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distemper with two different inactivated canine distemper virus (CDV) vaccines. Vaccine, 7, 521-526. Wahren, B., Nordlund, S., Akesson, A., Sundqvist, V.A.
& Morein, B. (1987), Monocyte and iscom enhancement of cell-mediated response to cytomegalovirus. Med. Microbial. Immunol., 176, 13-19. Weiss, H.P., Stitz, I. & Becht, H. (1990), Immunogenic properties of iscom prepared with influenza virus nucleoprotein. Arch. Viral., 114, 109-120. Watson, D.L., Liivgren, K., Watson, N.A., Fossum, C., Morein, B. & HGglund, S. (1989), The inflammatory
response and antigen localization following immuni-
zation with influenza virus iscoms. Inflammation, 13,
641-649. Winter, A.J., Rowe, G.E., Duncan, J.R., Eis, M.J., Widsom, J., Ganem, B. & Morein, B. (1988), Effectivenessof natural and synthetic complexes of porin and 0 polysaccaride as vaccines against Bruce/la abortus in mice. Infect. Immun., 56, 2808-2817. Yewdell, J.W. & Bennink, J.R. (1990), The binary logic of antigen processing and presentation to T cells. Cell, 62, 203-206.
as antigen carriers and adjuvants Buiting
(I), N. van Rooijen
(I) and E. Claassen
‘I) Dept. Celbiology, Med. Fat., Vrije Universiteit, POB 7161, 1007-MC, Amsterdam ‘2) Dept. Immunology and Med. Microbiology, Rijswijk (The Netherlands)
Liposomes are artificially prepared spheres composed of concentric phospholipid bilayers, separated by aqueous compartments. Hydrophobic interactions of the phospholipids are the driving force of formation of liposomes when phospholipids are confronted with water. Liposomes may differ in their dimension, composition (different phospholipid and cholesterol content), charge (resulting from the charges of the component phospholipids), and structure (multi- vs unilamellar). These physical properties and the location of antigens in liposomes determine the adjuvanticity of the liposome. There are a number of important advantages of liposomes when used as immunoadjuvant : they are biodegradable, nontoxic, immunologically inert, simple to prepare and their composition can be varied to obtain the most efficient antigen-liposome preparation. In combination with other immunoadjuvants, liposomes may induce an extremely strong immune response. The toxicity of certain antigens can be reduced by their incorporation into liposomes, and liposomes can be used to modulate the immune response (reviewed by Van Rooijen, 1990a). Liposomes have the ability to elicit both a cellular mediated immune response (CMI; Garcon and Six, 1991) and a hu-
moral immune response (HI) to antigens administered by a variety of routes, and there is a potential for selective interaction with populations of immunocompetent cells (Gregoriadis, 1990). In some cases, liposomes can be stronger adjuvants than complete Freund’s adjuvant (CFA; Gregoriadis and Manesis, 1980; Brynestad et al., 1990), but when the antigens are e.g. immunogenic proteins, the immune response is slightly higher when CFA is used as adjuvant (Sanchez et al., 1980; Alving et al., 1986). Inclusion of cholesterol or sphingomyelin in liposomes increases the stability of their phospholipid bilayers and decreases their susceptibility to destruction by various serum components (Scherphof et al., 1978 ; Kirby et al., 1980; Claassen et al., 1988). Cholesterol inclusion also influences the interaction of liposomes with lymphoid and non-lymphoid cells involved in immune reactions (Van Rooijen and Van Nieuwmegen, 1982). Immune antigens
reaction against and haptens
The use of liposomes as immunoadjuvants was first described by Allison and Gregoriadis (1974). Since than, many studies have confirmed this capability to enhance the immune response to various antigens (Van Rooijen, 1988). Interest centred on the
M = mouse; G = guinea pig; R = rat ; sA = secretory IgA.
S. sobrinus A. scopania
BSA Lipid A
12.5 pg 7.5; 15 pg
0.18 mg 5 Pi% 7.6 kg 9.3 Ia 10 w 25 118
10 PFU o-o.1 pg
ip im iv iv
10 I43 2-10 pg 0.2; 1; 5 pg
50 I% 75 w 25 c(g 25 pg 0.5; 2.0 pg
1w 5 mg lipid 5 mg lipid
G,, G,, G,,, G,, M
M M/G G,/M G G/M G/M
ip ip ip
ip b ip id
iv iv iv iv iv
I. sendai S. mutants
Diphtheria toxoid Cholera toxoid Cyclosporin Sporozoite
Glycolipid Lipid Lipid Virus Virus
Protein Peptide Protein Protein Protein Protein
(foreign) (foreign) (foreign) (foreign)
3-(p-azobenzene arsonate)-Nacetyl-L-tyrosyl glycylglycine DNP BSA BSA DNP BSA BSA BSA BSA Hepatitis B Hepatitis B LPS Lipid A Lipid A HSVI HSVl Tetanus toxoid
Table I. Characteristics
T cells 1981
El Guink et al., 1989 Jackson et al., 1990; Wachsmann et al., 1986 Gregory et al., 1986 Arora and Gangal, 1990
Gregoriadis and Allison, Alving et al., 1986
Lawman et al., 1981 Davis and Gregoriadis,
Beatty et al., 1980 Shek and Sabiston, 1982a,b Thtrien et al., 1990 ThCrien and Shahum, 1988 Sanchez et al., 1980 Manesis et al., 1979 Desiderio and Campbell, 1985 Yasuda et al., 1982 Naylor et al., 1982
Claassen et al., 1987
M M M M M M G G M M M
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possible applicability of liposomes as adjuvant to the immune system. Haptens as well as antigens (e.g. proteins) can be coupled to liposomes. Haptens are not able to elicit an immune response when given without a carrier. When haptens are coupled to liposomes, the immune response will be a thymusindependent IgM response, without the generation of immunological memory (Yasuda et al., 1977 ; Van Houte et al., 1979; Claassen et al., 1987). When antigen molecules are associated with liposomes, the immune response will be of the same type as when the antigen is given free in solution, but the response will be greatly enhanced. Liposome-associated antigens elicit a thymus-dependent immune response, including the production of both IgM and IgG antibodies and the generation of immunological memory (Van Rooijen, 1988). Immunity to antigens may be drastically improved through the administration of liposomes together with other adjuvants. For example, when liposomes were administered together with IL2, antibody responses were changed above or below levels attained with the liposomal antigen alone. Results depended on the amount and source of mediator given, its mode of presentation with the liposomal antigen and the IgG subclass tested (Tan and Gregoriadis, 1989; Davis and Gregoriadis, 1989). Lipid A influenced not only the adjuvanticity of liposomes (Alving et al., 1986) but could also switch a T-cellindependent antigen to a T-cell-dependent one (Zigterman et al., 1986); liposomes containing lipid A may also prevent the immunosuppression that is sometimes found after immunization with soluble antigens (Alving and Richards, 1990) (see also table I). Association
of antigens to liposomes
Antigens can be associated with liposomes in two ways. The antigenic determinant is (1) masked (encapsulated within the internal aqueous spaces of the liposomes) or (2) exposed (attached to the outer surface or reconstituted within the lipid bilayer of the liposomes; see Therien ef al., 1991). Surface exposition of antigens on the liposomes can be achieved by different methods of covalent binding of the antigen to the phospholipids used for preparation of the liposomes, or by non-specific adsorption of (partly) hydrophobic antigens (cf. Claassen and Van Rooijen, 1983). Hydrophilic antigens can be converted to hydrophobic (partly) antigens by their association with small hydrophobic molecules such as fatty acids. If the coupling of antigen and phospholipid is preformed before the preparation of the liposomes, the association of antigens with the liposomal bilayers will result. Liposomes can be taken up by non-receptormediated endocytosis, Fc-mediated endocytosis,
phagocytosis, or complement-dependent phagocytosis (Wassef and Alving, 1987). It is also known that liposomes can be coated with many other plasma proteins, and some of these proteins might have opsonic activities (Bonte and Juliano, 1986). Protein antigens that are either encapsulated in or conjugated to liposomes incubated in mixed lymphocyte cultures, in which macrophages are also present, are presented to T helper cells (Alving and Richards, 1983; Burakoff et al., 1984). Antigen presentation to T cells is probably improved by the increased internalization of hydrophobic antigen by macrophages. When antigen is covalently coupled to the surface of the liposomes rather than being encapsulated within the vesicles, the antigen can be presented, although inefficiently, by B cells (Dal Monte and Szoka, 1989b).
Role of macrophages in the processing of liposomes The substantial enhancement of the humoral immune response to a liposome-associated T celldependent antigen (the liposomal adjuvant effect) has raised considerable interest in the use of liposomeencapsulated antigens as vaccines (AIving, 1987 ; Van Rooijen, 1990a). Since liposomes are avidly phagocytosed by macrophages, it has been proposed that macrophages play a central role in the processing and presentation of liposome-encapsulated (masked) antigens. Indeed, methods for depletion of macrophages in vivo or in vitro (resp. : Claassen et al., 1987 ; Claassen et al., 1990) have shown that macrophages are essential for the liposomal adjuvant effect. Intraperitoneal injection of carageenan in mice before antigen administration suppresses macrophage functions, and plaque-forming cell responses to liposome-encapsulated albumin were strongly decreased (Shek and Lubovich, 1982). Mice were intravenously treated with liposome-encapsulated dichloromethylene diphosphonate to deplete the splenic macrophages (but not lymphocytes and dendritic cells, Van Rooijen et al., 1988). Such pretreatment suppressed the primary immune response to a subsequent injection of liposomal albumin antigen (Claassen et al., 1987; Su and Van Rooijen, 1989) and other TI-2 antigens (Claassen et al., 1986). Reappearance of the splenic macrophages was accompanied by a simultaneous reappearance of the immune response to the liposomal albumin (Delemarre, 1990; Van Rooijen, 1992). An enhanced immune response to albumin was obtained after injection into mice of macrophages, that had previously ingested liposomeencapsulated albumin in vitro (Beatty et al., 1984). Finally, in vitro antigen presentation experiments have shown that macrophages are necessary and sufficient for antigen presentation of liposomeencapsulated antigens to T cells whereas B cells are
incapable of presenting a liposome-encapsulated antigen (Dal Monte and Szoka, 1989a; Harding et al., 1991). Based on the current status of research in this area, one could predict that in vivo processing would predominantly involve uptake, ingestion and fragmentation of liposomes by macrophages, with less common or less important contributions to the initiation of the immune response by other cell types such as B lymphocytes or dendritic cells (Claassen, 1992a,b). However it may well be that macrophages transfer the (pre)processed antigens to B cells or dendritic cells for further processing and final presentation to T cells, as suggested before (Van Rooijen, 1990b). It is possible that antigens which are bound to the liposomal membrane are processed and presented by nonphagocytic cells. Under circumstances of high antigen epitope density on the liposome surface, presentation of liposomal antigen may not necessarily be MHC-restricted (Walden et al., 1986; Dal Monte and Szoka, 1989b). Interactions of liposomes with cells in vivo
Liposomes containing both antigenic epitopes (peptides) and major histocompatibility gene complex molecules can stimulate T lymphocytes. Cytotoxic T lymphocytes (CTL) use class I MHC molecules as restriction elements, T helper cells see antigens in association with class II MHC molecules (Schwartz, 1984). In the case of CD4+ T lymphocytes, dendritic cells can deliver all signals required for complete stimulation, as can macrophages and (activated) B cells. The function of CTL also depends on the presence of specialized accessory cells. It has been shown that these accessory cells can behave like scavenger cells (Debrick et al., 1991). The accessory cells use foreign antigen, in the form of cellular debris, as immunogen. These cells are also crucial for CTL induction because in vivo depletion of phagocytic cells completely inhibits CTL responses. In these animals, CTL activity could be restored by transfer of macrophages. All of the reappearing CTL used MHC restriction elements expressed by the infused macrophages. It was suggested that a cognate interaction between macrophages and CTL precursors initiates class I MHC-restricted immune responses. Activated T cells do not have to interact with viable accessory cells (Watts et al., 1984; Haskins et al., 1983; Staerz et al., 1984). Resting T cells are not stimulated unless they encounter Ag on the surface of appropriate antigen-presenting cells (Staerz and Bevan, 1986). Evidence has been given that macrophages act as accessory cells for CD8+ CTL during in vivo priming (Debrick et al., 1991). The authors also showed that a cognate interaction between ma-
ture T cells and macrophages initiates class I MHCrestricted responses. Targeting of Ag via liposomes in vivo (tissue)
Parenterally injected liposomes are rapidly ingested by macrophages, particularly in the liver and spleen, where they are degraded in lysosomal vacuoles (Segal et al., 1974; Claassen, 1992b). The distribution of intravenously administered liposomes in the body is strongly influenced by the liposomal characteristics (Gregoriadis, 1988), which can now be studied with a new carbocyanin-based labelling technique (Claassen, 1992a). The distribution of liposomes between liver and spleen depends on their cholesterol content. Cholesterol-poor liposomes were mainly taken up by Kupffer cells and cholesterol-rich liposomes by spleen macrophages. It has been postulated that cholesterol influences the binding of serum opsonins on the liposomes. This may in turn influence the uptake by macrophages (Moghimi and Patel, 1988). Uptake of liposomes in the spleen can also be manipulated by liposomal size and by the inclusion of ganglioside GM1 in the lipid composition of liposomes (Liu et al., 1991). Lysozyme entrapped in the splenotropic liposomes composed of PC/chol/GMl (elevated accumulation in the red pulp after i.v. injection) showed higher efficiency in potentiating the humoral response than that of either free lysozyme or lysozyme entrapped in hepatotropic liposomes composed of PC/chol (Liu et al., 1992). So, antigen delivery by liposomes to the splenic macrophage instead of the liver Kupffer cells is important in the liposomal adjuvanticity. These higher concentrations of antigen in the spleen are supposed to enhance the possibility of uptake, processing and presentation of the antigen by antigen-presenting cells, resulting in an elevated level of antibody production in the immunized animal. In our own studies, we found no marked differences in the distribution patterns in the murine spleen of intravenously injected liposomes with different membrane compositions. The level of cholesterol and charge of the bilayers of the liposomes were varied as well as the antigens/haptens that were coupled to the liposomes (Buiting et al., 1992). A more specific targeting of liposomes might be achieved by coupling of (monoclonal) antibodies to the surface of the liposomes (Connor et al., 1985), although we have found that targeting of antibody-enzyme conjugates was only possible when using monomeric antigenenzymatic conjugates (Van Rooijen et al., 1992). Evidence has been provided that synthetic muramyl dipeptide, liposomes and recombinant Gram-negative bacteria, exhibit adjuvant activity when given by oral route (reviewed by Mestecky,
USE OF NE W-GENERA
1987). Local secretory IgA antibodies and the serum antibodies were induced (Michalek ef al., 1984; Pierce et al., 1984; Gregory et al., 1986; Ogawa et al., 1986; Wachsmann et al., 1986; Jackson et al., 1990 ; Alving et al., 1986). The increase in secretory IgA is probably the result of vesicle interaction with the gut lymphoid tissue (Gregoriadis, 1988). Recently, a liposomal vaccine for use in humans has been developed (Alving and Richards, 1990). There are indications that it may be considered a safe vaccine which induces extremely high levels of antibody against a poorly immunogenic antigen (Alving et al., 1992). Intracellular
targeting of liposomes
Complexes of antigens and class I MHC glycoproteins can either be generated from the degradation of endogenously synthesized proteins or result from exposure of the antigen-presenting cells (APC) to exogenous peptides (reviewed by Townsend and Bodmer, 1989). Peptides in the cytosol of APC enter the endoplasmic reticulum and complex with newly synthesized class I proteins, which are then processed and transported to the cell surface via the golgi apparatus (Nuchtern et al., 1989; Cox et al., 1990). Exogenously supplied proteins are usually taken up and processed in the endosomes and the peptides generated complex with class II, but not usually with class I MHC proteins (Germain, 1986; Bevan, 1987). pH-sensitive liposomes have been developed that destabilize and become fusion-active at mildly acidic pH (Ellens et al., 1984; Straubinger et al., 1985; Connor et al., 1984; Collins et al., 1989). Significantly increased cytosolic levels of the entrapped contents have been observed when using these liposomes, as compared to contents delivered in conventional pHinsensitive liposomes (Connor and Huang, 1985). The mechanism of the enhanced delivery is probably related to the fusion of liposome membranes with the endosome membrane in response to the acidification of the endosomes (Collins ef al., 1990). References Allison, A.C. & Gregoriadis, G. (1974), Liposomes as immunological adjuvants. Nature (Lond.), 252, 252. Alving, C.R. (1987), Liposomes as carriers for vaccines, in “Liposomes from biophysics to therapeutics” (M.J. Ostro) (pp. 195218). Marcel Dekker, Inc., New York. Alving, C.R. & Richards, R.L. (1983), Immunologic aspects of liposomes, in “Liposomes” (M.J. Ostro) (pp. 209-287). Marcel Dekker, Inc., New York. Alving, C.R. & Richards, R.L. (1990), Liposomes containing lipid A : a potent nontoxic adjuvant for a human malaria sporozoite vaccine. Immunol. Letters, 25, 275-279.
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as novel adjuvants
W.G. Bessler (I) and G. Jung c2) I’) Institut fiir Immunbiologie der Universitiit, 07800 Freiburg (Germany) and “j Institut fir Organ&he Chemie der Universitiit, 07400 Tiibingen (Germany)
Lipopeptides constitute potent novel immunoadjuvants in mice, rabbits and other species, enhancing markedly the immune response when given in mixture with antigens. Lipopeptides are non-toxic, non-pyrogenic and do not induce tissue damage when injected. They are thus well suited to replace Freund’s adjuvant avoiding its side effects; the antibody titres obtained using lipopeptide analogues are in most cases comparable to the titres obtained by administering Freund’s adjuvant. Lipopeptides also improve the efficiency of vaccines, which is important in decreasing the amount of vaccine required. Lipopeptides covalently coupled to low molecular weight haptens, e.g. peptides or toxins, are able to elicit high antigenspecific antibody titres in mice and rabbits. Conjugates containing B or T helper cell epitopes constitute novel synthetic vaccines which protect against viral infections by inducing virus-specific antibodies. When coupled to CTL epitopes, the conjugates are able to induce cy-totoxic T lymphocytes in vivo which eliminate virus-infected cells. Thus, due to their efficacy and their lack of side effects, these novel lipopeptide adjuvants provide a substitute for many conventional adjuvants.
Immunoadjuvants are substances enhancing the antigen-specific humoral or cellular immune response when given together with antigen. They are expected to show high effectivity and to lack side effects such as the induction of autoimmune or allergic reactions, cytotoxicity or tumour growth. Many immunoadjuvants consist of two components: one component acts as a deposit substance to protect the antigen from degradation. The most commonly used deposit substances are - in the human system - aluminum compounds (phosphate or hydroxide), and - in animal systems - Freund’s incomplete adjuvant in which the antigen is incorporated into the aqueous phase of a stabilized water in paraffin oil emulsion. Alternative deposit systems include liposomes or synthetic surfactants. The second component, present in many effective adjuvants, consists of substances of mostly bacterial origin which stimulate the humoral or cellular immune response, or which activate monocytes/macrophages. These stimulants either consist of killed bacteria or fragments thereof, or of defined constituents of the bacterial cell wall like muramyl dipeptide (MDP), lipopolysaccharide (LPS), or lipid A analogues. In