- Email: [email protected]
[6] Use of liposomes as biodegradable and harmless adjuvants
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matography of QUA tumor extrct on Sephadex G-200 columns and subsequent precipitin analysis of each fraction as described) Conclusions The methodology described herein has resulted in the preparation of large quantities of apparently monospecific antiserum to 7-FA. Compared to the original anti-3,-FA serum, polyclonal antiserum to y-FA immune complexes had a greater specificity (since it did not require absorption) and higher titer of reactivity to the serologically and physicochemically defined antigen y-FA. The availability of this serum will provide the basis for future directions in y-FA research and is currently being utilized for the affinity purification of 7-FA for subsequent RIA development. It is anticipated that the use of more sensitive methods for 7-FA detection may necessitate a reevaluation of current ideas, particularly in regard to presumptive 7-FA-negative cancers (e.g., certain leukemias and tumors of neural ectoderm origin7). The present data indicate that antigenically stable agarose-immobilized immune complexes can be produced using small volumes of primary precipitating antibody. After processing, such complexes appear to act (at least for y-FA) as suitable immunogens leading to the formation of large quantities of a more highly specific antiserum. This technique is of particular relevance to situations where it is not possible or desirable either to process solution-precipitated complexes or to dissociate the antigen-antibody complex prior to immunization. Acknowledgments This work was supported by Grant 5 R23 CA25285-02from thc National Cancer Institute.
[6] U s e o f L i p o s o m e s as B i o d e g r a d a b l e a n d Harmless Adjuvants
By Nico VAN ROOIJEN and RIA VAN NIEUWMEGEN Introduction to Liposomes
Liposomes are artificially prepared spheres of concentric phospholipid bilayers separated by aqueous compartments. They form when waterinsoluble phospholipids are confronted with water. The phospholipid molMETHODS IN ENZYMOLOGY, VOL, 93
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181993-0
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E
group ~ =,= Hydrophilic Hydrophobic fatty acid chains X m Aqueous
compartments
FIG. 1. Schematic representation of a phosphatidylcholine liposome with entrapped aqueous compartments.
ecules try to reach a conformation in which their hydrophobic fatty acid groups are not in direct contact with water. The formation of phospholipid bilayers, in which the relatively hydrophilic head groups are localized on both outer parts of the bilayers, and the hydrophobi~ fatty acid groups are localized directly opposite to each other in the inner part of the bilayer, is a logical consequence (Fig. 1). Liposomes may differ with respect to their dimensions, composition (different phospholipids), charge (neutral, positive, or negative liposomes), and structure (multilamellar and unilamellar liposomes). Liposome-entrapped or associated compounds may be targeted to different sites in or on living cells, and their application in biomedical research increases continuously. 1,2 Immunology is one of the research areas in which liposomes are frequently applied. 3,4 Their use as an adjuvant in immune reactions against various antigens is one of the most promising immunological applications of liposomes. 5 G. Gregoriadis, in "Liposomes in Biological Systems" (G. Gregoriadis and A. C. Allison, eds.), p. 25. Wiley, New York, 1980. 2 B. E. Ryman and D. A. Tyrrell, Essays Biochem. 18, 49 (1980). 3 B. H. Tom, in "Liposomes and Immunobiology" (B. H. Tom and H. R. Six, eds.), p. 3. Elsevier, Amsterdam, 1980. 4 C. R. Alving and R. L. Richards, in "The Liposomes" (M. Ostro, ed.), Marcel Dekker, New York (in press). 5 N. van Rooijen and R. van Nieuwmegen, in "Targeting of Drugs", (G. Gregoriadis, ed.), NATO Adv. Study Inst. Ser., Set. A 47, 301 (1982).
[6]
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=
85
Phosphatidylcholine molecules
• = Antigen
molecules Masked
Black parts:
Open p a r t s : U n m a s k e d
FIG. 2. Schematic representation of a part of a phosphatidylcholine liposome with exposed and masked antigen molecules.
Immune Responses against Antigen Exposed on the Surfaces of Liposomes and Entrapped Antigen When liposomes are prepared in an aqueous solution of antigen, the antigen may be entrapped in the aqueous compartments of the liposomes, and it may be associated with the phospholipid bilayers themselves. If antigen is bound to the phospholipids of the liposomes, part of the antigen probably will be exposed on the outer surface of the liposomes. This may be recognized by lymphocytes with surface receptors for that antigen, and an immune response can be initiated. When antigen is entrapped in the aqueous compartments of the liposomes without any association with the phospholipid bilayers, recognition by antigen-specific lymphocytes is impossible, since the antigen is completely masked (Fig. 2). In this case macrophages are required for processing of the liposome entrapped antigen, since no other way than lysosomal digestion of the liposomal membranes seems to exist for unmasking the antigen. 6 We have studied whether exposure of the antigens on the surfaces of liposomes, entrapment of the antigens in their aqueous compartments, or both surface-exposed and entrapped antigen are responsible for the adjuvant activity of liposomes in the immune response against associated antigen. 5,7 In these experiments we compared the immune response against antigen administered free in solution, coated on the surfaces of empty liposomes, or both coated on and entrapped in liposomes. For reasons mentioned before 5 we were not able to incorporate in the experiments a group of animals injected with antigen, given completely entrapped in liposomes without any surface interaction. 6 N. van Rooijen and R. van Nieuwmegen, Immunol. Commun. 8, 381 (1979). 7 N. van Rooijen and R. van Nieuwmegen, Cell. Immunol. 49, 402 (1980).
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Detailed information about the association of antigen with liposomes will be given later in this chapter. The results of the experiments showed a strong adjuvant effect of liposomes both in the immune responses against the antigens human serum albumin (HSA) and bovine y-globulin (BGG) in rabbits. The immune response elicited by empty liposomes coated with the antigens after their preparation was a little stronger than that elicited by liposomes that had in addition to their (relatively low amount of) surface-exposed antigen also (a relatively high amount of) antigen entrapped in their inner compartments. Clearly, entrapped antigen did not contribute to the immune response, in spite of the fact that the bulk of the liposome-associated antigen had been entrapped. The finding that entrapped antigen seemed to inhibit the immune response against liposomeassociated HSA or BGG could be explained by assuming that antigen in the liposomes, especially the part that was fixed to the inner half of the outer phospholipid bilayer, hindered binding of the antigen to the outer surfaces of the liposomes. Unlike albumin or y-globulin antigens, 5,7 nonspecifically associated horseradish peroxidase (HRP) appeared not to elicit a primary immune response after intravenous injection in rabbits. It has been shown that a primary anti-HRP antibody response could be elicited when HRP covalently attached to the liposomes was injected. Nonspecific association of the antigen HRP with the liposomes occurs to a very low degree, contrary to albumin and y-globulin antigens. Nonspecific HRP association with liposomes takes place, as concluded from the finding that rabbits injected with such HRP-liposomes appeared to be primed for a secondary response against the antigen, but it occurred to such a small extent that no exact determination of the amount of liposome-associated HRP could be performed. 8 We also compared the priming effect of empty liposomes nonspecifically associated with HRP and liposomes with HRP both entrapped and nonspecifically associated with their surfaces. It appeared that entrapped HRP clearly contributed to the establishment of immunological memory, since higher peak titers were reached and a higher percentage of rabbits appeared to be primed, when HRP had been entrapped in the injected liposomes. 8 Although this finding seems to contradict our earlier results that entrapped HSA or BGG does not contribute to the adjuvant effect of liposomes with both entrapped and surface-associated albumin or 3'globulin antigens, there is probably no real discrepancy between these results. When antigen can be nonspecifically bound to liposomes to a sufficient degree, as found with albumin and y-globulin antigens: ,7 entrapped antigen does not contribute to the adjuvant effect of liposomes. s N. van Rooijen, R. v a n N i e u w m e g e n , N. Kors, in preparation.
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However, when only an extremely small amount of antigen is bound to the surfaces of liposomes, as found with HRP, entrapped antigen can markedly contribute to the adjuvant effect. In this case we must assume that macrophages play a, role in the releasing and processing of the liposome-entrapped antigen, as we suggested earlier. 6 It has been shown that a small part of the antigen, ingested by macrophages, is prevented from complete digestion by lysosomal enzymes, and it has been suggested that this macrophage-associated antigen is particularly immunogenic. 9 Summarizing the results obtained until now, we conclude that the adjuvant effect of liposomes is primarily dependent on antigen presentation, and it is not unlikely that liposomes can replace macrophages with respect to this antigen presentation. However if antigen is not exposed on the surfaces of liposomes, either nonspecifically coated on or covalently attached to the phospholipid bilayers, entrapped antigen may initiate an immune response after ingestion of the liposomes by macrophages, digestion of the liposomal membranes, and unmasking the antigenic determinants. Although the mechanism by which liposomes exert their adjuvant effect is relatively unimportant for various practical applications, it may be important for application of liposomes as adjuvants in more fundamental immunological studies. Preparation of Liposomes Multilamellar liposomes can be prepared from one single phospholipid, e.g., phosphatidylcholine (lecithin), or from mixtures of different lipids. By adding negatively charged phosphatidic acid or dicetylphosphate or positively charged stearylamine to the mixtures, the charge of the resulting liposomes can be influenced correspondingly. For a comparative study on adjuvant activity of various liposomes,~° we prepared neutral liposomes composed of phosphatidylcholine only, positive liposomes composed of'phosphatidylcholine and stearylamine, and negative liposomes composed of phosphatidylcholine, phosphatidic acid, and cholesterol. All lipids were obtained from Sigma Chemical Company, St. Louis, Missouri: 150 mg of phosphatidylcholine, 150 mg of phosphatidylcholine, and 6 mg of stearylamine, or 150 mg of phosphatidylcholine, 22 mg of cholesterol, and 21.2 mg of phosphatidic acid, were dissolved in 25 ml of chloroform in a round-bottom flask. The thin film that formed on the walls of the flask after rotary evaporation at 37° was dispersed by gentle shaking for 10 min in 20 ml of 10 mM sodium phosphate buffer for 2 hr, sonicated 9 G. Weissmannand P. Dukor, Adv. Immunol. 12, 283 (1970). ~0N. van Rooijenand R. van Nieuwmegen,Immunol. Commun. 9, 243 (1980).
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for 3 min at 20 ° in a Sonicor (50 Hz), and kept at room temperature for another 2 hr. The liposomes were centrifuged at 17,500 g for 30 min in a Beckman J-21 Centrifuge. The pellet containing the liposomal fraction was washed in PBS and finally resuspended in 8 ml of PBS. Another way to prepare multilamellar liposomes is by the ether evaporation method. Ether solutions of phospholipids or phospholipid mixtures are injected in warm aqueous solutions. After vaporization of the ether, liposomes are formed. When vacuum is drawn over the aqueous solution rapid vaporization of the ether may be obtained at a lower temperature in order to prevent degradation of temperature-sensitive molecules. 11 Several lipids may be combined for preparation of liposomes. 1,12Phosphatidylethanolamine may be an important component of liposomes, since this phospholipid contains a free amino group, which can be used for covalent attachment of antigens. Using combinations of lipids that have shown their use for preparation of liposomes, 1 it is not necessary to check the formed liposomal structures. The adjuvant activities of the three types of liposomes, of which the compositions have been mentioned above are shown in the table (with respect to the immune response against HSA). In order to check the character of liposomes prepared from previously uninvestigated lipid mixtures or their dimensions, liposomes may be stained with fluorochromes and studied with the fluorescence micros c o p e . 13 Staining can be performed simply by incubation of the multilamellar liposomes in a 1% aqueous solution of the fluorochromes acridine orange or eosin Y for several minutes. They have to be centrifuged and washed afterward, and after resuspending the last pellet, a drop can be observed under the fluorescence microscope (1000x magnification). Entrapment of Antigens in Liposomes For entrapment of antigens in the aqueous compartments of liposomes, the antigens must be dissolved in the PBS in which the phospholipid film, formed on the walls of the round-bottom flask after rotary evaporation, was dispersed (see preparation of liposomes). Organic solvent vaporization methods for preparation of liposomes cannot be recommended for liposomal entrapment of antigen, since the organic solvents may alter the (protein) antigens. Although entrapment of antigen may be required when the antigen shows only a very low degree of nonspecific binding to the outer surfaces of liposomes, and methods for covalent t1 D. Deamer and A. D. Bangham, Biochim. Biophys. Acta 443, 629 (1976). 12 R. E. Pagano and J. N. Weinstein, Annu. Rev. Biophys. Bioeng. 7, 435 (1978). z3 N. van Rooijen and R. van Nieuwmegen, Stain Technol. 43, 307 (1978).
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attachment of the antigen to liposomes are lacking, the adjuvant effect of liposomes is particularly useful when sufficient exposure of the antigens on the surfaces of liposomes can be obtained. Nonspecific Association of Antigens with Surfaces of Liposomes Antigens, which can be coated on the surfaces of liposomes after their preparation, must be dissolved in PBS. The " e m p t y " liposomes are suspended in this antigen solution. Since, depending on the nature of the antigen, a high concentration of it may be required, the total volume of the suspension must be kept as small as possible. This is particularly important in cases where large amounts of antigen are too expensive or difficult to prepare. Whereas albumin antigen concentrations of 25 mg/ml were used for preparation of antigen-associated liposomes in most of our studies, a concentration of 250/~g/ml still appeared to be sufficient to demonstrate the adjuvant effect of liposomes. 5 After preparation of liposomeassociated antigens, the liposomes can be washed in order to remove free antigen. This will be necessary only when there is concern that free antigen may inhibit the adjuvant effect of the liposome-associated antigen, although there are no data available on a possible inhibition of the adjuvant effect of liposomes by free antigen. For practical purposes, the same antigen solution may be used several times for preparation of antigenassociated liposomes when only a small fraction of the antigen is bound to the surfaces of liposomes, as we found with albumin and 3J-globulin antigens.5,7,10 Another way to prepare liposomes with antigen nonspecifically associated with their surfaces is to dissolve the antigens in the PBS in which the phospholipid film has been dispersed, i.e., the same method as that recommended for entrapment of antigens in liposomes. Liposomes prepared in this way may have the antigen both entrapped in their aqueous compartments and nonspecifically associated with their surfaces. When sonication of the antigen-liposome mixture is necessary for binding immunoglobulin G (used as an antigen) to liposomes, as suggested by Huang and Kennel, ]4 the latter method is required in order to get sufficient antigen-liposome association. Alteration of Originally Hydrophilic Proteins to Hydrophobic Proteins and Their Incorporation in Liposomal Membranes It is not surprising that hydrophobic proteins show more tendency to bind to liposomes than hydrophilic proteins. Interaction between 3,-globu14 L. Huang and S. J. Kennel, Biochemistry 18, 1702 (1979).
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lins and liposomes may well depend on a change of their conformation that allows a hydrophobic binding, as suggested by Vanderbranden et al.~5 Evidence was given that the immunoglobulin region interacting with the lipid acyl chains is located in the heavy chain. Koelsch et a l ) 6 reported on the chemical modification of originally hydrophilic proteins to hydrophobic proteins in order to incorporate these in liposomal membranes. The proteins were modified by acylation with fatty acid chlorides. " H y d r o p h o b i z a t i o n " of protein antigens as a general method to enhance their binding to liposomes seems to offer a promising approach. Several studies are available on the use of lipid-conjugated albumin antigens, 17-2°and such lipid-conjugated proteins may be expected to show an enhanced affinity for liposomal membranes. Covalent Attachment of Antigens to Liposomes When nonspecific interaction of antigens with liposomes does not result in a sufficient binding of antigen to the liposomes, the antigen may be attached to the liposomes covalently in order to get a strong adjuvant effect of liposomes. As mentioned before, covalent attachment of H R P to the outer surfaces of liposomes results in a H R P - l i p o s o m e complex, which, after intravenous injection in rabbits, evokes a primary immune response against HRP. 8 Nonspecifically liposome-associated and/or liposome-entrapped H R P appeared not to elicit a primary immune response, but priming for a secondary response occurred. 2~ HRP, injected free in solution, neither elicited a primary immune response, nor primed for a secondary response in most animals. Apart from the method for covalent attachment of H R P to phosphatidylethanolamine as a component of liposomes suggested by Heath et al., 22 covalent attachment of immunoglobulins to liposomes via glycosphingolipids has been described. 23 Immunoglobulins may also be coupled with fatty acids covalently, followed by incorporation of these amphipathic antibodies in liposomal membranes 15M. Vanderbranden, J. L. de Coen, R. Jeener, L. Kanarck, and J. M. Ruyschaert, Mol. lmmunol. 18, 621 (1981). 16R. Koelsch, J. Lasch, A. L. Klibanov, and V. P. Torchilin, Acta Biol. Med. Germ. 40, 331 (1981). 17M. O. Dailey and R. L. Hunter, J. Immunol. 118, 957 (1977). is S. B. Singh and S. Leskowitz, J. lmmunol. 120, 734 (1978). 19G. Drach and J. Chen-Marotel, Int. Arch. Allergy Appl. lmmunol. 59, 28 (1979). 2oj. M. Stark, N. Matthews, and J. Locke, Immunology 39, 353 (1980). 2~N. van Rooijen, R. van Nieuwmegen, and N. Kors, lmmunol. Commun. 10, 59 (1981). 22T. D. Heath, D. Robertson, M. S. C. Birbeck, and A. J. S. Davies, Biochim. Biophys. Acta 599, 42 (1980). 23T. D. Heath, B. A. Macher, and D. Papahadjopoulos, Biochim. Biophys. Acta 640, 66 (1981).
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by a detergent-dialysis method. 24 Another method for covalent coupling of a soluble proteins to liposomes uses the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl-3-(2-pyridyldithio)propionate (SPDP, Pharmacia) as suggested by L e s e r m a n e t al. 25 Finally, antibody can be covalently coupled to liposomes by a glutaraldehyde reaction. 26 Attachment of Antigen to Liposomes via an Inserted Receptor Alving e t al. studied the immune r e s p o n s e against liposome-associated cholera toxin. A t t a c h m e n t of cholera toxin to the liposomes was performed by incorporation of a receptor, i.e., ganglioside GMI in the liposomal bilayers. 27,28 Incorporation of a receptor molecule into liposomal bilayers in order to attach an antigen to their surfaces is another form of association of antigens with liposomes. Methods to Enhance the I m m u n o a d j u v a n t Activity of Liposomes The relative p o t e n c y o f various adjuvants differs markedly according to the antigen used. 29 The adjuvant effect of liposomes appears to depend primarily on antigen presentation, as we discussed earlier. N o stimulation effect of liposomes t h e m s e l v e s on proliferation of l y m p h o c y t e s has been observed. On the contrary, they rather inhibit the proliferation of lymphocytes as measured by [3H]thymidine incorporation into cellular D N A . 3°-32 The fact that different adjuvants enhance the immune response by different effects suggests that the adjuvant effect of liposomes obtained by antigen presentation, m a y be enhanced by combination with other adjuvants. Endotoxin and endotoxin-derived c o m p o u n d lipid A m a y enhance the adjuvant effect of liposomes against liposome-associated antigens. 27,2s,33 When a weak albumin antigen and endotoxin were associated with the 24A. Huang, L. Huang, and S. J. Kennel, J. Biol. Chem. 255, 8015 (1980). L. D. Leserman, J. Barbet, and F. Kourilsky, Nature (London) 288, 602 (1980). 26V. P. Torchilin, B. A. Khaw, V. N. Smirnov, and E. Haber, Bioehem. Biophys. Res. Commun. 89, lll4 (1979). 27C. R. Alving, B. Banerji, J. D. Clements, and R. L. Richards, in "Liposomes and Immunobiology" (B. H. Tom and H. R. Six, eds.), p. 67. Elsevier, Amsterdam, 1980. 2s C. R. Alving, B. Banerji, T. Shiba, S. Kotani, J. D. Clements, and R. L. Richards, Prog. Clin. Biol. Res. 47, 339 (1980). 29R. Bomford, Clin. Exp. lmmunol. 39, 426 (1980). 3oS. Shi-Hua Chen and R. M. Keenan, Biochem. Biophys. Res. Commun. 79, 852 (1977). 3~B. Rivnay, A. Globerson, and M. Shinitzky, Eur. J. lmmunol. 8, 185 (1978). 3zM. H. Ng, W. S. Ng, W. K. K. Ho, K. P. Fung, and J. P. Lamelin, Exp. Cell Res. 116, 387 (1978). 33N. van Rooijen and R. van Nieuwmegen, lmmunol. Commun. 9, 757 (1980).
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same liposomes, antibody production in rabbits could be detected within 3 days after injection of the antigen-liposome-endotoxin preparation. When antigen and endotoxin were associated with different liposomes and these antigen-liposome and endotoxin-liposome preparations were injected simultaneously, the adjuvant effect of the liposomes was enhanced also, but it took 1 day longer before antibodies could be detected, whereas peak titers were not markedly different. 33 However, it is not unlikely that antigen-containing liposomes and endotoxin-containing lip0somes had been aggregated or fused in v i v o resulting in liposomes containing both endotoxin and antigen, since endotoxin may form bridges between different liposomes. The structure of liposomes is not markedly affected by endotoxin or lipid A, since these compounds do not increase their permeability to divalent anions 34or nonionic substances. 35 However, injection of liposomes containing lipid A may provoke an immune response not only against the associated antigen, but also against lipid A and the phospholipids of which the liposomes are composed) 9 Other lipid substances that might be incorporated into liposomes to enhance the immunogenicity of liposome-associated antigens include acylated derivatives of muramyl dipeptide. 36 A very powerful adjuvant results if antigen containing liposomes are combined with Freund's complete adjuvant. 37 Advantages of Liposomes as an Immunoadjuvant, Particularly with Respect to Preparation of Vaccines The fact that liposomes, composed of phosphatidylcholine only, can be used successfully as an immunoadjuvant (see the table) increases their value for this purpose. Phosphatidylcholine is a normal ingredient of cell membranes and is biodegradable. Moreover, it is a harmless compound when administered as liposomes. One of the most important advantages of phosphatidylcholine liposomes as an adjuvant is that, in contrast with certain other phospholipids, such as cardiolipin, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid, phosphatidylcholine is a very poor antigen. 3s Schuster et al. 39 confirmed that liposomes by them34M. Davies, D. E. S. Stewart-Tull,and D. M. Jackson,Biochim. Biophys. Acta 508, 260 (1978). 35S. Rottem, FEBS Lett. 95, 121 (1978). W. A. Siddiqui,D. W. Taylor,S. C. Kan, K. Kramer, S. M. Richmond-Crum,S. Kotani, T. Shiba, S. Kusumoto,Science 201, 1237(1978). 37D. Gerlier and J. F. Dor6, in "Targetingof Drugs" (G. Gregoriadis, ed.), NATO Adv. Study Inst. Ser., Ser. A, Vol. 47, 1982. 38C. R. Alving,in "The Antigens"(M. Sela, ed.), Vol. 4, p. 1. AcademicPress, New York (1977). 39B. G. Schuster, M. Neidig, B. M. Alving,and C. R. Alving,J. Immunol. 122, 900 (1979).
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selves do not evoke a detectable immune response in rabbits, even in the presence of incomplete Freund's adjuvant. It may be concluded that in multilamellar phosphatidylcholine liposomes we have a biodegradable, harmless, easy obtainable immunoadjuvant, which has no immunogenic activity of its own and may be applied for intravenous administration. Dependent on the antigen to be incorporated, the phospholipid composition of liposomes may be varied in order to obtain the most efficient antigen-liposome preparation. Edelman 4° composed a list of factors that are important with respect to the safety of any adjuvanted vaccine. Comparing the characteristics of liposomes as an immunoadjuvant, it is clear that liposomes may be considered a promising vaccine adjuvant. Apart from their application as vaccine adjuvant 36 liposomes may be useful as an adjuvant in the immune response against antigens that may be harmful, especially when they have to be injected intravenously or in a relatively high concentration, when liposomes are omitted.
Aspects of Liposome-Mediated Immune Responses The first studies, in which adjuvant properties of liposomes have been described, concerned the humoral immune response. 4~-43 It has been shown in recent studies that liposomes can be used to induce cell-mediated immunity. 44,45 Allison and Gregoriadis 41 showed that both the primary and secondary immune response were enhanced when the antigen had been associated with liposomes before injection. They studied the secondary response after both priming and booster injections with liposome-associated antigen. We have studied the effect of liposomes on the secondary response against antigens in rabbits. 21 In all experiments, booster injections were given as antigen free in solution, whereas priming injections with either free antigen or liposome-associated antigen were given in order to study the specific effect of liposomes on the generation of immunological memory. It appeared that apart from an enhanced primary immune response, liposomes also enhanced the generation of immunological memory to associated antigens, 4o R. Edelman, Rev. Infect. Dis. 2, 370 (1980). 41 A. C. Allison and G. Gregoriadis, Nature (London) 252, 252 (1974). 42 T. D. Heath, D. C. Edwards, and B. E. Ryman, Biochem. Soc. Trans. 4, 129 (1976). 43 N. van Rooijen and R. van Nieuwmegen, Imrnunol. Commun. 6, 489 (1977). 44 M. J. P. Lawman, P. T. Naylor, L. Huang, R. J. Courtney, and B. T. Rouse, J. Irnmunol. 126, 304 (1981). 45 y. Sanchez, I. Ionescu-Matiu, G. R. Dreesman, W. Kramp, H. R. Six, F. B. Hollinger, and J. L. Melnick, Infect. Immun. 30, 728 (1980).
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Finally, liposomes are able to alter not only the strength, but also the type, of response to associated antigens, since the IgG response may be enhanced more than the IgM response. 46 Conclusions Immunoadjuvant activity of liposomes has been demonstrated in the immune response against various antigens. Evidence is presented that a strong immunoadjuvant activity of liposomes is mediated by antigen exposed on the outer surfaces of liposomes. Direct contact between antigen exposing liposomes and lymphocytes with specific surface receptors for that antigen may well be involved in the liposome-mediated immune response. When no antigen is bound to the liposomes, entrapped antigen may also enhance the immune response to some extent, probably after ingestion by macrophages, digestion of the liposomal membranes leading to unmasking, and macrophage processing of the antigen. Depending on the antigen to be used, several methods can be followed to produce liposomes with surface-exposed antigen. Antigens can be coated onto liposomes nonspecifically, or they can be changed from hydrophilic to hydrophobic in order to enhance their association with liposomes. Some antigens can be attached to liposomes covalently, and others may be bound via a receptor inserted in the phospholipid bilayers. Endotoxin and lipid A, compounds that have adjuvant activity by themselves, may be incorporated in the phospholipid bilayers in order to enhance the immunoadjuvant activity of liposomes. In phosphatidylcholine liposomes, we have a biodegradable, harmless, and easy obtainable immunoadjuvant, which has no immunogenic activity of its own and may be applied for intravenous administration. For that reason, liposomes have already been suggested as promising vehicles for vaccines. 46 E. Ruttkowski and K. Nixdorff, J. Immunol. 124, 2548 (1980).
[7] Role of Diffusion R e g u l a t i o n in R e c e p t o r - L i g a n d Interactions By CHARLES DELISI
The purpose of this chapter is to describe how the physical environment of a receptor influences its interaction with a ligand, and how the effects of that influence can be quantitatively characterized. The developMETHODS IN ENZYMOLOGY, VOL. 93
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181993-0
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matography of QUA tumor extrct on Sephadex G-200 columns and subsequent precipitin analysis of each fraction as described) Conclusions The methodology described herein has resulted in the preparation of large quantities of apparently monospecific antiserum to 7-FA. Compared to the original anti-3,-FA serum, polyclonal antiserum to y-FA immune complexes had a greater specificity (since it did not require absorption) and higher titer of reactivity to the serologically and physicochemically defined antigen y-FA. The availability of this serum will provide the basis for future directions in y-FA research and is currently being utilized for the affinity purification of 7-FA for subsequent RIA development. It is anticipated that the use of more sensitive methods for 7-FA detection may necessitate a reevaluation of current ideas, particularly in regard to presumptive 7-FA-negative cancers (e.g., certain leukemias and tumors of neural ectoderm origin7). The present data indicate that antigenically stable agarose-immobilized immune complexes can be produced using small volumes of primary precipitating antibody. After processing, such complexes appear to act (at least for y-FA) as suitable immunogens leading to the formation of large quantities of a more highly specific antiserum. This technique is of particular relevance to situations where it is not possible or desirable either to process solution-precipitated complexes or to dissociate the antigen-antibody complex prior to immunization. Acknowledgments This work was supported by Grant 5 R23 CA25285-02from thc National Cancer Institute.
[6] U s e o f L i p o s o m e s as B i o d e g r a d a b l e a n d Harmless Adjuvants
By Nico VAN ROOIJEN and RIA VAN NIEUWMEGEN Introduction to Liposomes
Liposomes are artificially prepared spheres of concentric phospholipid bilayers separated by aqueous compartments. They form when waterinsoluble phospholipids are confronted with water. The phospholipid molMETHODS IN ENZYMOLOGY, VOL, 93
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181993-0
84
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[6]
E
group ~ =,= Hydrophilic Hydrophobic fatty acid chains X m Aqueous
compartments
FIG. 1. Schematic representation of a phosphatidylcholine liposome with entrapped aqueous compartments.
ecules try to reach a conformation in which their hydrophobic fatty acid groups are not in direct contact with water. The formation of phospholipid bilayers, in which the relatively hydrophilic head groups are localized on both outer parts of the bilayers, and the hydrophobi~ fatty acid groups are localized directly opposite to each other in the inner part of the bilayer, is a logical consequence (Fig. 1). Liposomes may differ with respect to their dimensions, composition (different phospholipids), charge (neutral, positive, or negative liposomes), and structure (multilamellar and unilamellar liposomes). Liposome-entrapped or associated compounds may be targeted to different sites in or on living cells, and their application in biomedical research increases continuously. 1,2 Immunology is one of the research areas in which liposomes are frequently applied. 3,4 Their use as an adjuvant in immune reactions against various antigens is one of the most promising immunological applications of liposomes. 5 G. Gregoriadis, in "Liposomes in Biological Systems" (G. Gregoriadis and A. C. Allison, eds.), p. 25. Wiley, New York, 1980. 2 B. E. Ryman and D. A. Tyrrell, Essays Biochem. 18, 49 (1980). 3 B. H. Tom, in "Liposomes and Immunobiology" (B. H. Tom and H. R. Six, eds.), p. 3. Elsevier, Amsterdam, 1980. 4 C. R. Alving and R. L. Richards, in "The Liposomes" (M. Ostro, ed.), Marcel Dekker, New York (in press). 5 N. van Rooijen and R. van Nieuwmegen, in "Targeting of Drugs", (G. Gregoriadis, ed.), NATO Adv. Study Inst. Ser., Set. A 47, 301 (1982).
[6]
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=
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Phosphatidylcholine molecules
• = Antigen
molecules Masked
Black parts:
Open p a r t s : U n m a s k e d
FIG. 2. Schematic representation of a part of a phosphatidylcholine liposome with exposed and masked antigen molecules.
Immune Responses against Antigen Exposed on the Surfaces of Liposomes and Entrapped Antigen When liposomes are prepared in an aqueous solution of antigen, the antigen may be entrapped in the aqueous compartments of the liposomes, and it may be associated with the phospholipid bilayers themselves. If antigen is bound to the phospholipids of the liposomes, part of the antigen probably will be exposed on the outer surface of the liposomes. This may be recognized by lymphocytes with surface receptors for that antigen, and an immune response can be initiated. When antigen is entrapped in the aqueous compartments of the liposomes without any association with the phospholipid bilayers, recognition by antigen-specific lymphocytes is impossible, since the antigen is completely masked (Fig. 2). In this case macrophages are required for processing of the liposome entrapped antigen, since no other way than lysosomal digestion of the liposomal membranes seems to exist for unmasking the antigen. 6 We have studied whether exposure of the antigens on the surfaces of liposomes, entrapment of the antigens in their aqueous compartments, or both surface-exposed and entrapped antigen are responsible for the adjuvant activity of liposomes in the immune response against associated antigen. 5,7 In these experiments we compared the immune response against antigen administered free in solution, coated on the surfaces of empty liposomes, or both coated on and entrapped in liposomes. For reasons mentioned before 5 we were not able to incorporate in the experiments a group of animals injected with antigen, given completely entrapped in liposomes without any surface interaction. 6 N. van Rooijen and R. van Nieuwmegen, Immunol. Commun. 8, 381 (1979). 7 N. van Rooijen and R. van Nieuwmegen, Cell. Immunol. 49, 402 (1980).
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Detailed information about the association of antigen with liposomes will be given later in this chapter. The results of the experiments showed a strong adjuvant effect of liposomes both in the immune responses against the antigens human serum albumin (HSA) and bovine y-globulin (BGG) in rabbits. The immune response elicited by empty liposomes coated with the antigens after their preparation was a little stronger than that elicited by liposomes that had in addition to their (relatively low amount of) surface-exposed antigen also (a relatively high amount of) antigen entrapped in their inner compartments. Clearly, entrapped antigen did not contribute to the immune response, in spite of the fact that the bulk of the liposome-associated antigen had been entrapped. The finding that entrapped antigen seemed to inhibit the immune response against liposomeassociated HSA or BGG could be explained by assuming that antigen in the liposomes, especially the part that was fixed to the inner half of the outer phospholipid bilayer, hindered binding of the antigen to the outer surfaces of the liposomes. Unlike albumin or y-globulin antigens, 5,7 nonspecifically associated horseradish peroxidase (HRP) appeared not to elicit a primary immune response after intravenous injection in rabbits. It has been shown that a primary anti-HRP antibody response could be elicited when HRP covalently attached to the liposomes was injected. Nonspecific association of the antigen HRP with the liposomes occurs to a very low degree, contrary to albumin and y-globulin antigens. Nonspecific HRP association with liposomes takes place, as concluded from the finding that rabbits injected with such HRP-liposomes appeared to be primed for a secondary response against the antigen, but it occurred to such a small extent that no exact determination of the amount of liposome-associated HRP could be performed. 8 We also compared the priming effect of empty liposomes nonspecifically associated with HRP and liposomes with HRP both entrapped and nonspecifically associated with their surfaces. It appeared that entrapped HRP clearly contributed to the establishment of immunological memory, since higher peak titers were reached and a higher percentage of rabbits appeared to be primed, when HRP had been entrapped in the injected liposomes. 8 Although this finding seems to contradict our earlier results that entrapped HSA or BGG does not contribute to the adjuvant effect of liposomes with both entrapped and surface-associated albumin or 3'globulin antigens, there is probably no real discrepancy between these results. When antigen can be nonspecifically bound to liposomes to a sufficient degree, as found with albumin and y-globulin antigens: ,7 entrapped antigen does not contribute to the adjuvant effect of liposomes. s N. van Rooijen, R. v a n N i e u w m e g e n , N. Kors, in preparation.
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However, when only an extremely small amount of antigen is bound to the surfaces of liposomes, as found with HRP, entrapped antigen can markedly contribute to the adjuvant effect. In this case we must assume that macrophages play a, role in the releasing and processing of the liposome-entrapped antigen, as we suggested earlier. 6 It has been shown that a small part of the antigen, ingested by macrophages, is prevented from complete digestion by lysosomal enzymes, and it has been suggested that this macrophage-associated antigen is particularly immunogenic. 9 Summarizing the results obtained until now, we conclude that the adjuvant effect of liposomes is primarily dependent on antigen presentation, and it is not unlikely that liposomes can replace macrophages with respect to this antigen presentation. However if antigen is not exposed on the surfaces of liposomes, either nonspecifically coated on or covalently attached to the phospholipid bilayers, entrapped antigen may initiate an immune response after ingestion of the liposomes by macrophages, digestion of the liposomal membranes, and unmasking the antigenic determinants. Although the mechanism by which liposomes exert their adjuvant effect is relatively unimportant for various practical applications, it may be important for application of liposomes as adjuvants in more fundamental immunological studies. Preparation of Liposomes Multilamellar liposomes can be prepared from one single phospholipid, e.g., phosphatidylcholine (lecithin), or from mixtures of different lipids. By adding negatively charged phosphatidic acid or dicetylphosphate or positively charged stearylamine to the mixtures, the charge of the resulting liposomes can be influenced correspondingly. For a comparative study on adjuvant activity of various liposomes,~° we prepared neutral liposomes composed of phosphatidylcholine only, positive liposomes composed of'phosphatidylcholine and stearylamine, and negative liposomes composed of phosphatidylcholine, phosphatidic acid, and cholesterol. All lipids were obtained from Sigma Chemical Company, St. Louis, Missouri: 150 mg of phosphatidylcholine, 150 mg of phosphatidylcholine, and 6 mg of stearylamine, or 150 mg of phosphatidylcholine, 22 mg of cholesterol, and 21.2 mg of phosphatidic acid, were dissolved in 25 ml of chloroform in a round-bottom flask. The thin film that formed on the walls of the flask after rotary evaporation at 37° was dispersed by gentle shaking for 10 min in 20 ml of 10 mM sodium phosphate buffer for 2 hr, sonicated 9 G. Weissmannand P. Dukor, Adv. Immunol. 12, 283 (1970). ~0N. van Rooijenand R. van Nieuwmegen,Immunol. Commun. 9, 243 (1980).
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for 3 min at 20 ° in a Sonicor (50 Hz), and kept at room temperature for another 2 hr. The liposomes were centrifuged at 17,500 g for 30 min in a Beckman J-21 Centrifuge. The pellet containing the liposomal fraction was washed in PBS and finally resuspended in 8 ml of PBS. Another way to prepare multilamellar liposomes is by the ether evaporation method. Ether solutions of phospholipids or phospholipid mixtures are injected in warm aqueous solutions. After vaporization of the ether, liposomes are formed. When vacuum is drawn over the aqueous solution rapid vaporization of the ether may be obtained at a lower temperature in order to prevent degradation of temperature-sensitive molecules. 11 Several lipids may be combined for preparation of liposomes. 1,12Phosphatidylethanolamine may be an important component of liposomes, since this phospholipid contains a free amino group, which can be used for covalent attachment of antigens. Using combinations of lipids that have shown their use for preparation of liposomes, 1 it is not necessary to check the formed liposomal structures. The adjuvant activities of the three types of liposomes, of which the compositions have been mentioned above are shown in the table (with respect to the immune response against HSA). In order to check the character of liposomes prepared from previously uninvestigated lipid mixtures or their dimensions, liposomes may be stained with fluorochromes and studied with the fluorescence micros c o p e . 13 Staining can be performed simply by incubation of the multilamellar liposomes in a 1% aqueous solution of the fluorochromes acridine orange or eosin Y for several minutes. They have to be centrifuged and washed afterward, and after resuspending the last pellet, a drop can be observed under the fluorescence microscope (1000x magnification). Entrapment of Antigens in Liposomes For entrapment of antigens in the aqueous compartments of liposomes, the antigens must be dissolved in the PBS in which the phospholipid film, formed on the walls of the round-bottom flask after rotary evaporation, was dispersed (see preparation of liposomes). Organic solvent vaporization methods for preparation of liposomes cannot be recommended for liposomal entrapment of antigen, since the organic solvents may alter the (protein) antigens. Although entrapment of antigen may be required when the antigen shows only a very low degree of nonspecific binding to the outer surfaces of liposomes, and methods for covalent t1 D. Deamer and A. D. Bangham, Biochim. Biophys. Acta 443, 629 (1976). 12 R. E. Pagano and J. N. Weinstein, Annu. Rev. Biophys. Bioeng. 7, 435 (1978). z3 N. van Rooijen and R. van Nieuwmegen, Stain Technol. 43, 307 (1978).
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attachment of the antigen to liposomes are lacking, the adjuvant effect of liposomes is particularly useful when sufficient exposure of the antigens on the surfaces of liposomes can be obtained. Nonspecific Association of Antigens with Surfaces of Liposomes Antigens, which can be coated on the surfaces of liposomes after their preparation, must be dissolved in PBS. The " e m p t y " liposomes are suspended in this antigen solution. Since, depending on the nature of the antigen, a high concentration of it may be required, the total volume of the suspension must be kept as small as possible. This is particularly important in cases where large amounts of antigen are too expensive or difficult to prepare. Whereas albumin antigen concentrations of 25 mg/ml were used for preparation of antigen-associated liposomes in most of our studies, a concentration of 250/~g/ml still appeared to be sufficient to demonstrate the adjuvant effect of liposomes. 5 After preparation of liposomeassociated antigens, the liposomes can be washed in order to remove free antigen. This will be necessary only when there is concern that free antigen may inhibit the adjuvant effect of the liposome-associated antigen, although there are no data available on a possible inhibition of the adjuvant effect of liposomes by free antigen. For practical purposes, the same antigen solution may be used several times for preparation of antigenassociated liposomes when only a small fraction of the antigen is bound to the surfaces of liposomes, as we found with albumin and 3J-globulin antigens.5,7,10 Another way to prepare liposomes with antigen nonspecifically associated with their surfaces is to dissolve the antigens in the PBS in which the phospholipid film has been dispersed, i.e., the same method as that recommended for entrapment of antigens in liposomes. Liposomes prepared in this way may have the antigen both entrapped in their aqueous compartments and nonspecifically associated with their surfaces. When sonication of the antigen-liposome mixture is necessary for binding immunoglobulin G (used as an antigen) to liposomes, as suggested by Huang and Kennel, ]4 the latter method is required in order to get sufficient antigen-liposome association. Alteration of Originally Hydrophilic Proteins to Hydrophobic Proteins and Their Incorporation in Liposomal Membranes It is not surprising that hydrophobic proteins show more tendency to bind to liposomes than hydrophilic proteins. Interaction between 3,-globu14 L. Huang and S. J. Kennel, Biochemistry 18, 1702 (1979).
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lins and liposomes may well depend on a change of their conformation that allows a hydrophobic binding, as suggested by Vanderbranden et al.~5 Evidence was given that the immunoglobulin region interacting with the lipid acyl chains is located in the heavy chain. Koelsch et a l ) 6 reported on the chemical modification of originally hydrophilic proteins to hydrophobic proteins in order to incorporate these in liposomal membranes. The proteins were modified by acylation with fatty acid chlorides. " H y d r o p h o b i z a t i o n " of protein antigens as a general method to enhance their binding to liposomes seems to offer a promising approach. Several studies are available on the use of lipid-conjugated albumin antigens, 17-2°and such lipid-conjugated proteins may be expected to show an enhanced affinity for liposomal membranes. Covalent Attachment of Antigens to Liposomes When nonspecific interaction of antigens with liposomes does not result in a sufficient binding of antigen to the liposomes, the antigen may be attached to the liposomes covalently in order to get a strong adjuvant effect of liposomes. As mentioned before, covalent attachment of H R P to the outer surfaces of liposomes results in a H R P - l i p o s o m e complex, which, after intravenous injection in rabbits, evokes a primary immune response against HRP. 8 Nonspecifically liposome-associated and/or liposome-entrapped H R P appeared not to elicit a primary immune response, but priming for a secondary response occurred. 2~ HRP, injected free in solution, neither elicited a primary immune response, nor primed for a secondary response in most animals. Apart from the method for covalent attachment of H R P to phosphatidylethanolamine as a component of liposomes suggested by Heath et al., 22 covalent attachment of immunoglobulins to liposomes via glycosphingolipids has been described. 23 Immunoglobulins may also be coupled with fatty acids covalently, followed by incorporation of these amphipathic antibodies in liposomal membranes 15M. Vanderbranden, J. L. de Coen, R. Jeener, L. Kanarck, and J. M. Ruyschaert, Mol. lmmunol. 18, 621 (1981). 16R. Koelsch, J. Lasch, A. L. Klibanov, and V. P. Torchilin, Acta Biol. Med. Germ. 40, 331 (1981). 17M. O. Dailey and R. L. Hunter, J. Immunol. 118, 957 (1977). is S. B. Singh and S. Leskowitz, J. lmmunol. 120, 734 (1978). 19G. Drach and J. Chen-Marotel, Int. Arch. Allergy Appl. lmmunol. 59, 28 (1979). 2oj. M. Stark, N. Matthews, and J. Locke, Immunology 39, 353 (1980). 2~N. van Rooijen, R. van Nieuwmegen, and N. Kors, lmmunol. Commun. 10, 59 (1981). 22T. D. Heath, D. Robertson, M. S. C. Birbeck, and A. J. S. Davies, Biochim. Biophys. Acta 599, 42 (1980). 23T. D. Heath, B. A. Macher, and D. Papahadjopoulos, Biochim. Biophys. Acta 640, 66 (1981).
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by a detergent-dialysis method. 24 Another method for covalent coupling of a soluble proteins to liposomes uses the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl-3-(2-pyridyldithio)propionate (SPDP, Pharmacia) as suggested by L e s e r m a n e t al. 25 Finally, antibody can be covalently coupled to liposomes by a glutaraldehyde reaction. 26 Attachment of Antigen to Liposomes via an Inserted Receptor Alving e t al. studied the immune r e s p o n s e against liposome-associated cholera toxin. A t t a c h m e n t of cholera toxin to the liposomes was performed by incorporation of a receptor, i.e., ganglioside GMI in the liposomal bilayers. 27,28 Incorporation of a receptor molecule into liposomal bilayers in order to attach an antigen to their surfaces is another form of association of antigens with liposomes. Methods to Enhance the I m m u n o a d j u v a n t Activity of Liposomes The relative p o t e n c y o f various adjuvants differs markedly according to the antigen used. 29 The adjuvant effect of liposomes appears to depend primarily on antigen presentation, as we discussed earlier. N o stimulation effect of liposomes t h e m s e l v e s on proliferation of l y m p h o c y t e s has been observed. On the contrary, they rather inhibit the proliferation of lymphocytes as measured by [3H]thymidine incorporation into cellular D N A . 3°-32 The fact that different adjuvants enhance the immune response by different effects suggests that the adjuvant effect of liposomes obtained by antigen presentation, m a y be enhanced by combination with other adjuvants. Endotoxin and endotoxin-derived c o m p o u n d lipid A m a y enhance the adjuvant effect of liposomes against liposome-associated antigens. 27,2s,33 When a weak albumin antigen and endotoxin were associated with the 24A. Huang, L. Huang, and S. J. Kennel, J. Biol. Chem. 255, 8015 (1980). L. D. Leserman, J. Barbet, and F. Kourilsky, Nature (London) 288, 602 (1980). 26V. P. Torchilin, B. A. Khaw, V. N. Smirnov, and E. Haber, Bioehem. Biophys. Res. Commun. 89, lll4 (1979). 27C. R. Alving, B. Banerji, J. D. Clements, and R. L. Richards, in "Liposomes and Immunobiology" (B. H. Tom and H. R. Six, eds.), p. 67. Elsevier, Amsterdam, 1980. 2s C. R. Alving, B. Banerji, T. Shiba, S. Kotani, J. D. Clements, and R. L. Richards, Prog. Clin. Biol. Res. 47, 339 (1980). 29R. Bomford, Clin. Exp. lmmunol. 39, 426 (1980). 3oS. Shi-Hua Chen and R. M. Keenan, Biochem. Biophys. Res. Commun. 79, 852 (1977). 3~B. Rivnay, A. Globerson, and M. Shinitzky, Eur. J. lmmunol. 8, 185 (1978). 3zM. H. Ng, W. S. Ng, W. K. K. Ho, K. P. Fung, and J. P. Lamelin, Exp. Cell Res. 116, 387 (1978). 33N. van Rooijen and R. van Nieuwmegen, lmmunol. Commun. 9, 757 (1980).
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same liposomes, antibody production in rabbits could be detected within 3 days after injection of the antigen-liposome-endotoxin preparation. When antigen and endotoxin were associated with different liposomes and these antigen-liposome and endotoxin-liposome preparations were injected simultaneously, the adjuvant effect of the liposomes was enhanced also, but it took 1 day longer before antibodies could be detected, whereas peak titers were not markedly different. 33 However, it is not unlikely that antigen-containing liposomes and endotoxin-containing lip0somes had been aggregated or fused in v i v o resulting in liposomes containing both endotoxin and antigen, since endotoxin may form bridges between different liposomes. The structure of liposomes is not markedly affected by endotoxin or lipid A, since these compounds do not increase their permeability to divalent anions 34or nonionic substances. 35 However, injection of liposomes containing lipid A may provoke an immune response not only against the associated antigen, but also against lipid A and the phospholipids of which the liposomes are composed) 9 Other lipid substances that might be incorporated into liposomes to enhance the immunogenicity of liposome-associated antigens include acylated derivatives of muramyl dipeptide. 36 A very powerful adjuvant results if antigen containing liposomes are combined with Freund's complete adjuvant. 37 Advantages of Liposomes as an Immunoadjuvant, Particularly with Respect to Preparation of Vaccines The fact that liposomes, composed of phosphatidylcholine only, can be used successfully as an immunoadjuvant (see the table) increases their value for this purpose. Phosphatidylcholine is a normal ingredient of cell membranes and is biodegradable. Moreover, it is a harmless compound when administered as liposomes. One of the most important advantages of phosphatidylcholine liposomes as an adjuvant is that, in contrast with certain other phospholipids, such as cardiolipin, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid, phosphatidylcholine is a very poor antigen. 3s Schuster et al. 39 confirmed that liposomes by them34M. Davies, D. E. S. Stewart-Tull,and D. M. Jackson,Biochim. Biophys. Acta 508, 260 (1978). 35S. Rottem, FEBS Lett. 95, 121 (1978). W. A. Siddiqui,D. W. Taylor,S. C. Kan, K. Kramer, S. M. Richmond-Crum,S. Kotani, T. Shiba, S. Kusumoto,Science 201, 1237(1978). 37D. Gerlier and J. F. Dor6, in "Targetingof Drugs" (G. Gregoriadis, ed.), NATO Adv. Study Inst. Ser., Ser. A, Vol. 47, 1982. 38C. R. Alving,in "The Antigens"(M. Sela, ed.), Vol. 4, p. 1. AcademicPress, New York (1977). 39B. G. Schuster, M. Neidig, B. M. Alving,and C. R. Alving,J. Immunol. 122, 900 (1979).
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selves do not evoke a detectable immune response in rabbits, even in the presence of incomplete Freund's adjuvant. It may be concluded that in multilamellar phosphatidylcholine liposomes we have a biodegradable, harmless, easy obtainable immunoadjuvant, which has no immunogenic activity of its own and may be applied for intravenous administration. Dependent on the antigen to be incorporated, the phospholipid composition of liposomes may be varied in order to obtain the most efficient antigen-liposome preparation. Edelman 4° composed a list of factors that are important with respect to the safety of any adjuvanted vaccine. Comparing the characteristics of liposomes as an immunoadjuvant, it is clear that liposomes may be considered a promising vaccine adjuvant. Apart from their application as vaccine adjuvant 36 liposomes may be useful as an adjuvant in the immune response against antigens that may be harmful, especially when they have to be injected intravenously or in a relatively high concentration, when liposomes are omitted.
Aspects of Liposome-Mediated Immune Responses The first studies, in which adjuvant properties of liposomes have been described, concerned the humoral immune response. 4~-43 It has been shown in recent studies that liposomes can be used to induce cell-mediated immunity. 44,45 Allison and Gregoriadis 41 showed that both the primary and secondary immune response were enhanced when the antigen had been associated with liposomes before injection. They studied the secondary response after both priming and booster injections with liposome-associated antigen. We have studied the effect of liposomes on the secondary response against antigens in rabbits. 21 In all experiments, booster injections were given as antigen free in solution, whereas priming injections with either free antigen or liposome-associated antigen were given in order to study the specific effect of liposomes on the generation of immunological memory. It appeared that apart from an enhanced primary immune response, liposomes also enhanced the generation of immunological memory to associated antigens, 4o R. Edelman, Rev. Infect. Dis. 2, 370 (1980). 41 A. C. Allison and G. Gregoriadis, Nature (London) 252, 252 (1974). 42 T. D. Heath, D. C. Edwards, and B. E. Ryman, Biochem. Soc. Trans. 4, 129 (1976). 43 N. van Rooijen and R. van Nieuwmegen, Imrnunol. Commun. 6, 489 (1977). 44 M. J. P. Lawman, P. T. Naylor, L. Huang, R. J. Courtney, and B. T. Rouse, J. Irnmunol. 126, 304 (1981). 45 y. Sanchez, I. Ionescu-Matiu, G. R. Dreesman, W. Kramp, H. R. Six, F. B. Hollinger, and J. L. Melnick, Infect. Immun. 30, 728 (1980).
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Finally, liposomes are able to alter not only the strength, but also the type, of response to associated antigens, since the IgG response may be enhanced more than the IgM response. 46 Conclusions Immunoadjuvant activity of liposomes has been demonstrated in the immune response against various antigens. Evidence is presented that a strong immunoadjuvant activity of liposomes is mediated by antigen exposed on the outer surfaces of liposomes. Direct contact between antigen exposing liposomes and lymphocytes with specific surface receptors for that antigen may well be involved in the liposome-mediated immune response. When no antigen is bound to the liposomes, entrapped antigen may also enhance the immune response to some extent, probably after ingestion by macrophages, digestion of the liposomal membranes leading to unmasking, and macrophage processing of the antigen. Depending on the antigen to be used, several methods can be followed to produce liposomes with surface-exposed antigen. Antigens can be coated onto liposomes nonspecifically, or they can be changed from hydrophilic to hydrophobic in order to enhance their association with liposomes. Some antigens can be attached to liposomes covalently, and others may be bound via a receptor inserted in the phospholipid bilayers. Endotoxin and lipid A, compounds that have adjuvant activity by themselves, may be incorporated in the phospholipid bilayers in order to enhance the immunoadjuvant activity of liposomes. In phosphatidylcholine liposomes, we have a biodegradable, harmless, and easy obtainable immunoadjuvant, which has no immunogenic activity of its own and may be applied for intravenous administration. For that reason, liposomes have already been suggested as promising vehicles for vaccines. 46 E. Ruttkowski and K. Nixdorff, J. Immunol. 124, 2548 (1980).
[7] Role of Diffusion R e g u l a t i o n in R e c e p t o r - L i g a n d Interactions By CHARLES DELISI
The purpose of this chapter is to describe how the physical environment of a receptor influences its interaction with a ligand, and how the effects of that influence can be quantitatively characterized. The developMETHODS IN ENZYMOLOGY, VOL. 93
Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181993-0