adjuvant for anti-tumor immunity. LIPOSOMES AS CARRIERS Advances in our understanding of the physical and biological characteristics of liposomal drug delivery vehicles have allowed us to overcome numerous technological and physiological obstacles and design liposomal carriers with well-deﬁned biological targets (Gabizon and Papahadjopoulos, 1992; Holland et al., 1996). This developmental process has culminated in the recent FDA approval of three different liposome-based drug formulations for treatment of certain cancers and microbial infections. While this is an encouraging sign, these initial liposomal systems represent only the ﬁrst level of sophistication that can be designed into such carriers. Speciﬁcally, liposome use has historically focused on carriers that are retained in the circulation for extended periods of time and exhibit slow release of entrapped contents. Ideally, however, liposomal systems should be designed speciﬁcally to optimize interactions between the biological target and the carrier-associated antigen. It is important to review the present generation of liposomal carriers, typically considered as optimal drug delivery systems, in order to understand how they differ from formulations being developed for induction of T-cell response. The same advances in liposome technology that gave rise to the ﬁrst generation of clinically-proven drug formulations unfortunately limit therapeutic activity. Highly stable carriers that show optimal retention of a drug in the circulation, and hence maximal delivery to 342 Chikh and Schutze-Redelmeir the tumor, may not release the encapsulated drug at an adequate rate to optimize tumor cell killing. Signiﬁcant advances in the use of liposomes for intracellular delivery of biopharmaceuticals, such as peptides, antisense oligonucleotides and genes, will require the development of multifunctional liposomes that contain features speciﬁc for stability to biological ﬂuids (blood, interstitial ﬂuids, lymph), controlled distribution characteristics, site-speciﬁc targeting, and controlled-content release. FIRST GENERATION LIPOSOMAL VACCINES Liposomes as carriers of antigens have been used extensively in developing strategies to induce CTLs in ûitro (Nair et al., 1992; Zhou et al., 1994) and in ûiûo (Babu et al., 1995; White et al., 1995) and Alving et al., 1995, for review. In ûitro studies on proteins encapsulated within conventional liposomes and pH-sensitive liposomes have demonstrated that some encapsulated material can penetrate into the cytosol and enter the class I processing pathway by ‘‘spilling’’ from endosomes (Alving and Wassef, 1994; Alving et al., 1995). The role of pH-sensitive liposomes for the improvement of antigen delivery to the cytosol and subsequent CTLs has been reported by numerous laboratories (Harding et al., 1991; Reddy et al., 1991; Nair et al., 1992; Nair et al., 1993; Zhou et al., 1994). The mechanism proposed is that, upon exposure to the low pH of the endosome, liposomes disrupt and fuse with the endosome membrane, allowing for some contents to leak into the cytosol. Despite some encouraging results in experimental animal models, the efﬁcacy of the CTL response induced was typically dependent on the nature of the antigen (Reddy et al., 1991; Zhou et al., 1992; Alving and Wassef, 1994). The efﬁcacy of pH liposomes as immunoadjuvant has been recently re-evaluated by Chang et al., 2001. The study reported that immunization of mice with CTL epitopes derived from Hantaan nucleocapsid protein (M6) or human papilloma virus E7 encapsulated in pH-sensitive liposomes induced effective antigen-speciﬁc CTL responses. The CTL responses induced by M6 peptide blocked the formation of tumor mass from Hantaan NP transfected B16 melanoma cells in mice and delayed the growth of pre-inoculated melanoma cells. The major advantages offered by pH liposomes are that they are less toxic and more efﬁcient in terms of processing by the MHC class I pathway than conventional liposomes or free peptide. One limitation is that their success does not apply to all antigens so far. Most of the earlier work done with liposomes was approached under the assumption that macrophages were serving as the principal APCs for liposomal antigen presentation. Since the emergence of the central role of DCs as professional APCs, DCs have been analyzed more intensively for their ability to present antigens encapsulated in liposomes. Numerous reports have demonstrated that DCs loaded with antigen in liposomes in ûitro are able to present in a very efﬁcient manner the antigen in the context of the MHC class I molecules and prime CTLs (Nair et al., 1993; Mulders et al., 1999; Ludewig et al., 2000; Chikh et al., 2001a). Human DCs generated from peripheral blood of patients with metastasic renal carcinoma (RCC) and loaded with autologous tumor lysate (TuLy) using liposomes were shown to display increased expression of HLA class I molecules and a persistent high Dendritic Cells and Liposomes 343 expression of class II molecules. Interestingly, these mature DCs-TuLy could activate immunosuppressed tumor inﬁltrating lymphocytes (TILs) via an induction of enhanced anti-tumor CTL responses associated with production of Th1 cells (Muldlers et al., 1999). It is important however to note that with conventional liposomes, although they have been shown to be active in ûitro, their efﬁcacy in ûiûo has not been clearly demonstrated (Toes et al., 1998; Chikh et al., 2001a). Studies on vaccination with CTL epitopes reported few years ago by Toes et al., 1998, might have important implications for peptide liposome-based vaccine development. The immunization with CTL epitopes from Ad5E1 incorporated into liposomes did not actually induce an immune response but speciﬁcally tolerized T cell. Interestingly, the in ûiûo CTL-tolerizing potential of these peptides was converted to speciﬁc immunostimulation when loaded in ûitro on DCs and then re-injected in ûiûo. This was, however, not conﬁrmed by (Ludewig et al. 2000) studies that demonstrated that DCs could be directly loaded in ûiûo with peptides in liposomes following intradermal immunization in LCMV model, achieving protective anti-tumor and anti-viral immunity. It is not clear whether these contradictory ﬁndings result from the type of antigen or the route of immunization used. NOVEL CLASS OF LIPOSOMAL VACCINES During the last ﬁve years a large number of different approaches have been made to improve the immunoadjuvant action of liposomes. Approaches currently tested include modiﬁcation of the liposome structure, combination other molecules, with surface ligands for targeting to increase the immunogenicity of peptide and protein vaccines. Cationic Liposomes There is clear evidence that liposome composition plays a critical role in the induction of CTL response. Investigations on the relationship between liposomal surface charge and adjuvant action have been conducted and recent studies have showed that positively-charged vesicles were taken up efﬁciently by macrophages and DCs, while neutral liposomes were weakly internalized. Consistent with this process, potent in ûiûo CTL and humoral responses were reported using cationic compared to anionic and neutral liposomes (Nakanishi et al., 1997; Nakanishi et al., 1999; Chikh et al., 2001a, b). In the HIV model, delivery of HIV-1 Gag, Pol, and Env proteins to DCs by cationic liposomes such as lipofectin stimulated greater anti-HIV-1 memory CTL responses in cells from HIV-1-infected subjects than those induced by DCs loaded with protein alone (Zheng et al., 1999). Antigen presentation was enhanced by chloroquine, but blocked by brefeldin A, indicating that the classic MHC class I cytosolic pathway was used for processing and presentation of HIV-1 protein by the DCs. This was conﬁrmed more recently with a murine immature DC line DC2.4 treated with lipofectin OVA. Surprisingly, lipofectin did not initiate DC maturation (Okada et al., 2001). Another example illustrating the high capacity of cationic liposomes to induce robust immune response, was the administration of 344 Chikh and Schutze-Redelmeir vesicles containing cationic cholesterol derivative 3β[N-(N′,N′-dimethylaminoethane)-carbomyl] cholesterol in mice and macaques. Both CTL and antibodies were induced against inﬂuenza and helicobacter pylori infections, while neutral liposomes displayed virtually no stable antigen and no adjuvant effect (Guy et al., 1998; Guy et al., 2001). Poly(ethylene glycol)-Modiﬁed Liposomes Most types of conventional liposomes lack clinical applicability mainly because of the absence of stability of liposomes in ûiûo and the rapid uptake by mononuclear phagocyte system (MPS). Improving stability of the liposomes is one of the key elements for induction of efﬁcient T-cell mediated response. Poly(ethylene glycol) (PEG)-modiﬁed lipids are known to provide an effective steric barrier against surface-macromolecules interaction, circulation time of liposomes in ûiûo (Lasic et al., 1991; Ishiwata et al., 1995), and uptake by phagocytic cells (Ishiwata et al., 1998; Chikh et al., 2001b). Recent in ûiûo studies using sterically-stabilized liposomes (SL), termed as stealth liposomes showed a greater CD8+ T cell response when the antigen was given in SL liposomes than when given in soluble form or in conventional or positively charged liposomes (Ignatius et al., 2000). SL were shown to be internalized into neutral or mildly acidic compartments unrelated to endocytic vacuoles by both immature and mature DCs. These DCs were able to present SL-encapsulated OVA to both CD4C and CD8+ T cells in ûitro. Immunization of mice with SL-encapsulated OVA led to antigen presentation by DCs in ûiûo. Adjuvants Given in Combination with Liposomes Although liposomes are potent carrier systems for delivering antigens to APCs and clearly enhance their immunogenicity, additional features may be included for better adjuvanticity and therapeutic effect. Such features include antigen aggregation and ‘‘depot’’ at the site of administration, leading to slow release and to local inﬂammation co-delivery of other immuno-stimulating agents such as CpG, lipid A, cholera toxin or cytokines (for review, see Cox and Coulter, 1997). In this regard, it is important to report that a phase II clinical trial for non-small-cell lung cancer MUC-1 peptide-based vaccine BLP-25 is currently ongoing for the potential treatment of cancer. The introduction of interleukine 2 (IL-2) in liposomes reverses the T-cell suppression caused by MUC-1 mucin and enhances the cellular immune response (Morse, 2001). Amongst the other immuno-stimulatory agents under studies with liposomes are the CpG DNA sequences consisting of an unmethylated CpG dinucleotide mimicking natural ‘‘danger signals’’ usually provided by infectious agents. These motifs have been shown to trigger immune response by stimulating B cells, T cells, NK cells and phagocytic cells to secrete pro-inﬂammatory cytokines (Klinman et al., 1999 # 142; Bauer et al., 2001). Immunostimulatory CpG oligonucleotides (ODN) show promise as immune adjuvants and immunoprotective agents: by increasing the bioavailability and duration of action of CpG ODN following coencapsulation with antigen in sterically stabilized cationic liposomes improves their therapeutic utility. This was recently illustrated in the study of Gursel et al., 2001, Dendritic Cells and Liposomes 345 where cationic liposomes provided protection to CpG ODN from serum nucleases and facilitated uptake by B cells, DCs and macrophages. In a pathogen challenge model, these sterically stabilized liposomes encapsulation doubled the duration of CpG ODN-induced immune protection. Co-encapsulation of CpG ODN with protein antigen (OVA) magniﬁed the resultant antigen speciﬁc IFNγ and IgG responses by 15- to 40-fold compared with antigen plus CpG ODN alone. Flt3 ligand (Flt3 L), a ligand for the fms-like tyrosine kinase receptor has been shown to induce the differentiation, mobilization and in ûiûo expansion of DCs (Lynch 1998). Immunization with HGP-30 peptide vaccine (a synthetic 30-aminoacid peptide homologous to a conserved region of HIV-1 p17) induces strong Tcell responses and co-administration of the vaccine with Fl3t and or incorporation within liposomes improves the response (Pisarev et al., 2000). The efﬁcacy of an additional adjuvant with liposomes, such as lipid A has also been reported. Intracellular delivery of antigen containing CTL epitopes RTS,S (a particular malaria antigen consisting of hepatitis-B particles co-expressed with epitopes from the Plasmodium falciparum circumsporozoite protein) was achieved to the trans-Golgi apparatus of murine bone marrow-derived macrophages and resulted in markedly increased humoral and CTL responses in ûiûo in mice (Richards et al., 1998). The general use of lipid A has not been however clearly established for induction of CTL responses. Single modiﬁcation of HIV-1 env (312–327) and (302–335) by a lipidic aminoacid, (N-palmitoyl-L-Lysylamide) preferably in C-terminal region creating lipopeptides, has enhanced their immunogenicity and facilitated their encapsulation in liposomes (Deprez et al., 1996; Mora and Tam, 1998; Loing et al., 2000). Activation of APC via CD40-CD40 ligand pathway plays an important role in up-regulating co-stimulatory molecules such as B7 and production of IL–12: antiCD40 mAb and anti-CTLA-4 mAb function as immunomodulators that may be applicable to speciﬁc cancer immunotherapy with anti-tumor peptide vaccine. Recently, Ito et al., 2000 demonstrated that s.c. administration of OVA peptide encapsulated in multilamellar liposomes in presence of anti-CD40 mAb or antiCTLA-4 mAb generates enhanced CTL response against the antigen. In addition, administration of both anti-CD40 and anti-CTLA-4 mAb enhanced strongly the CTL response, and prolonged survival of mice upon immunization with pRL1a peptide, a tumor-associated antigen of RLmale symbol1 leukemia cells, was observed following RLmale symbol1 inoculation. A Novel Class of Liposomes: The Archaeosomes Novel lipid vesicles termed archaeosomes consisting of liposomes prepared with new lipid isolated from Archaea, microorganisms with unique polar lipid structures, have been developed very recently (Krishnan et al., 2000; Conlan et al., 2001; Krishnan et al., 2001). The distinct structures of archael lipids confer considerable stability to liposomes formulated from total polar lipids of different archaea. This includes enhanced stability against extreme pH, oxidation, elevated temperatures and action of lipases, that might be expected to impart superior properties to them for certain biotechnology applications compared with conventional liposomes. A 346 Chikh and Schutze-Redelmeir potent antigen-speciﬁc CD8+ T-cell response after immunization of mice with OVA entrapped in archaeosomes has been reported where administration of the same antigen with aluminium hydroxide or entrapped in conventional liposomes failed to induce signiﬁcant CTL response (Krishnan et al., 2001). APCs treated with archaeosomes exhibit increased TNF production and functional ability to stimulate allogenic T-cell proliferation. The exposure of J774A.1 macrophages to archaeosomes in ûitro has been shown to up-regulate B7.1, B7.2, and MCH class II molecules to an extent comparable to that achieved with LPS. Similarly, the incubation of bone marrowderived DCs with archaeosomes resulted in enhanced expression of MHC class II and B7.2 molecules. More interestingly, archaeosomes enhance APCs’ recruitment and activation in ûiûo. Overall, the activation of APCs correlated to the ability of archaeosomes to induce strong humoral, T helper, and CTL responses to entrapped antigen. Thus, the recruitment and activation of professional APCs by archaeosomes constitutes an efﬁcient self-adjuvanting’’ process for induction of antigen-speciﬁc responses (CTL, T helper and humoral responses) to entrapped antigen. Immunization of mice with these lipid vesicles containing a lipidic immunodominant epitope of listeriolysin lipidic has resulted in rapid and prolonged speciﬁc protective immunity against infection with Listeria monocytogenes (Conlan et al., 2001). In this regard, all of the tested archaeosomes were superior to conventional liposomes. Fusogenic Liposomes Another approach described to efﬁciently deliver the antigenic peptide to the cytosol is the utilization of fusogenic liposomes. Fusogenic liposomes based on Sendai virus were ﬁrst described for gene transfer into mammalian cells (Mizuguchi et al., 1996). They constitute a unique system that delivers their content efﬁciently into animal cells in ûitro and in ûiûo. Recently Nakanishi et al., 2000, have used the same procedure to prepare liposomes in order to avoid the classical endocytosis phagocytosis pathway but to allow fusion with the cell membrane and to deliver content directly into the cytoplasm of cells in a more efﬁcient manner. They demonstrated that fusogenic liposomes containing OVA can deliver the encapsulated soluble protein directly into the cytosol of cells and introduce it into the conventional MHC class I antigen-presentation pathway. In an immunization model these studies show that single administration with OVA encapsulated in fusogenic liposomes but not conventional liposomes resulted in potent priming of OVA-speciﬁc CTLs. A Novel Hybrid Delivery System: Antennapedia Vector Formulated in Liposomes We have described a novel approach to deliver deﬁned CTL epitopes into the cytosol of DCs (Chikh et al., 2001b). It involves the combination of liposomes and a recombinant peptide which has the ability to translocate through plasma membrane of a variety of cells. A peptide sequence referred to as antennapedia homeodomain (AntpHD) can effectively introduce CTL epitopes into the class I processing pathway and induce CTL in ûiûo but only in the presence of sodium dodecyl sulfate (SDS) as stabilizing factor (Schutze-Redelmeier et al., 1996). Further development of this technology has been limited because the recombinant peptide is very sensitive Dendritic Cells and Liposomes 347 to degradation in serum. Chikh et al., 2001b, have demonstrated that the encapsulation AntpHD recombinant protein in liposomes protects the peptide from degradation and importantly, the liposomes do not modify the function of AntpHDrecombinant peptide. Still, when in liposomes AntpHD recombinant protein can deliver a CTL epitope to the class I MHC pathway of DCs very effectively and sensitize target cells for CTL lysis. In contrast with the free AntpHD-recombinant protein which is directly delivered in the cytosol, AntpHD-recombinant protein encapsulated in liposomes is initially delivered in the endosomes of cells and subsequently the peptide enters the cytosol from the endosomes. We found that AntpHD-recombinant protein could induce CTL response in mice, response which was strongly enhanced when it was delivered by cationic liposomes (Chikh et al., 2001a). The crucial role of AntpHD at directing epitopes in the cytosol was demonstrated by the absence of response to the CTL peptide encapsulated in liposomes without AntpHD. One of the main advantages of using this hybrid delivery system: antennapedia vector formulated in liposomes, is the absence of toxicity of the peptide vector. Because of the high sequence homology of AntpHD with mammalian homeodomains of Hox 7 group (98%), no adverse immune response against AntpHD has been observed. Amongst the different proteins able to deliver macromolecules in the cytosol from the endosomes, we can cite the earlier work of Lee et al., 1996, demonstrating the efﬁcient delivery of macromolecules into the cytosolic space of macrophages from liposomes that contain listeriolysin O (LLO), the haemolytic protein of Listeria monocytogenes. LLO was puriﬁed and encapsulated inside pH-sensitive liposomes, along with OVA to be delivered. In ûitro delivery was signiﬁcantly better than that obtained by other currently available liposome formulations. Delivery of antigens in ûiûo had not been tested. Targeting APCs with Liposomes Current research is developing strategies to target speciﬁcally the DC system in ûiûo as an alternative approach to circumvent the infusion of ex ûiûo DC loaded with antigen used in the current clinic trials (Nestle et al., 1998) and (Tarte and Klein, 1999) for review. The recent identiﬁcation of surface receptors expressed by APCs allows for speciﬁc targeting of this cell population. Receptors such as FCγ RI (Serre et al., 1998), mannose (Sallusto et al., 1995), α Mβ2 integrin (CD11b CD18) (Guermonprez et al., 2001), CD36 and α vβ5 (receptor for engulfment of apoptotic cells) (Albert et al., 2000) are attractive candidates for surface molecules targeting. Through these receptors it has clearly been shown that internalization of antigens can be achieved efﬁciently and presented to T cells. It is believed that liposomal formulations can be designed to engender speciﬁc targeting to deﬁned cell types following localization, for example Fc-fragments of IgG antibodies directed against certain cell surface molecules can be coupled to liposomes facilitating speciﬁc interactions with the target cell (Serre et al., 1998; Machy et al., 2000). HEL (hen egg lysozyme)-containing liposomes targeted by an antibody to either surface Ig, Fc receptor or MHC class I and II molecules of DCs and B cells augmented the antigen presentation 1000 to 10,000-fold and 100 fold respectively, compared to free antigen or liposomes with no targeting feature. 348 Chikh and Schutze-Redelmeir Table 1. Overview of Pre-clinical Studies Evaluating Novel Lipid Based Delivery Vehicles Liposome formulation Antigen HIV-1 (env-2-3SF2) Immune response stronger Ab and CTL response in presence of MTP-PE and interleukine 7 than alum CTL in presence of lipid A CTL in presence of lipid A CD4C, CD8C T cell response in presence of liposomal IL2 strong CTL response in presence of CpG and T helper epitope strong and long lasting CTL response tumor delay, Th1 cytokines Ab and Th1 and Th2 immune response stronger CTL and Ab in comparison to neutral and anionic liposomes robust CTL and Ab in response in mice and macaques TILs proliferation following incubation of DCs loaded with TuLy in liposomes synergistic effect with Il2 anti-HIV-1 memory CTL responses in cells from HIV-1 infected subjects anti Cw3 CTL response adjuvant effect of CpG ODN CTL responses enhanced in the presence of antiCD40 and anti-CTLA-4 mAbs prolonged survival of mice Ref. (Bui et al. 1994) Neutral liposomes EPC C DMPC DMPG C DMPC DMPG C DMPC HIV peptide derived from V3 loop of gp120 Plasmodium falciparum (RTS,S) lymphoma Ig (White et al. 1995) (Richards et al. 1998) (Kwak et al. 1998) SPC C DL tocopherol LCMV (GP33, P13) peptides (Ludewig et al. 2000) pH sensitive liposomes POPE CHOH Hantaan NP (M6) MPL Human Papilloma Virus (E7) Cationic liposomes DC-Chol derivative EPC SA C (Chang et al. 2001) Hepatitis B virus (HbsAg) OVA (Brunel et al. 1999) (Nakanishi et al. 1997) (Guy et al. 2001) DC-Chol DOPE trivalent split inﬂuenza virus (HA, NA, NP peptides) kidney cancer (TuLy) tumor Ag from tumor lysate DDAP DOPC (Mulders et al. 1999) lipofectin HIV-1 Gag, Pol, and Env loaded on DCs AntpHD-Cw3 (Zheng et al. 1999) DOP DOTAP C (Chikh et al. 2001b) Anionic liposomes DOPS DOPC OVA peptide (257–264) pRL1a peptide of RL male symbol1 leukemia (Ito et al. 2000) Dendritic Cells and Liposomes Table 1. Continued. Liposome formulation Sterically stabilized liposomes DOPE PEG 349 Antigen Immune response Ref. Listeria monocytogenes survival of mice after infection in presence of CpG enhanced CD8C and CD4C T cell responses priming CTL response compared to no response with conventional liposomes strong humoral, T helper and CTL responses, up regulation of MHC class II, B7.2on DCs, DCs recruitment and activation in ûiûo rapid and prolonged speciﬁc immunity enhanced CTL responses with lipopeptide antigen accumulation of liposomes in lymph nodes (Gursel et al. 2001) (Ignatius et al. 2000) (Nakanishi et al. 2000) POPE PEG Fusogenic liposomes EPC DMPA C and Sendai virus OVA OVA Archaeosomes OVA (Krishnan et al. 2000) (Krishnan et al. 2001) Listeria monocytogenes (lipidic listeriolysin epitope) Targeted liposomes DPPC DPPG PEG-MPB containing HLA.DR targeting motif DPMC C DNP-cap PE targeted to Fcγ R of DCs (Conlan et al. 2001) none (Bestman-Smith et al. 2000) OVA efﬁcient presentation by DCs to CTLs in ûitro (Machy et al. 2000) Since DCs speciﬁcally recognize carbohydrates moieties of pathogens (saccharides, glycoproteins, lectins) and use these pattern recognition receptors to internalize large amounts of antigens (Sallusto et al., 1995), speciﬁc targeting of antigen-loaded liposomes to these receptors might increase the adjuvanticity of liposomal vaccines. Modiﬁcation of a liposomal HIV vaccine, containing an immunodominant peptide of the envelope glycoprotein gp120 of HIV-1 with different oligomannose residues, has induced a major histocompatibility complex class I-restricted CD8+ CTL response in mice after a single subcutaneous immunization, whereas non-coated liposomes did not (Fukasawa et al., 1998). Targeting to APCs with mannosylated liposomes has been reported elsewhere (Sasaki et al., 1997; Toda et al., 1997) and it is assumed that the beneﬁcial effect of mannose ligand can be attributed to a better delivery to DCs. 350 Chikh and Schutze-Redelmeir Using HLA-DR Fab′ fragments to target DCs represents an interesting strategy which has been recently tested, liposomes bearing anti-HLA-DR Fab′ fragments at the end termini of polyethyleneglycol chains have a level of accumulation into lymphoid organs which is enhanced after a single subcutaneous injection to mice compared to conventional immunoliposomes (Bestman-Smith et al., 2000). PERSPECTIVES Recent development in peptide-based vaccines formulated in liposomes indicate clearly that liposomal antigen delivery to DCs in ûiûo appears to be a promising approach to elicit strong T-cell mediated immune responses. It appears likely that such a delivery system together with speciﬁc targeting to DCs may be well suited to achieve protection and therapy against viral infections or malignancies. There is still a lot of work required to further study novel liposomal peptide-based vaccines and test their protective effect in cancer and viral diseases. It is believed that combination of liposome vaccines containing multiple tumoral- or viral-associated epitopes together with immunostimulatory agents will be needed. ACKNOWLEDGMENTS This research has been funded by a Canadian Institute of Health Research operating grant # MOP-36394 and the Cancer Research Society Inc. M-P SR is a Research Scholar of the British Columbia Health Research Foundation. REFERENCES Aiba, S. 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