Liposomes as immunological adjuvants: approaches to immunopotentiation including ligand-mediated targeting to macrophages

178

41st FOR UA4 IN IMMUNOLOGY

important accessory cells for the processing of liposome-associated antigens. Contributions by Gregoriadis, Szoka, Harding et al., Huang et al., and Alving et al., to the present Forum, focus on the role of macrophages in the processing and presentation of liposome-associated antigens. Taking advantage of the natural ability of liposomes to target, albeit passively, immunomodulators to cells of the mononuclear phagocyte system (MPS), Fidler and colleagues were able to activate MPS cells to the tumoricidal or virocidal state. Systemic administration of liposomes containing various immunomodulators have been shown to bring about regression of lymph node, lung and liver metastases, and promote effective prophylaxis of viral infections in rodents. Contributions by Fidler, Phillips and Daemen to the present Forum focus on the activation of macrophages by liposome-associated immunomodulators. Macrophages perform a wide variety of functional activities in immune reactions, from processing and presentation of antigens to appropriate Tcells, to production of soluble mediators. To study such functional aspects of macrophages in vivo, several macrophage elimination techniques have been developed. Elimination of macrophages is often performed by the administration of silica particles. This method also influences other lymphoid- and non-lymphoid

cells, and silica particles are not biodegradable. For that reason, we have developed a new liposomemediated “macrophage suicide technique” based on the intracellular toxicity of the drug dichloromethylene diphosphonate. Macrophages ingest the liposome-encapsulated drug and consecutively digest the phospholipid bilayers using their lysosomal phospholipases. The drug is released within the cell and the phagocyte is killed. By liposomal encapsulation of the drug, it is targeted to macrophages (contrary to the free drug) and, at the same time, it cannot interact with other cells of the body. Contributions by van Rooijen, Bogers et al., van Lent et al., and De Rijk et al. to the present Forum deal with the applications of this macrophage suicide technique in studies on macrophage function. Closely related to the use of liposomes for manipulating and studying macrophage function are studies which are devoted to the fate of liposomes in the body and those focussing on attempts to influence this fate by altering the composition and structure of the liposomal bilayers. Contributions by Claassen and Pate1 to the present Forum focus on these aspects of liposomes respectively. An introduction to liposomes is given in the first contribution by Gregoriadis. N. van Rooijen

Liposomes as immunological adjuvants : approaches to immunopotentiation including ligand-mediated targeting to macrophages G. Gregoriadis Centre for Drug Delivery Research, The School of Pharmacy, University 29/39 Brunswick Square, London WCIN 1AX

Introduction A number of laboratories are now working towards the development of effective immunological adjuvants to meet the challenges of subunit and peptide vaccines and on new insights into the ways by which immunity is generated (Gregoriadis et al.,

of London,

1989, 1991). One of the adjuvant systems currently under investigation is liposomes (Gregoriadis, 1990). This chapter will briefly discuss some of the properties of liposomes that are relevant to their immunoadjuvant action and conditions under which such action is enhanced, including ligand-mediated interaction of the system with macrophages in vivo.

LIPOSOMES

AND MACROPHAGE

FUNCTIONS

179

Phospholipids and other polar amphiphiles form closed concentric bilayer membranes (liposomes or vesicles) when confronted with excess water. In the process of their formation, liposomes entrap water and solutes dissolved in it. Alternatively, lipid-soluble agents and molecules coupled to lipids can be accommodated into or onto the liposomal membrane (fig. 1). Thus, almost any substance (including antigens), regardless of solubility, size, shape and electric charge can be incorporated into liposomes as long as they do not interfere with their formation (Gregoriadis, 1984). A large variety of phospholipids either alone or together with other lipids, such as those extracted from membranes, can be used to form liposomes. Depending on their gel-liquid crystalline transition temperature (Tc), phospholipids influence membrane fluidity and stability which, in turn control permeability to solutes in vitro and in vivo. Inclusion of sterols (e.g. cholesterol), also influences bilayer fluidity and stability. Furthermore, the presence of charged amphiphiles will render the liposomal surface positively or negatively charged and may augment the space (and therefore volume and solute entrapment) between bilayers (Gregoriadis, 1984). It is because of this unusually versatile nature of liposomes as well as their similarity to biological membranes that the concept of using the system in targeted drug delivery was first aired (for a historical background see Gregoriadis, 1976). During the intervening 21 years since the first animal experiments with liposomes were carried out (Gregoriadis and Ryman, 1972) a wide range of liposomal drugs including anti-tumour and antimicrobial agents, enzymes, hormones, vitamins, metal chelators, genetic material, immunomodulators and vaccines has been used therapeutically in both experimental animals and man (Gregoriadis, 1988a).

As a result, there now exists a considerable body of knowledge on the behaviour of liposomes and their drug contents within the biological milieu and on the means by which such behaviour can be controlled. Revelant to the role of liposomes as immunoadjuvants is their interaction with the biological fluids (with which liposomes first come into contact on injection) and uptake by tissues. In both instances, liposomal behaviour is dictated by their structural characteristics (Gregoriadis, 1988b). Thus, whereas, plasma high-density lipoproteins (I-IDL) will attack and destabilize or disintegrate liposomal bilayers made of, say, egg phosphatidylcholine (PC) only, substitution of PC with distearoyl phosphatidylcholine and/or equimolar cholesterol will reduce or abolish HDL action. This type of stable liposome is expected to retain their antigen content quantitatively en route to their destination. The latter is determined mainly by the size of the vesicles with large ones ending up mostly in the fixed macrophages of

Fig. 1. A section of an electron micrograph of a negativelystained multilamellar liposome showing lipid bilayers alternating with electron-opaque aqueous channels. Three of these bilayers are enlarged. Polar heads of phospholipids face the water phase and acyl chains form the hydrophobic regions of the leaflet. Open circles denote drugs entrapped in the aqueous channels. Oblong shapes are cholesterol (filled) and membrane soluble drugs (open) sandwiched between phospholipid molecules. Liposomes can very in size from about 20 nm (small unilamellar vesicles, SUV) to several microns in diameter. The latter can have one (large unilamellar vesicles, LUV) or more (multilamellar vesicles, MLV) bilayers. Depending on the methodology and the type of drug, entrapment values can very from less than 1% to 100% of the drug used (from Gregoriadis, 1990).

41si FORUM

180

IN IMMUNULOG

the liver and spleen. Smaller vesicles, capable of penetrating the fenestrations of the liver are taken up by the parenchymal cells of the tissue. In addition, liposomes which have a small size and a prolonged circulatory time are intercepted to a large extent by bone marrow macrophages. Uptake of liposomes by macrophages is endocytic with the vesicles ending up in the lysosomal apparatus of the cells. Although such observations on the liposomal fate in vivo have been made mostly with formulations given by the intravenous route, they are also relevant to situations where preparations were given intramuscularly, intradermally, subcut~eously or intraperitoneally : depending on vesicle size, composition and route of injection, a proportion of liposomes reaches the lymphatic circulation and eventually, the blood. Remaining vesicles are attacked by infiltrating macrophages and disintegrate locally or are intercepted by the regional lymph nodes (Gregoriadis, 1988a).

The immunological

adjuvant

activity of liposomes

Early attempts to circumvent immune responses to injected foreign therapeutic enzymes by entrapping them in liposomes, revealed that, contrary to expectations, antibody titres to the model protein (diphtheria toxoid) used were much higher than those obtained with the free protein (Allison and Gregoriadis, 1974; Gregoriadis and Allison, 1974). This was subsequently found to apply to other antigens derived from bacteria, protozoa, viruses, tumours and other sources (Gregoriadis, 1990). It is now established that a prerequisite for liposomal adjuvanticity to occur is physical association between liposomes and antigen (Shek, 1984). Thus, antigens must be entrapped into the aqueous or lipid phase of the vesicles, absorbed onto their surface electrostatically or hydrophobically or bound to it chemically... However, with the exception of certain liposomal viral subunits which form well organized structures (also known as virosomes ; Almeida et al., 1975), the spatial arrangement within liposomes of most of the antigens studied so far is largely undefined. It is, indeed, likely that in the process of incorporation into liposomes, many antigens are partly entrapped in the aqueous phase, partly inserted into the lipid phase or, with some of them, absorbed to some extent on the bilayer surface. Furthermore, the role of a number of variables such as vesicle size, number of bilayers and surface charge, in liposomal adjuvanticity has not been clearly defined, On the basis of what has been published already, it appears likely that liposomal adjuvanticity does not depend crucially on any specific formulation or on the animal species, route and mode of immunization used (Gregoriadis, 1990). Although with most antigens

Y

studied the approach to liposomal formulation and immunization protocols has been largely empirical, primary and/or secondary responses were observed for all antigens tested for humoral immunity (HI), with T-cell dependency maintained in at least some cases (Shek, 1984; Beatty er al., 1984). Studies from this laboratory have also shown (Davis et al., 1987) that liposomal adjuvanticity is reflected in most IgG subclasses, with no apparent shift in subclass responses when compared with those observed after immunization with the free antigen. Available information on the fate of liposomes in vivo (as already briefly outlined), suggests that HI to liposomal antigens can be partly or wholly attributed to the system’s function as an antigen depot, providing antigenpresenting cells (APC) either with free (locally released) or liposome-entrapped antigen, or both. For those formulations which have been shown to be effective, supply of antigen to the cells is presumably carried out at rates or in a form conducive to efficient presentation.

Amplification

of liposomal

immunoadjuvant

action

A variety of approaches leading to the amplification of the adjuvant effect of liposomes have been tested and found successful. These include administration of liposomes together with other adjuvants, for instance interleukin-2 (IL2) (Tan and Gregoriadis, 1989), lipid A (Alving et al., 1986 ; Naylor et al., 1982), MDP and lipophilic derivatives (Kersten et al., 1988), saponin (Manesis el al., 1979), B. pertussis (Manesis et al., 1979), etc. Synergistic action of coadjuvants is obtained by co-entrapping the adjuvant together with the antigen in the same vesicles or entrapping the adjuvant (e.g. IL2; see table I) and antigen in separate vesicle populations which are mixed before injection, or by mixing the adjuvant (for example alum, saponin, B. pertussis) with antigen-containing liposomes.

Optimization

tif liposomal

adjuvanticity

Evidence from previous studies (Gregoriadis, 1990) suggests that liposomal adjuvanticity is an attribute of the vesicular structure of the system and, probably, its lipoid nature, rather than that of the identity of its lipid components or othet secondary physical characteristics. Nonetheless, the latter are known (Gregoriadis, 1988b) to control the behaviour of liposomes in vivo and could thus be instrumental in the expression of liposomal immunoadjuvant activity, both qualitatively and quantitatively. Studies on the extent to which bilayer fluidity (e.g. Davis and Gr~goriad~~, 1987; Bakouche et a/,, 1987), number of lamellae in bilayers (Shek, 1984), vesicle size (Fran-

LIPOSOMES

Table I. Effect of liposomal

AND MACROPHAGE

recombinant

IL2 on the immune

Liposomes A) Entrapped

toxoid only

B) Co-entrapped

toxoid and rIL2 (lo3 units)

C) Co-entrapped

toxoid and rIL2 (IO4 units)

D) Separately entrapped toxoid and rIL2 (10’ units)

E) Separately entrapped toxoid and rIL2 (lo4 units)

FUNCTIONS

181

response to entrapped tetanus toxoid.

IgGl

IgG2a

IgG2b

0.184 0.299 0.359 0.506 0.546 0.898 1.520 1.591 P < 0.01 1.740 (T = 15) 2.000

0.205 0.251 0.661 0.879 0.910 0.170 0.221 0.242 N.S. 0.368 (T = 21) 0.389

0.153 0.184 0.352 1.543 1.553 0.293 0.540 0.776 N.S. 0.790 (T = 29) 1.073

1.135 1.648 1.755 1.997 2.000 0.187 0.201 0.237 0.291 0.418

0.722 1.063 P < 0.01 1.484 (T = 15) 2.000 2.000 0.088 0.104 N.S. 0.130 (T = 24) 0.187 0.319

P < 0.05 (T = 17)

P < 0.05 (T = 17)

1.260 1.560 2.000 2.000 2.000 0.047 0.064 0.073 0.259 0.301

P < 0.05 (T = 17)

P < 0.05 (T = 17)

0.316 0.905 0.242 0.723. 1.215 0.283 1.234 N.S. 1.495 P < 0.05 0.986 N.S. 1.416 (T = 18) 2.000 (T = 16) 1.440 (T = 25) 1.490 2.000 2.000

BALB/c mice in groups of 5 were injected intramuscularly on day 0 and day 42 with I pg tetanus toxoid entrapped in DRV liposomes without (A) or with rIL2 (co-entrapped) (B and C), or entrapped alone in DRV which were mixed with similar DRV containing rIL2 (separately entrapped) (D’ and E). ELISA readings shown are from blood samples obtained 14 days after the booster injection (secondary response). The Mann-Whitney test was used to compare groups B, C, D and E with group A (control). Median readings are underlined. N.S. = not significant; T = lower sum of the ranks in pairs of groups compared (from Tan and Gregoriadis, 1989).

cis et al., 1985) and surface charge (Latif and Bacchawat, 1987) as well as lipid to antigen mass ratio (Davis and Gregoriadis, 1987, 1989) and mode of antigen localization within liposomes (Davis and Gregoriadis, 1987; van Rooijen and van Niewmegen, 1980) influence adjuvanticity, suggest that all such parameters have an effect. However, conclusions as to the role of individual parameters in augmenting or reducing adjuvanticity have often been contradictory or have not been confirmed (Gregoriadis, 1990). While some of these contradictions reflect differences in adopted experimental protocols, others can be explained on the basis of the anticipated diversity of interactions between liposomal antigens and APC in situ. For instance, depending on the nature of the liposomal formulation and its behaviour in the bio-

logical milieu, antigen may be taken by APC following local disintegration of the carrier, be transferred to the APC membranes following contact with and internalization of liposomes by the cells or catabolized intracellularly with ensuing migration in fragmented forms to the APC membrane (for a discussion see Gregoriadis, 1990).

Targeting

of liposomal

antigen to macrophages

One of the advantages of liposomes as an immunoadjuvant is their targeting to specific cells. This was originally demonstrated both in vitro and in vivo with a number of tumour cell lines and for hepatic parenchymal cells (Gregoriadis and Neerunium,

182

41st FOR WA4 IN IMMUNOLOGY

1975 ; Gregoriadis et al., 1977) and has been subsequently greatly expanded by numerous groups (Gregoriadis, 1988a). Since liposomes are taken avidly by fixed macrophages, the need for targeting to these cells would seem superfluous. Although this is probably true in situations where liposomes are injected intravenously, application of the system as a carrier of vaccines intramuscularly may benefit from specific targeting : liposomes, especially those of small size, are expected to reach the blood circulation rapidly through the lymphatics. Therefore, augmented uptake of liposomes by local macrophages (e.g. those of regional lymph nodes) through receptor-mediated endocytosis, may also augment adjuvanticity. Here, we provide evidence that anchoring of a mannosylated ligand on to the surface of liposomes containing antigen not only leads to more efficient binding of the vesicles to the marmose receptor on macrophages, it also enhances their adjuvanticity. Dehydration-rehydration vesicles (DRV) containing immunopurified tetanus toxoid were prepared as such or coated with albumin or mannosylated albumin. Coating was achieved by a technique (Garcon et al., 1988 ; Senior and Gregoriadis, 1989) which allows covalent coupling of a ligand to the surface of liposomes without exposing the entrapped antigen to potentially damaging coupling reagents. In brief, DRV were generated (Garcon et al., 1988; Kirby and Gregoriadis, 1984) from small unilamellar vesicles (SUV ; p;epared as in Kirby et al., 1980) in the presence of I-labelled tetanus toxoid. On the basis of previous studies (Davis and Gregoriadis, 1987) pertaining to conditions for optimal liposomal adjuvanticity for tetanus toxoid, PC was chosen as the phospholipid component of liposomes. For the same reason, the amount of tetanus toxoid used for entrapment was such that the final formulations had relatively high mass ratios (67/l to 120/l) of phospholipid to entrapped antigen. WV composed of PC mixed with tracer 3H-PC, cholesterol and N-baminophenyl) stearylamide (APSA) were used as such or after covalent linkage (Snyder and Vannier, 1984) \ via the APSA) to mannose-free or mannosylated ’ ‘I-labelled albumin, The latter (8 or 36 mannose moles per mole protein) was prepared (Kataoka and Tavassoli, 1984) from bovine serum albumin (BSA) and p-aminophenyl-a-D-mannopyranoside. As estimated from radioactivity measurements, 36.5 - 48.4 % (or 6.0-8.4 ug/umol PC) of the toxoid used was entrapped in DRV with or without ligand on their surface. Of the mannose-free or mannosylated albumin used for coupling, 42.0-49.6 % was recovered with the DRV (6.4-15.2 ug/umol PC), and of this about 44 % was found exposed on the liposomal surface following protease treatment (Garcon et al., 1986). Prior to immunization,

the ability of mannosy-

lated DRV (8 to 36 mannose moles per mole albumin) to interact with mouse peritoneal macrophages specifically was investigated. Data in figure 2a show that the two mannosylated liposomal preparations at the concentrations used bound to the cells similarly, although binding for both was significantly greater than for mannose-free vesicles. The specificity of the interaction with the cells was confirmed by the addition (together with the liposomes) of excess methyla-D-mannopyranoside : binding returned to levels obtained with DRV coated with mannose-free BSA (fig. 2b). In immunization studies, BALB/c mice were primed intramuscularly with two different doses (0.063 and 0.2 pg) of toxoid entrapped either in plain DRV or in DRV coated with mannosylated albumin (36 moles mannose per mole albumin). Three weeks later they were injected again with identical preparations and sera were analysed by ELISA for antitoxoid IgGl 9 days after the second injection. Results (Garcon et al., 1988) suggested that mannosylated DRV, compared to plain DRV, induced a stronger immune response for the lower but not for the high-

0.3.

a)

0.2

b) (Y methyl-0-Hannose

1 0.1

F I 01

0.4 Llposomal

l pld

(fig)

added

0.6

0.8

1

to cells

Fig. 2. Binding of mannosylated liposomes to macrophages. Peritoneal macrophages were harvested from male BALB/c mice weighing 20-25 g 4 days after i.p. injection with 1.0 ml of 10% thioglycolate per mouse. Cells were suspended in HBSS medium and grown at 37% in small petri dishes (IO6 cells per dish) at 5% CO, and 95% humidity. After 3 h, non-adherent cells were removed by washing the dishes twice with HBSS. At the end of their first day of growth, cells were incubated for 2 h in the absence (a) or presence (b) of 100 mmoles methyl-Dmannopyranoside with increasing amounts of radiolabelled, toxoid-containing DRV (IO“ d.p.m. ‘H-PC) coated with albumin only (0) or albumin coupled to 8 (v) or 36 ( n ) moles mannose per mole protein. Albumin content of DRV was lo-12 ng/pg PC. Following incubation, cells were detached with a rubber policeman and assayed for lipid IH radioactivity (from Garcon et al., 1988).

LIPOSOMES hln-Alb-DRV-(1.1.)

AND MACROPHAGE

Alb-DRV-(T.T.)

DRV-(T, T.)

10200 E

2z!%I:

&.. *.....

n:.' /.... ...

w i= 160z I

40-

10 1

) 1.25

, 5

I 5

1.25

Tetanus

Toxoid

(ug

, 1.25

, 5

x 10e2)

Fig. 3. Regression lines of IgGl antibody response against tetanus toxoid entrapped in mannosylated or nonmannosylated liposomes. BALB/c mice (in groups of 5) were immunized intramuscularly with 4-fold increasing doses of tetanus toxoid (1.25 x 10e2-20 x 10e2 t.tg) entrapped in DRV composed of PC, cholesterol and APSA (90 pg PC per 1 pg toxoid) (o), in similar DRV coated with albumin (120 pg PC and 2.4 pg albumin per 1 pg toxoid) (0) or in similar DRV coated with mannosylated albumin (36 moles mannose per mole albumin) (67 pg PC and 1.36 pg albumin per 1 pg toxoid) (w). Three weeks after primary immunization, mice received identical injections of the same preparations and bled 9 days later. ELISA values for IgGl anti-toxoid antibody were plotted against the dose (not shown). Sera from the mice injected with the lowest dose (0.0125 pg) of toxoid (all treatments), which gave an IgGI response, and sera from mice injected with a 4-fold higher dose (0.05 pg) of the toxoid (all treatments) were titrated for their IgGl anti-toxoid antibody (see figure). The least squares estimate of the regression line of IgGl antibody titre against antigen dose was derived separately for the 4 groups. The gradients of these lines were compared by the t-test and found not to be significantly different. Therefore, 3 parallel lines were fitted with a common gradient. The differences between the antibody titres at any antigen concentration were then examined using the f-test. Regression lines (H versus o or l ) were significantly different (P < 0.05). Dotted lines denote 95% confidence interval of the regression lines (from Garcon et al., 1988).

er dose. IgG2b responses to toxoid entrapped in mannosylated liposomes were, on the other hand, stronger for both doses of the antigen. In a second similar experiment (fig. 3) carried out using an appropriate antigen dosage range (0.0125-0.2 pg toxoid per mouse), a control preparation of liposomes coated with mannose-free albumin was used in addition to the two preparations in the initial experiment. Analysis of sera by ELISA (readings not shown) confirmed the absence of differences in responses (IgGl) in the 3 preparations for the higher (0.2 pg) dose and improvement of respon-

FUNCTIONS

183

ses when mannosylated liposomes with antigen doses of 0.05 pg or less were used. Sera from the 3 groups of mice injected with the lowest (0.0125 yg) and the highest (0.05 pg) dose of toxoid which led to an IgGl response, were titrated for their IgGl anti-toxoid antibody. Results (fig. 3) indicated that a given antibody titre achieved with either of the two control preparations (plain and BSA-coated DRV) could be equaled with an 8-fold reduced concentration of the antigen given in liposomes coated with mannosylated BSA. In subsequent dose-response experiments, mice were immunized (using the protocol of fig. 3) with toxoid-containing DRV liposomes coated with mannosylated albumin. The amounts of the protein on the liposomal surface corresponded to two different numbers of protein chains which,-however, had the same number (36) of mannose moles per mole albumin. Anti-toxoid IgGl responses (ELISA median readings) were found (Gregoriadis et al., 1988) to be dependent on the number of mannosylated albumin molecules on the vesicle surface: responses were significantly higher with all toxoid doses (0.003, 0.125,0.05 and 0.2 pg) used for DRV coupled to the higher amount (1.36 pg) of mannosylated albumin than for DRV coupled to the lesser (0.75 Kg) amount (fig. 4). Small but statistically significant differences between the two preparations were also seen for IgG2b (0.003 and 0.0125 pg toxoid) (fig. 4). In a separate dose-response experiment, however, where the two preparations used were coated with similar amounts of mannosylated albumin (i.e. similar number of protein chains) which had either 8 or 36 mannose moles per mole protein, no significant differences in anti-toxoid responses were observed (Gregoriadis et al., 1988). According to figure 3, these DRV preparations bind to macrophages to a similar extent. In conjunction with the data in figure 4 for DRV coated with two different amounts of ligand, it is probable that adjuvanticity of liposomes is related directly to the extent of vesicle association with macrophages. This, presumably, is likely to increase with increasing numbers of ligand molecules on the vesicle surface. Experimental evidence obtained so far indicates that DRV liposomes bearing mannosylated albumin bind to mouse peritoneal macrophages selectively and in greater numbers than mannose-free vesicles. By doing so, ligand-coated liposomes potentiate immune responses to the liposome-entrapped antigen in immunized animals to levels above those achieved through the immunoadjuvant action of conventional (plain) liposomes. Available data, limited as it is, suggests that it is the number of ligand molecules on the surface of liposomes rather than the extent of ligand mannosylation that influences the adjuvanticity of the system. Thus, immune responses to an antigen, augmented by an adjuvant, can be improved

184

IN IMWJNOLOGY

41st FORUM

0.31

1.25

5

20

0.31

1.25

5

20

Tetanus Toxold tug x 10-21

Fig. 4. Immuneresponses after immunization with tetanustoxoid containing DRV coated with two different amountsof mannosylatedalbumin. BALB/c mice(in groupsof 5) wereinjectedintramuscularlywith tetanustoxoid-containing DRV composedof equimolarPC and cholesteroland coatedwith 1.36pg (m) or 0.75 pg (L;) mannosylated albumin(36 mannoseresiduesper albumin molecule).The massratio of lipid to toxoid was 67/O/ 1. Three weeksafter priming, all animalsreceivedan identical secondinjection of the samepreparations. Seracollected9 days later wereanalysedfor IgG1 and IgG2b (from Cregoriadiset al., 1988).

further by receptor-mediated targeting of the adjuvant to APC such as peritoneal macrophages. It would be of interest to see whether this targeted approach to immunopotentiation could be applied more selectively. For instance, it has been suggested(Allison and Byars, 1986) that strong and persistent immune responses may be elicited by directing antigens to interdigitating or follicular dendritic cells. These, unlike macrophages, constitutively express major histocompatibility (MHC) class II molecules and specialize in presenting antigen to T and B lymphocytes, respectively. By employing ligands which could target antigen-containing liposomes to either cells, cellmediated or humoural immunity could be favoured. The same principle would apply to target liposomes to other APC provided that ligands can be found which associate in some specific manner with a particular APC target. Some of these cells (e.g. B or dentritic ceils) appear to be much less active in endocytosing and catabolizing liposomal antigens (Dal Monte and Szoka, 1989) compared to macrophages. However, it is conceivable that such processes may be augmented as as result of (hposomal) ligand interaction with the relevant receptor.

Acknowledgements The work in the author’s laboratory wassupportedby a Medical ResearchCouncil project grant. I thank Ms. Janie Saundersfor excellent secretarialassistance.

References Allison, A.C. & Byars, N. (1986),An adjuvant formulation that selectivelyelicits the formation of antibodies of protective isotypes and of cell-mediated immunity. J. Immunol. Methods, 95, 157-168. Allison, A.C. & Gregoriadis. G. (1974), Liposomesas immunologicaladjuvants. Nature (Lond.), 252, 252. Almeida et al. (1975), Formation of virosomes from influenza virus subunitsand liposomes.Lancer, II, 889-890. Alving, C.R., Richards,R.L., Moss, J ., Alving, L.I., Clemerits, J.D., Shiba, T., Kotani, S., Wirtz, R.A. & Hockmeyer, W.T. (1986),Effectivenessof liposomes aspotential carriersof vaccines:applicationsto cholera toxin and human malaria antigen. Vaccine, 4, 166-172. Bakouche,Q.. David, F. & Gerlier, D. (1987),Impairment of immunogenicity by antigen presentationin liposomesmadefrom dimyristoylphosphatidylethanolaminelinked to the secretionof prostaglandin.Europ. J. Immunol., 17, 1839-1842. Beatty, J.D., Beatty, B.G., Paraskevas,F. & Froese, E. (1984),Liposomesasimmuneadjuvants.T celldependence. Surgery. 96, 345-351. Dal Monte, P. & Szoka, F.C. (1989), Effect of liposome encapsulation on antigen presentation in vitro: comparisonof presentationsby peritonealmacrophagesand B cell tumours.J. Immunol., 142,1437-1443. Davis, D. & Gregoriadis, G. (1987), Liposomesas adjuvants with immunopurifiedtetanustoxoid : influence of liposomal characteristics. Immunology. 61, 229-234. Davis, D., Davies, A. & Gregoriadis, G. (1987), Liposomes as adjuvants with immunopurified tetanus toxoid : the immuneresponse.Immunol. Letters, 14, 341-348.

LIPOSOMES

AND

MACROPHAGE

Francis, M.J., Fry, C.M., Rowlands, D.J., Brown, F., Bittle, J.L., Houghten, R.A. & Lerner, R.A. (1985), Immunological priming with synthetic peptides of foot-and-mouth disease virus. J. gen. Viral., 66, 2347-2354. Garcon, N., Senior, J. & Gregoriadis, G. (1986), Coupling of ligands to liposomes before entrapment of agents sensitive to coupling procedures. Biochem. Sot. Trans., 14, 1038-1039. Garcon, N., Gregoriadis, G., Taylor, M. & Summerfield, J. (1988), Mannose-mediated targeted immunoadjuvant action of liposomes. Immunology, 64,743-745. Gregoriadis, G. (1976), The carrier potential of liposomes in biology and medicine. New Engl. J. Med., 295, 704-7 10 and 765-770. Gregoriadis, G. (1984), “Liposome technology” vol. 1, 2 & 3, CRC Press, Boca Raton, FL. Gregoriadis, G. (1988a), “Liposomes as drug carriers: recent trends and progress” John Wiley and Sons, Chichester. Gregoriadis, G. (1988b), Fate of injected liposomes: observations on entrapped solute retention, vesicle clearance and tissue distribution in vivo, in : “Liposomes as drug carriers: recent trends and progress” (G. Gregoriadis) (pp. 3-18). John Wiley and Sons, Chichester. Gregoriadis, G., Garcon, N., Senior, J. & Davis, D. (1988), The immunoadjuvant action of liposomes : nature of immune response and influence of liposomal characteristics, in “Liposomes as drug carriers: recent trends and progress” (G. Gregoriadis) (pp. 279-307). John Wiley and Sons, Chichester. Gregoriadis, G. (1990), Immunological adjuvants : a role for liposomes. Immunol. Today, 11, 89-97. Gregoriadis, G. &Allison, A.C. (1974), Entrapment of proteins in liposomes prevents allergic reactions in preimmunised mice FEBS Letters, 45, 71-74. Gregoriadis, G. & Neerunjun, D. (1975), Homing of liposomes to target cells. Biochem. biophys. Res. Commun., 65, 537-544. Gregoriadis, G. & Ryman, B.E. (1972), Fate of proteincontaining liposomes injected into rats. An approach to the treatment of storage diseases. Europ. J. Biothem., 240, 485-491. Gregoriadis, G., Neerunjun, D. & Hunt, R. (1977), Fate of a liposome-associated agent injected into normal and tumour-bearing rodents. Attempts to improve localization in tumour tissues. Life Sci., 21,357-370. Gregoriadis, G., Allison, A.C. & Poste, G. (1989), “hnmunological adjuvants and vaccines”. Plenum Press, New York.

FUNCTIONS

185

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