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Liposome adjuvanticity: Influence of dose and protein: Lipid ratio on the humoral response to encapsulated and surface-linked antigen
Liposome Adjuvanticity: Influence of Dose and Protein:Lipid Ratio on the Humoral Response to Encapsulated and Surface-Linked Antigen HI%J%E-MARIE
‘I‘III?RIEN.’
ELIAM.
SHAHUM,
AND
ANDR&
FORTIN
The humoral response to hovine Serum albumin either encapsulated m or surface-linked to hposomes was studied as a function of dose and protein:hpid ratio. Total immunogIobulin, total IgG. IgM. and the G isotypes. IgGJ. IgGZa. and IgG3 were measured during the plateau phase of production after a boosting injection. Although the adjuvant character of liposomes was confirmed regardless of the mode of antigen association, important differences in the response to the two types of liposomal formulations were observed. Our results suggestthat surface-linked antigen stimulates the immune system at lower doses than its encapsulated counterpart. is more sensitive to the proteinlipid ratios. and can stimulate the production of particular immunoglobulin isotypes in controlled conditions. Our data support the idea that different pathways of processing are utilized by the two forms of liposomal antigen. 1~ 1991 AG&III~C PW IIII
IiVTKODIJCTION Liposomes are artificially made membranous vesicles essentially composed of naturally occurring phosphohpids. Because of their ability to modulate membrane composition or function and to vehiculate in the organism, without significant toxicity. hydrophobic as well as hydrophilic substances, liposomes have been considered potential tools for a wide variety of in viva applications. Although many of their expected applications have been seriously hampered by their rapid removal from circulation by phagocytic cells and their inability to cross the endothelial barrier, they have repeatedly been shown to be highly efficient in the field of immunomodulation therapy and prophylaxy, whether it is in the design of synthetic and harmless vaccines or in the targeting of immunopotentiating drugs to specialized cells of the immune network (I, 2). In view of these observations. it appears worthwhile to more thoroughly characterize their immunopotentiating properties and to explore the possibility of manipulating more rationally, with their use, the fate of an immune response. ’ To whom correspondence should be addressed
LIPOSOME
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403
In our study on the potentiation of the humoral response, we used as a model system, a soluble antigen, bovine serum albumin (BSA)2 either encapsulated in or surface-linked to liposomes. The adjuvant character of liposomes together with the absolute requirement of a physical association between antigen and liposomes for this adjuvanticity to be observed were clearly demonstrated (3, 4), in agreement with observations made for several other protein antigens (5). More interestingly however, we also reported some differences that exist between the two types of antigen-liposome formulations in terms of isotype distribution pattern and duration of the antibody response (6). We suggested that these differences could be attributed to a differential processing of the antigen by antigen-presenting cells (APCs), leading to stimulation of distinct activation pathways and could be used with profit to orient the immune response. In the murine system, two different subsets of cloned T helper cells which respond to activation by producing specific lymphokines with the consequent induction of specific immunoglobulin isotypes have been identified (7, 8). Th 1 secrete interleukin2 (IL-2) and interferon-y (EN--r) which activate cellular immunity and stimulate IgG2a while Th2 produce interleukin4 (IL-4) which stimulates IgGl and IgE production (9, 10). Although both types of helper cells can be activated through interaction with different APCs, it has been proposed that presentation by B cells favors Th2 activation and presentation by macrophages, the Thl pathway (11). The existence of these separate T helper subsets has not yet been clearly demonstrated in vivo and this functional duality might potentially be induced, under normal conditions, by specific activation signals acting on a pluripotent T helper cell ( 12). In any case, evidence exists to suggest that Thl or Th2 types of responses can be generated in viva depending on the adjuvant used for immunization (12, 13). The influence of lymphokines on immunoglobulin isotype production has also been demonstrated in viva although the resultant effect is not as clear cut as it is in vitro (12). Besides these T-dependent effects, McKeam et al. associate T-independent B cell activation with a dominant IgM and IgG3 production (14) opening therefore the possibility or discrimination between different routes of activation through measurements of immunoglobulin isotypes. In turn, since the different immunoglobulin isotypes differ in their physiological properties such as complement fixation or binding to IgFc receptors on various cell types, all these observations suggest that the fate of an immune response can be influenced by modifying the pathways of antigen processing and/or presentation with the consequent induction of a specific pattern of isotype production. In this perspective, liposomes constitute a particularly interesting tool due to the ease in controlling the mode of antigen presentation to the immune network and this, independently of any gross chemical modification in the adjuvant vehicle. It is known that, in vim, liposomes are naturally targeted to macrophages of the reticuloendothelial system by which they are phagocytosed and degraded (15). This natural targeting of liposomes constitutes a very efficient mean to focus relatively large * Abbreviations used: ABTS, 2,2’-azinobis(3-ethylbenzthiazohne)sulfonic acid; APC, antigen-presenting cell; BSA, bovine serum albumin; DMPC, dimyristoylphosphatidylcholine; DPPE, dipalmitoylphosphatidylethanolamine; DTT, dithiothreitol; IFN-y, interferon gamma; IL-2, interleukin-2; IL-4, interleukin-4; PE-DTP. dipalmitoylphosphatidylethanolamine dithiopyridine; SPDP, 3-(2-pyridyldithio)propionic acid Nhydroxysuccinimide ester.
404
I-HERIEN, SHAHUM.
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quantities of antigen to cells specialized in the initiation of the immune response. This pathway of activation should lead to the induction of a T-dependent, macrophagedependent immune response. Although this pathway appears to be the main one accessible for the masked encapsulated antigen, additional interactions can be envisaged for the covalently linked antigen during the life span of the liposomal vehicle in body fluids, such as direct interaction with specific membrane receptors on immunocompetent cells of the T and B lineages. These differences in interactions with immunocompetent cells between the encapsulated and the surface-linked antigen could in turn lead to specific isotype production and should be influenced by the protein:lipid ratio as well as by the dose of antigen administered. In order to test these possibilities. we measured the production of total immunoglobulin, total IgG, IgG I, IgG2a, IgG3. and IgM in response to various doses of BSA either encapsulated in or covalently linked to liposomes at different protein:lipid ratios. MATERIALS
AND METHQDS
Bovine serum albumin (BSA), dimyristoylphosphatidylcholine (DMPC). dipalmitoylphosphatidylethanolamine (DPPE), cholesterol, 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP), 2,2’-azinobis(3-ethylbenzthiazoline) sulfonic acid (ABTS), dithiothreitol (DTT), and Hepes were purchased from Sigma Chemical Co. (St. Louis, MO). Mini filtration columns PD-10 prepacked with Sephadex G-25M and used for BSA modification were obtained from Pharmacia (Uppsala, Sweden). Microtest assay plates were from Becton-Dickinson (Oxnard, CA) and IrsI, protein iodination grade, from New England Nuclear Canada Ltd. (Lachine. Quebec). Horseradish peroxidase-labeled goat anti-mouse total immunoglobulin, IgG and IgM (Fc specific), IgG I, IgG2a, and IgG3 (subclass specific) were all obtained from Nordic Immunological Lab. (Tilburg, the Netherlands).
Colonies of BALB/c mice. originally purchased from Charles River Canada, were expanded in our animal house and kept with food and drink ad libitum. Experiments were performed on g- to 12-week-old male or female mice. Mice were injected twice with the different antigenic formulations at 32-day intervals. The injections were all given intraperitoneally in a total volume of 250 ~1 of Hepes buffer (10 m,M Hepes. pH 7.4, 0.9% NaCl). Antibody titers were measured on blood collected by cardiac puncture on Day 11 after the boosting injection. At this time point, the antibody titers are maximal for free BSA as well as for liposomal BSA as previously determined in a kinetic study of the response (4. 6). Liposome Preparatir)n Liposomes composed of DMPC. cholesterol, and DPPE in a molar ratio of 63:3 1: 6 were prepared using an extrusion technique (Lipex Biomembranes Inc., Vancouver. Canada) as previously described ( 16) except for the use of 0.2-pm pore size polycarbonate filters instead of the 0.4~pm ones. When BSA was covalently coupled to the liposomal surface. DPPE was replaced by its modified counterpart, dipalmitoylphos-
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phatidyl ethanolamine-dithiopyridine al. (17).
405
(PE-DTP), prepared according to Leserman et
Association of Antigen with Liposomes Encapsulation. BSA was incorporated into liposomes in the course of their formation by resuspension of the dry lipid film in Hepes buffer containing the protein. Encapsulated BSA was separated from free BSA by two ultracentrifugations carried out at 12°C on a LKB ultracentrifuge using a 70.1 Ti rotor run for 30 min at 35,000 rpm. Covalent linkage. Surface coupling of BSA to liposomal surface was achieved following modification of both DPPE and protein with the heterobifunctional reagent SPDP, according to the method described by Leserman et al. ( 17). BSA was modified at a molar ratio SPDP:protein of 10: 1 and then activated with 50 mM DTT. At the end of the incubation with modified liposomes, free protein was separated from covalently linked BSA by ultracentrifugation as described for the encapsulated protein. The amount of protein associated with liposomes was estimated by assaying rz51 radioactivity of labeled BSA used as tracer throughout the preparations. BSA was iodine-labeled by the iodogen method (18). BSA-Specific Serum Antibody Titers Anti-BSA total immunoglobulin, IgG, IgM, IgGl , IgG2a, and IgG3 were measured by a microtiter plate enzyme-linked immunosorbent assay (ELISA) as previously described ( 19). Antibody titers are expressed as log2 of reciprocal dilutions. RESULTS For these experiments, we first standardized our methods of liposomal preparations in order to control with reproducibility the levels of protein association in both cases of encapsulation and covalent linkage. The Fry technique (20) which we used previously to separate free from associated protein was omitted since it was found to yield grossly overestimated ratio of protein:lipid due to difficulty in column calibration with ensuing incomplete and variable separation. This technique was replaced by a double ultracentrifugation which is less susceptible to variations from preparation to preparation. In all cases, the standardization was carried out with a fixed concentration of lipids ( 10 pmol DMPC/ml). The results obtained for encapsulation show that the encapsulated volume decreases with increasing concentrations of BSA (Fig. 1) but that the linearity of the relationship makes it possible to set the conditions required to obtain some specific protein:lipid ratio. For priming and boosting preparations, we obtained, respectively, 49 and 56, 8.9 and 8.7 or, 1 and 1.3 kg protein/pmol DMPC. The efficiency of covalent linkage is shown in Fig. 2. In most of our experiments, modified BSA, in 10 mMHepes, pH 7.4,0.9% NaCl was transferred into 0.1 Macetate buffer, pH 4.5, 0.9% NaCl by gel filtration on G-25 Sephadex equilibrated in the acetate buffer, prior to activation with DTT. A maximum efficiency of 80 pg BSA/ pmol DMPC could be achieved using this procedure. However, in the course of these experiments, we observed that if the modified protein was directly acidified with onefifth of its volume of acetate buffer prior to activation, the yield of covalent linkage
1
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.-.---_--
--i
50
il3SAl (mg/mL) f-tc;. I. Dry hprd films were resuspended wtth fiepes buffer containing After elimination of unassociated BSA. encapsulated protein was evaluated
various concentrattons by gamma counting.
of BSA.
was greatly improved. In the reported experiments with surface-linked antigen. this particular method of BSA preparation was only used to obtain the highest protein lipid ratio. In Fig. 3, we compare the total anti-BSA antibody titers elicited by encapsulated BSA at different ratios of BSA:pmol DMPC and by free BSA, for different doses of immunizing antigen. At ratios of 1 and 8 pg BSA/pmol DMPC, the intensity of the response is logarithmically related to the dose injected for the range of tested doses.
Modified
BSA
(mg/mL)
F’tci. 2. Modified liposomes (IO ~mol DMPC‘/mf) were resuspended with Hepes buffer contammg vartous concentrations of modified and activated BSA. After efimination of unassociated BSA. epitope density was evaluated by gamma counting. (0) BSA activated with DTT after transfer in acetate buffer by gel filtration chromatography. (0) BSA activated with DTT after direct acidification of medium with concentrated acetate buffer.
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0.1
0.5
1
407
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5
Immunizing
10
50
doses
100
500
(pg)
FIG. 3. Total immunoglobulin anti-BSA antibody titers obtained the 1lth day postboosting injection in response to different doses of either free BSA (X) or encapsulated BSA at protein:lipid ratio of 53 (0) 8.8 (m) and I .2 (0) pg:rmol DMPC. The results are expressed as log, of serial dilutions and represent the mean response of five individual mice.
However, at the higher ratio (50 pg/pmole), a drop of the response was observed for injected doses below 1.5 pg. The behavior of surface-linked BSA in analogous conditions is shown in Fig. 4. In contrast to what is observed with encapsulated BSA, the logarithmic relationship between intensity of response and immunizing doses is maintained over the entire range of tested doses and for all protein:lipid ratios, including the higher ratio of 150 pg BSA:pmol DMPC. Although the results shown are those obtained for total specific immunoglobulin production, similar relationships between dose and
16.
Immunizing
doses
(pg)
FIG. 4. Total immunoglobulin anti-BSA antibody titers obtained the 1 Ith day postboosting injection in response to different doses of either free BSA (X) or covalently linked BSA at epitope density of 150 (0), 50 (O), 10 (0) and 1 (B) pg:prnol DMPC. Results are expressed as log, serial dilutions and represent the mean response of five individual mice.
Protein:iipid (pg BSAl~mole
ratio DMPC)
FE 5. Dose-rcsponsc curves obtained with encapsulated ([I) and covalently linked(m) BSA were analyzed by linear regression statistics. Ail responses could be approximated to a straight line with correlation coefficient greater than 0.95. The slopes ofthesc linear approximations were calculated for each type of immunoglobulin measured at the different protein:lipid ratios. Results represent the mean slope I? standard deviation obtained for total lg. IgG. IgG I, and IgGZa at one specilic protein:lipid ratio. All these immunoglobulin isotypes were shown to behave similarly and were therefore treated together in the calculations presented.
response were obtained for all ofthe immunoglobulin isotypes studied, including total IgG. IgGl. IgGZa, IgG3, and IgM. When the linear part of the dose-response curves is considered, one observes no influence of the protein:lipid ratio on the dose-dependency of the response to encapsulated BSA. This observation holds true for all types of immunoglobulin tested as
or 3 ;:
m
4-l
33
i 1 '
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f-----1
5
I 10
Protein:lipid (Kg BSA/&mole
I 50
I 100
1 500
ratio DMPC)
FIG. 6. The data obtained for IgM (open symbols) and Igti3 (dark symbols) for encapsulated surface-linked (squares) antigen were analyzed as explained in the legend to Fig. 5
(cu-cles)
and
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shown in Figs. 5 and 6 where the slopes of the linearized dose-response curve have been plotted as a function of the protein:lipid ratio. Under the same conditions, a more complex relationship is seen for the surface-linked antigen. The productions of total Ig, IgG 1, and IgG2a are all markedly influenced as indicated by the increased dependency of the response on the dose administered with increasing protein:lipid ratios (Fig. 5) while that of IgM and IgG3 are not (Fig. 6). If one now considers the absolute titer values and compares the isotype distribution pattern obtained in the various tested conditions for both modes of antigen formulations (Fig. 7), three main observations can be noted. First, the difference in IgG3 production between encapsulated and covalently linked antigen increases with increasing ratio of protein:lipid as well as with increasing dose of antigen. At a ratio of 50 pg BSA/pmol, IgG3 production is always significantly higher for surface-linked than for encapsulated antigen and the level of significance increases with increasing dose of BSA. At the intermediate ratio of 10 pg/pmol, the difference in IgG3 production is only significant at the higher immunizing dose and at the ratio of 1 pg/pmol, no difference is observed at any of the doses tested. The reverse is true for IgG2a production which increases in response to surface-linked antigen as the protein:lipid ratio and the dose-administered decrease. Third, in the low dose-high ratio situation where the response to encapsulated BSA drastically drops for all immunoglobulin isotypes measured, covalently linked antigen still behaves as a potent immunogen with an immunoglobulin production which is always significantly different from that obtained with its encapsulated or free counterpart. In these controlled comparative studies carried out under similar conditions of doses and protein:lipid ratios during the plateau phase of immunoglobulin production after the boosting injection, no significant differences in the relative production of total IgG and IgM were observed between the two forms of liposomal antigen (Table 1). DISCUSSION This comparative study on the humoral response to encapsulated and covalently linked antigen puts light on several of the homologies and differences that exist between the two forms of antigen formulations. Both types of liposomal systems behave as potent stimulators of the humoral response relative to free antigen but differ in the conditions required for this potentiation to be expressed. These behavioral differences, as expected, are more exacerbated in the extreme conditions tested of either dose or protein:lipid ratio. The potentiation of the humoral response by liposomes is apparently neither related to an increased immunogenicity of the associated protein nor to a stimulatory effect of lipids since response of comparable intensity could be induced by free BSA, provided immunizing doses are sufficiently elevated. Our observations more strongly support the idea that adjuvanticity of liposomes mainly resides in their more efficient focusing of antigen to APCs. On the other hand, if this is really the case, one would expect to observe a magnified liposomal effect with increasing concentration of antigen in individual vesicle. However, such an effect is not observed, in agreement with previous data reported by Davis and Gregoriadis working with encapsulated immunopurified tetanus toxoid (2 1). On the basis of our results obtained with low doses of encapsulated
410 A.
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FIG. 7. Specific anti-B% IgCl I ~IgCi2a, and igG.3 response to various doses of encapsulated (plain columns) or covalently linked (dotted column) antigen associated to iiposomes at protein:lipid ratio of either SO(A), IO (B), or I (C) fig BSA:Gmol DMPC. Results are expressed as log2 serial dilution and represent the mean response + SEM of five individual mice. The data were subjected to Student I test statistical analysis and the number of stars indicates the level of significance of the difference between encapsulated and surfacclinked antigen, *P 41 0.05: **P c 0.01: ***I’ c 0 001.
antigen at diKerent protein:lipid ratios. we suggest, as a possible explanation to these apparently conflicting hypotheses, that a threshold dose must be delivered to a minimal number of APCs in order for the response to be maximally triggered. Increasing the dose or the number of APCs activated above the threshold level only slightly affects
LIPOSOME
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ADJUVANTICITY TABLE I
Antibody titers Encapsulated Ratio (rg/mnol DMPC)
Dose (4
T
G
Covalently linked BSA M
T
G
M
50
1 11.4 -+ 0.4 11.2 i 0.6 7.9 + 0.5 12.9 f 1.6* 1.5 9.9 kO.9 9.8 f 0.3 5.8 zk 0.7 9.3 2 0.1 0.15 2.8 f 1.7 2.0 * 1.4 N.D. 6.8 f l.O**
13.3 f 1.5* 9.5 + 0.5 6.2 2 0.4**
7.4 f 0.3 5.9 + 1.2 4.4 -c 0.6**
IO
15 13.2?0.7 11.6+0.4 7.OkO.5 14.1+1.3 1.5 10.3 + 0.6 9.4 + 0.5 5.3 + 0.3 12.1 + 0.3** 0.15 8.9?0.8 8.6kO.3 4.4kl.l 10.2k1.2
13.7 f 0.7*** 11.5 t 0.9*** 10.1 + 2.0
8.7 f 0.4*** 7.9 + 0.6** 5.2 f 1.0
I
13.2f1.4 13.1kl.l 7.3kO.7 13.3kO.8 15 1.5 11.0 + 1.2 10.3 + 0.8 6.7 f 1.0 12.4 +- 0.6 0.15 9.5kO.5 8.8kl.O 5.8kl.O 11.7+2.0**
12.3 -t 0.3 Il.5 f 1.2 10.3 * 0.5*
8.2 f 0.7 7.5 * 0.9 6.8 + 0.9
Total Ig, IgG, and IgM anti-BSA antibody titers were measured after immunization with various doses of encapsulated or covalently linked BSA associated to liposomes at three distinct protein:lipid ratios. The results are expressed as log, serial dilution and represent the mean response of five individual mice + standard deviation. Symbols refer to the level of significance of the difference between encapsulated and covalently linked antigen as determined by Student t test analysis. * P < 0.05; **p < 0.001; ***P < 0.01.
the intensity of the response as can be seen by the relatively low dose-response and protein:lipid ratio dependencies. Factors in addition to those previously mentioned might operate in the response to surface-linked antigen which, in analogous conditions, exhibits a much more complex behavior. At the lower ratio tested, the dose-dependency is almost totally abolished and one observes a change in isotype distribution pattern as the protein:lipid ratio is varied from 150 to 1 pg/pmol DMPC. This particular behavior supports our initial proposal that pathways of activation in addition to those accessible to the encapsulated antigen can be used by surface-linked antigen. According to our present and previously reported data (3, 6) these circuits appear to be of higher performance since they are more rapidly turned on, remain activated for a longer period, and respond to lower doses of antigen. These overall characteristics make surface-linked antigen a better adjuvant than its encapsulated counterpart, in agreement with the general conclusions of Vannier and Snyder (22). However, the poor efficiency of encapsulated antigen they reported contrasts with our observations as well as with those of several workers (5) and may simply have resulted from the different levels of association they achieved for the two modes of antigen presentation. As mentioned, the response to surface-linked antigen differs from that of encapsulated antigen not only by its greater intensity over a wider range of experimental conditions but also by its privileged induction of specific immunoglobulin isotypes, depending on the characteristics of the liposomal antigen. The surprising finding was that this isotype pattern was influenced by the protein:lipid ratio, exhibiting increased IgG3 and increased IgG2a at high and low protein:lipid ratios, respectively. The relative
412
i‘HiRIEN.
SHAHUM.
AND
FOR’L’Iti
contribution of IgG1 or 1gM was not. m contrast, profoundly influenced under the same conditions and is identical to that observed in response to the encapsulated form of the antigen. This latter observation differs from that which we previously reported and was attributed to the longer lag time between the priming and boosting injections (32 versus 21 days), since. in a kinetic study, we noticed a long-lasting primary IgM response to surface-linked antigen that might have significantly contributed to the IgM production measured after the shorter lag-time period (6). These results emphasize the importance of specifying measurement conditions in the analysis of liposome adjuvanticity. The lack of effect of our experimental conditions and of the mode of antigen association on IgG I production agrees with data reported by Davis and Gregoriadis (2 1). Although our data support the involvement of different routes of antigen processing and presentation for encapsulated and surface-linked antigen. the identification of these pathways remains at the moment a matter of speculation. Since it has been repeatedly demonstrated that liposomes are naturally targeted in biro to phagocytic cells of the reticuloendothelial system ( 15). it is difficult to exclude the participation of macrophages in the adjuvant effect of liposomes whether the antigen is associated by encapsulation or covalent-linkage at the surface. However, if macrophages can be considered a priori as the principal if not the unique route accessible to encapsulated antigen, various types of liposome-immune cell interactions can be hypothesized for the surface-linked protein, These potential interactions include such nonmutually exclusive phenomena as a direct interaction of the exposed antigen with surface receptors such as membrane immunoglobulins or, once the immune response is initiated. an interaction through IgFc receptors. The high IgM production ofthe primary response and the observed increased production of IgG3 after boosting injections in conditions of high doses, high protein:lipid ratio suggest the involvement of B cells as accessory cells in the response to surface-linked protein. This conclusion is consistent with the works of M&earn e2 al. (14) who associated a high production of IgM and IgG3 with a T-independent B cell activation and those of Walden C/ uf. (23) who demonstrated that specific immune cells can be triggered by surface liposomal antigen presented at sufficiently high epitope density. The gradual changes in isotype production leading to increased production of IgG2a that are observed with decreasing dose and decreasing protein:lipid ratio in response to surface-linked antigen might reflect the balanced interactions between stimulatory lymphokines. Whatever the precise mechanisms involved in antigen processing and presentation depending on the mode of association with a liposomal vehicle, our data demonstrate that the type of association has profound influence on the intensity as well as on the quality of the finally induced immune response. It should therefore be possible to design liposomal systems capable of stimulating the type of response that is wanted. depending on the desired goal. AC.‘KNUWL.EDGMENTS We are grateful to Ms. Lyne Hourhard and S$VIC Christian for thexr skillful techmcal asslstancc. work was supported by grants from the Natural Sciences and Engineering Council of Canada.
fins
REFERENCES I
Ostro. M. J.. td.. 19x7.
“Liposomcs:
born
Biophysics
to Thcrapcutics.
Marcel
Dekker
Inc..
Kew
York.
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2. Gregoriadis, G., Ed., “Liposomes as Drug Carriers. Recent Trends and Progress.” Wiley, Chichester Sussex, 1988. 3. Shahum, E., and ThCrien, H.-M., Immunology 65,3 15, 1988. 4. ThCrien, H.-M., and Shahum, E., Immunol. Lett. 22, 253, 1989. 5. Van Rooijen, N., and Su, D., In “Immunological Adjuvants and Vaccines” (G. Gregoriadis, Ed.), pp. 159-170. Plenum, New York, 1989. 6. Therien, H.-M., Lair, D., and Shahum, E., Vaccine& 558, 1990. 7. Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A., and Coffman, R. L., J. Immunol. 136, 2348, 1986. 8. Kurt-Jones. E. A., Hamberg, S., Ohara, J., Paul, W. E., and Abbas, A. K., J. Exp. Med. 166, 1774. 1987. 9. Snapper, C. M., and Paul, W. E., Science 236,944, 1987. 10. Vitetta, E. S., Bossie, A., Fernandez-Botran, R., Myers, C. D., Oliver, K. G., Sanders, V. M., and Stevens, T. L., Immunol. Rev. 99, 193, 1987. 1 I. Abbas. A. K., Amer. J. Pathol. 129, 6, 1987. 12. Finkelman, F. D., Holmes, J., Katona, I. M., Beckmann, M. P., Park, L. S., Schooley, K. E., Coffmann, R. L., Mosmann, T. R., and Paul, W. E., Annu. Rev. Immunol. 8, 303, 1990. 13. Grun, J. L., and Maurer, P. H., Cell. Immunol. 121, 134, 1989. 14. McKearn, J. P., Paslay, J. W., Slack, J., Baum, C., and Davie, J. M., Immunol. Rev. 64, 5, 1982. 15. Hwang, K. L. In “Liposomes. From Biophysics to Therapeutics.” (G. Gregoriadis, Ed.), pp. 109-156. Marcel Dekker Inc., New York, 1987. 16. Shahum, E., and Therien, H.-M., Cell. Immunol. 123, 36, 1989. 17. Leserman, L. D., Machy, P., and Barbet, J., In “Liposome Technology” (G. Gregoriadis, Ed.) pp. 2940. CRC Press, Boca Raton FL, 1984. 18. Salacinski, P. R. P., McLean, C., Sykes, J. E. C., Clement-Jones, V. V., and Lowry, P. J., Anal. Biochem. 117, 136, 1981.
19. 20. 2 I. 22. 23.
ThCrien, H.-M., and Shahum, E., Cell. Immunol. 116, 320, 1988. Fry, D. W., White, J. C., and Goldman, I. D., Anal. Biochem. 90, 809, 1978. Davis, D., and Gregoriadis, G., Immunology 61, 229, 1987. Vannier, W. E., and Snyder, S. L., Immunol. Lett. 19, 59, 1988. Walden, P.. Nagy, Z. A., and Klein, J., J. Mol. Cell Immunol. 2, 191. 1986.
‘I‘III?RIEN.’
ELIAM.
SHAHUM,
AND
ANDR&
FORTIN
The humoral response to hovine Serum albumin either encapsulated m or surface-linked to hposomes was studied as a function of dose and protein:hpid ratio. Total immunogIobulin, total IgG. IgM. and the G isotypes. IgGJ. IgGZa. and IgG3 were measured during the plateau phase of production after a boosting injection. Although the adjuvant character of liposomes was confirmed regardless of the mode of antigen association, important differences in the response to the two types of liposomal formulations were observed. Our results suggestthat surface-linked antigen stimulates the immune system at lower doses than its encapsulated counterpart. is more sensitive to the proteinlipid ratios. and can stimulate the production of particular immunoglobulin isotypes in controlled conditions. Our data support the idea that different pathways of processing are utilized by the two forms of liposomal antigen. 1~ 1991 AG&III~C PW IIII
IiVTKODIJCTION Liposomes are artificially made membranous vesicles essentially composed of naturally occurring phosphohpids. Because of their ability to modulate membrane composition or function and to vehiculate in the organism, without significant toxicity. hydrophobic as well as hydrophilic substances, liposomes have been considered potential tools for a wide variety of in viva applications. Although many of their expected applications have been seriously hampered by their rapid removal from circulation by phagocytic cells and their inability to cross the endothelial barrier, they have repeatedly been shown to be highly efficient in the field of immunomodulation therapy and prophylaxy, whether it is in the design of synthetic and harmless vaccines or in the targeting of immunopotentiating drugs to specialized cells of the immune network (I, 2). In view of these observations. it appears worthwhile to more thoroughly characterize their immunopotentiating properties and to explore the possibility of manipulating more rationally, with their use, the fate of an immune response. ’ To whom correspondence should be addressed
LIPOSOME
ADJUVANTICITY
403
In our study on the potentiation of the humoral response, we used as a model system, a soluble antigen, bovine serum albumin (BSA)2 either encapsulated in or surface-linked to liposomes. The adjuvant character of liposomes together with the absolute requirement of a physical association between antigen and liposomes for this adjuvanticity to be observed were clearly demonstrated (3, 4), in agreement with observations made for several other protein antigens (5). More interestingly however, we also reported some differences that exist between the two types of antigen-liposome formulations in terms of isotype distribution pattern and duration of the antibody response (6). We suggested that these differences could be attributed to a differential processing of the antigen by antigen-presenting cells (APCs), leading to stimulation of distinct activation pathways and could be used with profit to orient the immune response. In the murine system, two different subsets of cloned T helper cells which respond to activation by producing specific lymphokines with the consequent induction of specific immunoglobulin isotypes have been identified (7, 8). Th 1 secrete interleukin2 (IL-2) and interferon-y (EN--r) which activate cellular immunity and stimulate IgG2a while Th2 produce interleukin4 (IL-4) which stimulates IgGl and IgE production (9, 10). Although both types of helper cells can be activated through interaction with different APCs, it has been proposed that presentation by B cells favors Th2 activation and presentation by macrophages, the Thl pathway (11). The existence of these separate T helper subsets has not yet been clearly demonstrated in vivo and this functional duality might potentially be induced, under normal conditions, by specific activation signals acting on a pluripotent T helper cell ( 12). In any case, evidence exists to suggest that Thl or Th2 types of responses can be generated in viva depending on the adjuvant used for immunization (12, 13). The influence of lymphokines on immunoglobulin isotype production has also been demonstrated in viva although the resultant effect is not as clear cut as it is in vitro (12). Besides these T-dependent effects, McKeam et al. associate T-independent B cell activation with a dominant IgM and IgG3 production (14) opening therefore the possibility or discrimination between different routes of activation through measurements of immunoglobulin isotypes. In turn, since the different immunoglobulin isotypes differ in their physiological properties such as complement fixation or binding to IgFc receptors on various cell types, all these observations suggest that the fate of an immune response can be influenced by modifying the pathways of antigen processing and/or presentation with the consequent induction of a specific pattern of isotype production. In this perspective, liposomes constitute a particularly interesting tool due to the ease in controlling the mode of antigen presentation to the immune network and this, independently of any gross chemical modification in the adjuvant vehicle. It is known that, in vim, liposomes are naturally targeted to macrophages of the reticuloendothelial system by which they are phagocytosed and degraded (15). This natural targeting of liposomes constitutes a very efficient mean to focus relatively large * Abbreviations used: ABTS, 2,2’-azinobis(3-ethylbenzthiazohne)sulfonic acid; APC, antigen-presenting cell; BSA, bovine serum albumin; DMPC, dimyristoylphosphatidylcholine; DPPE, dipalmitoylphosphatidylethanolamine; DTT, dithiothreitol; IFN-y, interferon gamma; IL-2, interleukin-2; IL-4, interleukin-4; PE-DTP. dipalmitoylphosphatidylethanolamine dithiopyridine; SPDP, 3-(2-pyridyldithio)propionic acid Nhydroxysuccinimide ester.
404
I-HERIEN, SHAHUM.
4ND FORTIN
quantities of antigen to cells specialized in the initiation of the immune response. This pathway of activation should lead to the induction of a T-dependent, macrophagedependent immune response. Although this pathway appears to be the main one accessible for the masked encapsulated antigen, additional interactions can be envisaged for the covalently linked antigen during the life span of the liposomal vehicle in body fluids, such as direct interaction with specific membrane receptors on immunocompetent cells of the T and B lineages. These differences in interactions with immunocompetent cells between the encapsulated and the surface-linked antigen could in turn lead to specific isotype production and should be influenced by the protein:lipid ratio as well as by the dose of antigen administered. In order to test these possibilities. we measured the production of total immunoglobulin, total IgG, IgG I, IgG2a, IgG3. and IgM in response to various doses of BSA either encapsulated in or covalently linked to liposomes at different protein:lipid ratios. MATERIALS
AND METHQDS
Bovine serum albumin (BSA), dimyristoylphosphatidylcholine (DMPC). dipalmitoylphosphatidylethanolamine (DPPE), cholesterol, 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP), 2,2’-azinobis(3-ethylbenzthiazoline) sulfonic acid (ABTS), dithiothreitol (DTT), and Hepes were purchased from Sigma Chemical Co. (St. Louis, MO). Mini filtration columns PD-10 prepacked with Sephadex G-25M and used for BSA modification were obtained from Pharmacia (Uppsala, Sweden). Microtest assay plates were from Becton-Dickinson (Oxnard, CA) and IrsI, protein iodination grade, from New England Nuclear Canada Ltd. (Lachine. Quebec). Horseradish peroxidase-labeled goat anti-mouse total immunoglobulin, IgG and IgM (Fc specific), IgG I, IgG2a, and IgG3 (subclass specific) were all obtained from Nordic Immunological Lab. (Tilburg, the Netherlands).
Colonies of BALB/c mice. originally purchased from Charles River Canada, were expanded in our animal house and kept with food and drink ad libitum. Experiments were performed on g- to 12-week-old male or female mice. Mice were injected twice with the different antigenic formulations at 32-day intervals. The injections were all given intraperitoneally in a total volume of 250 ~1 of Hepes buffer (10 m,M Hepes. pH 7.4, 0.9% NaCl). Antibody titers were measured on blood collected by cardiac puncture on Day 11 after the boosting injection. At this time point, the antibody titers are maximal for free BSA as well as for liposomal BSA as previously determined in a kinetic study of the response (4. 6). Liposome Preparatir)n Liposomes composed of DMPC. cholesterol, and DPPE in a molar ratio of 63:3 1: 6 were prepared using an extrusion technique (Lipex Biomembranes Inc., Vancouver. Canada) as previously described ( 16) except for the use of 0.2-pm pore size polycarbonate filters instead of the 0.4~pm ones. When BSA was covalently coupled to the liposomal surface. DPPE was replaced by its modified counterpart, dipalmitoylphos-
LIPOSOME
ADJUVANTICITY
phatidyl ethanolamine-dithiopyridine al. (17).
405
(PE-DTP), prepared according to Leserman et
Association of Antigen with Liposomes Encapsulation. BSA was incorporated into liposomes in the course of their formation by resuspension of the dry lipid film in Hepes buffer containing the protein. Encapsulated BSA was separated from free BSA by two ultracentrifugations carried out at 12°C on a LKB ultracentrifuge using a 70.1 Ti rotor run for 30 min at 35,000 rpm. Covalent linkage. Surface coupling of BSA to liposomal surface was achieved following modification of both DPPE and protein with the heterobifunctional reagent SPDP, according to the method described by Leserman et al. ( 17). BSA was modified at a molar ratio SPDP:protein of 10: 1 and then activated with 50 mM DTT. At the end of the incubation with modified liposomes, free protein was separated from covalently linked BSA by ultracentrifugation as described for the encapsulated protein. The amount of protein associated with liposomes was estimated by assaying rz51 radioactivity of labeled BSA used as tracer throughout the preparations. BSA was iodine-labeled by the iodogen method (18). BSA-Specific Serum Antibody Titers Anti-BSA total immunoglobulin, IgG, IgM, IgGl , IgG2a, and IgG3 were measured by a microtiter plate enzyme-linked immunosorbent assay (ELISA) as previously described ( 19). Antibody titers are expressed as log2 of reciprocal dilutions. RESULTS For these experiments, we first standardized our methods of liposomal preparations in order to control with reproducibility the levels of protein association in both cases of encapsulation and covalent linkage. The Fry technique (20) which we used previously to separate free from associated protein was omitted since it was found to yield grossly overestimated ratio of protein:lipid due to difficulty in column calibration with ensuing incomplete and variable separation. This technique was replaced by a double ultracentrifugation which is less susceptible to variations from preparation to preparation. In all cases, the standardization was carried out with a fixed concentration of lipids ( 10 pmol DMPC/ml). The results obtained for encapsulation show that the encapsulated volume decreases with increasing concentrations of BSA (Fig. 1) but that the linearity of the relationship makes it possible to set the conditions required to obtain some specific protein:lipid ratio. For priming and boosting preparations, we obtained, respectively, 49 and 56, 8.9 and 8.7 or, 1 and 1.3 kg protein/pmol DMPC. The efficiency of covalent linkage is shown in Fig. 2. In most of our experiments, modified BSA, in 10 mMHepes, pH 7.4,0.9% NaCl was transferred into 0.1 Macetate buffer, pH 4.5, 0.9% NaCl by gel filtration on G-25 Sephadex equilibrated in the acetate buffer, prior to activation with DTT. A maximum efficiency of 80 pg BSA/ pmol DMPC could be achieved using this procedure. However, in the course of these experiments, we observed that if the modified protein was directly acidified with onefifth of its volume of acetate buffer prior to activation, the yield of covalent linkage
1
,“,---.--.-.--,“,--;-.--------~--~~-
.-.---_--
--i
50
il3SAl (mg/mL) f-tc;. I. Dry hprd films were resuspended wtth fiepes buffer containing After elimination of unassociated BSA. encapsulated protein was evaluated
various concentrattons by gamma counting.
of BSA.
was greatly improved. In the reported experiments with surface-linked antigen. this particular method of BSA preparation was only used to obtain the highest protein lipid ratio. In Fig. 3, we compare the total anti-BSA antibody titers elicited by encapsulated BSA at different ratios of BSA:pmol DMPC and by free BSA, for different doses of immunizing antigen. At ratios of 1 and 8 pg BSA/pmol DMPC, the intensity of the response is logarithmically related to the dose injected for the range of tested doses.
Modified
BSA
(mg/mL)
F’tci. 2. Modified liposomes (IO ~mol DMPC‘/mf) were resuspended with Hepes buffer contammg vartous concentrations of modified and activated BSA. After efimination of unassociated BSA. epitope density was evaluated by gamma counting. (0) BSA activated with DTT after transfer in acetate buffer by gel filtration chromatography. (0) BSA activated with DTT after direct acidification of medium with concentrated acetate buffer.
LIPOSOME
0.1
0.5
1
407
ADJUVANTICITY
5
Immunizing
10
50
doses
100
500
(pg)
FIG. 3. Total immunoglobulin anti-BSA antibody titers obtained the 1lth day postboosting injection in response to different doses of either free BSA (X) or encapsulated BSA at protein:lipid ratio of 53 (0) 8.8 (m) and I .2 (0) pg:rmol DMPC. The results are expressed as log, of serial dilutions and represent the mean response of five individual mice.
However, at the higher ratio (50 pg/pmole), a drop of the response was observed for injected doses below 1.5 pg. The behavior of surface-linked BSA in analogous conditions is shown in Fig. 4. In contrast to what is observed with encapsulated BSA, the logarithmic relationship between intensity of response and immunizing doses is maintained over the entire range of tested doses and for all protein:lipid ratios, including the higher ratio of 150 pg BSA:pmol DMPC. Although the results shown are those obtained for total specific immunoglobulin production, similar relationships between dose and
16.
Immunizing
doses
(pg)
FIG. 4. Total immunoglobulin anti-BSA antibody titers obtained the 1 Ith day postboosting injection in response to different doses of either free BSA (X) or covalently linked BSA at epitope density of 150 (0), 50 (O), 10 (0) and 1 (B) pg:prnol DMPC. Results are expressed as log, serial dilutions and represent the mean response of five individual mice.
Protein:iipid (pg BSAl~mole
ratio DMPC)
FE 5. Dose-rcsponsc curves obtained with encapsulated ([I) and covalently linked(m) BSA were analyzed by linear regression statistics. Ail responses could be approximated to a straight line with correlation coefficient greater than 0.95. The slopes ofthesc linear approximations were calculated for each type of immunoglobulin measured at the different protein:lipid ratios. Results represent the mean slope I? standard deviation obtained for total lg. IgG. IgG I, and IgGZa at one specilic protein:lipid ratio. All these immunoglobulin isotypes were shown to behave similarly and were therefore treated together in the calculations presented.
response were obtained for all ofthe immunoglobulin isotypes studied, including total IgG. IgGl. IgGZa, IgG3, and IgM. When the linear part of the dose-response curves is considered, one observes no influence of the protein:lipid ratio on the dose-dependency of the response to encapsulated BSA. This observation holds true for all types of immunoglobulin tested as
or 3 ;:
m
4-l
33
i 1 '
j
f-----1
5
I 10
Protein:lipid (Kg BSA/&mole
I 50
I 100
1 500
ratio DMPC)
FIG. 6. The data obtained for IgM (open symbols) and Igti3 (dark symbols) for encapsulated surface-linked (squares) antigen were analyzed as explained in the legend to Fig. 5
(cu-cles)
and
LIPOSOME
ADJUVANTICITY
409
shown in Figs. 5 and 6 where the slopes of the linearized dose-response curve have been plotted as a function of the protein:lipid ratio. Under the same conditions, a more complex relationship is seen for the surface-linked antigen. The productions of total Ig, IgG 1, and IgG2a are all markedly influenced as indicated by the increased dependency of the response on the dose administered with increasing protein:lipid ratios (Fig. 5) while that of IgM and IgG3 are not (Fig. 6). If one now considers the absolute titer values and compares the isotype distribution pattern obtained in the various tested conditions for both modes of antigen formulations (Fig. 7), three main observations can be noted. First, the difference in IgG3 production between encapsulated and covalently linked antigen increases with increasing ratio of protein:lipid as well as with increasing dose of antigen. At a ratio of 50 pg BSA/pmol, IgG3 production is always significantly higher for surface-linked than for encapsulated antigen and the level of significance increases with increasing dose of BSA. At the intermediate ratio of 10 pg/pmol, the difference in IgG3 production is only significant at the higher immunizing dose and at the ratio of 1 pg/pmol, no difference is observed at any of the doses tested. The reverse is true for IgG2a production which increases in response to surface-linked antigen as the protein:lipid ratio and the dose-administered decrease. Third, in the low dose-high ratio situation where the response to encapsulated BSA drastically drops for all immunoglobulin isotypes measured, covalently linked antigen still behaves as a potent immunogen with an immunoglobulin production which is always significantly different from that obtained with its encapsulated or free counterpart. In these controlled comparative studies carried out under similar conditions of doses and protein:lipid ratios during the plateau phase of immunoglobulin production after the boosting injection, no significant differences in the relative production of total IgG and IgM were observed between the two forms of liposomal antigen (Table 1). DISCUSSION This comparative study on the humoral response to encapsulated and covalently linked antigen puts light on several of the homologies and differences that exist between the two forms of antigen formulations. Both types of liposomal systems behave as potent stimulators of the humoral response relative to free antigen but differ in the conditions required for this potentiation to be expressed. These behavioral differences, as expected, are more exacerbated in the extreme conditions tested of either dose or protein:lipid ratio. The potentiation of the humoral response by liposomes is apparently neither related to an increased immunogenicity of the associated protein nor to a stimulatory effect of lipids since response of comparable intensity could be induced by free BSA, provided immunizing doses are sufficiently elevated. Our observations more strongly support the idea that adjuvanticity of liposomes mainly resides in their more efficient focusing of antigen to APCs. On the other hand, if this is really the case, one would expect to observe a magnified liposomal effect with increasing concentration of antigen in individual vesicle. However, such an effect is not observed, in agreement with previous data reported by Davis and Gregoriadis working with encapsulated immunopurified tetanus toxoid (2 1). On the basis of our results obtained with low doses of encapsulated
410 A.
,
,--ISfig
-
-1.5119
-
1 2-
a-
4.
EL
12-
a-
4.
a
C. I 2-
a-
4-
A
FIG. 7. Specific anti-B% IgCl I ~IgCi2a, and igG.3 response to various doses of encapsulated (plain columns) or covalently linked (dotted column) antigen associated to iiposomes at protein:lipid ratio of either SO(A), IO (B), or I (C) fig BSA:Gmol DMPC. Results are expressed as log2 serial dilution and represent the mean response + SEM of five individual mice. The data were subjected to Student I test statistical analysis and the number of stars indicates the level of significance of the difference between encapsulated and surfacclinked antigen, *P 41 0.05: **P c 0.01: ***I’ c 0 001.
antigen at diKerent protein:lipid ratios. we suggest, as a possible explanation to these apparently conflicting hypotheses, that a threshold dose must be delivered to a minimal number of APCs in order for the response to be maximally triggered. Increasing the dose or the number of APCs activated above the threshold level only slightly affects
LIPOSOME
411
ADJUVANTICITY TABLE I
Antibody titers Encapsulated Ratio (rg/mnol DMPC)
Dose (4
T
G
Covalently linked BSA M
T
G
M
50
1 11.4 -+ 0.4 11.2 i 0.6 7.9 + 0.5 12.9 f 1.6* 1.5 9.9 kO.9 9.8 f 0.3 5.8 zk 0.7 9.3 2 0.1 0.15 2.8 f 1.7 2.0 * 1.4 N.D. 6.8 f l.O**
13.3 f 1.5* 9.5 + 0.5 6.2 2 0.4**
7.4 f 0.3 5.9 + 1.2 4.4 -c 0.6**
IO
15 13.2?0.7 11.6+0.4 7.OkO.5 14.1+1.3 1.5 10.3 + 0.6 9.4 + 0.5 5.3 + 0.3 12.1 + 0.3** 0.15 8.9?0.8 8.6kO.3 4.4kl.l 10.2k1.2
13.7 f 0.7*** 11.5 t 0.9*** 10.1 + 2.0
8.7 f 0.4*** 7.9 + 0.6** 5.2 f 1.0
I
13.2f1.4 13.1kl.l 7.3kO.7 13.3kO.8 15 1.5 11.0 + 1.2 10.3 + 0.8 6.7 f 1.0 12.4 +- 0.6 0.15 9.5kO.5 8.8kl.O 5.8kl.O 11.7+2.0**
12.3 -t 0.3 Il.5 f 1.2 10.3 * 0.5*
8.2 f 0.7 7.5 * 0.9 6.8 + 0.9
Total Ig, IgG, and IgM anti-BSA antibody titers were measured after immunization with various doses of encapsulated or covalently linked BSA associated to liposomes at three distinct protein:lipid ratios. The results are expressed as log, serial dilution and represent the mean response of five individual mice + standard deviation. Symbols refer to the level of significance of the difference between encapsulated and covalently linked antigen as determined by Student t test analysis. * P < 0.05; **p < 0.001; ***P < 0.01.
the intensity of the response as can be seen by the relatively low dose-response and protein:lipid ratio dependencies. Factors in addition to those previously mentioned might operate in the response to surface-linked antigen which, in analogous conditions, exhibits a much more complex behavior. At the lower ratio tested, the dose-dependency is almost totally abolished and one observes a change in isotype distribution pattern as the protein:lipid ratio is varied from 150 to 1 pg/pmol DMPC. This particular behavior supports our initial proposal that pathways of activation in addition to those accessible to the encapsulated antigen can be used by surface-linked antigen. According to our present and previously reported data (3, 6) these circuits appear to be of higher performance since they are more rapidly turned on, remain activated for a longer period, and respond to lower doses of antigen. These overall characteristics make surface-linked antigen a better adjuvant than its encapsulated counterpart, in agreement with the general conclusions of Vannier and Snyder (22). However, the poor efficiency of encapsulated antigen they reported contrasts with our observations as well as with those of several workers (5) and may simply have resulted from the different levels of association they achieved for the two modes of antigen presentation. As mentioned, the response to surface-linked antigen differs from that of encapsulated antigen not only by its greater intensity over a wider range of experimental conditions but also by its privileged induction of specific immunoglobulin isotypes, depending on the characteristics of the liposomal antigen. The surprising finding was that this isotype pattern was influenced by the protein:lipid ratio, exhibiting increased IgG3 and increased IgG2a at high and low protein:lipid ratios, respectively. The relative
412
i‘HiRIEN.
SHAHUM.
AND
FOR’L’Iti
contribution of IgG1 or 1gM was not. m contrast, profoundly influenced under the same conditions and is identical to that observed in response to the encapsulated form of the antigen. This latter observation differs from that which we previously reported and was attributed to the longer lag time between the priming and boosting injections (32 versus 21 days), since. in a kinetic study, we noticed a long-lasting primary IgM response to surface-linked antigen that might have significantly contributed to the IgM production measured after the shorter lag-time period (6). These results emphasize the importance of specifying measurement conditions in the analysis of liposome adjuvanticity. The lack of effect of our experimental conditions and of the mode of antigen association on IgG I production agrees with data reported by Davis and Gregoriadis (2 1). Although our data support the involvement of different routes of antigen processing and presentation for encapsulated and surface-linked antigen. the identification of these pathways remains at the moment a matter of speculation. Since it has been repeatedly demonstrated that liposomes are naturally targeted in biro to phagocytic cells of the reticuloendothelial system ( 15). it is difficult to exclude the participation of macrophages in the adjuvant effect of liposomes whether the antigen is associated by encapsulation or covalent-linkage at the surface. However, if macrophages can be considered a priori as the principal if not the unique route accessible to encapsulated antigen, various types of liposome-immune cell interactions can be hypothesized for the surface-linked protein, These potential interactions include such nonmutually exclusive phenomena as a direct interaction of the exposed antigen with surface receptors such as membrane immunoglobulins or, once the immune response is initiated. an interaction through IgFc receptors. The high IgM production ofthe primary response and the observed increased production of IgG3 after boosting injections in conditions of high doses, high protein:lipid ratio suggest the involvement of B cells as accessory cells in the response to surface-linked protein. This conclusion is consistent with the works of M&earn e2 al. (14) who associated a high production of IgM and IgG3 with a T-independent B cell activation and those of Walden C/ uf. (23) who demonstrated that specific immune cells can be triggered by surface liposomal antigen presented at sufficiently high epitope density. The gradual changes in isotype production leading to increased production of IgG2a that are observed with decreasing dose and decreasing protein:lipid ratio in response to surface-linked antigen might reflect the balanced interactions between stimulatory lymphokines. Whatever the precise mechanisms involved in antigen processing and presentation depending on the mode of association with a liposomal vehicle, our data demonstrate that the type of association has profound influence on the intensity as well as on the quality of the finally induced immune response. It should therefore be possible to design liposomal systems capable of stimulating the type of response that is wanted. depending on the desired goal. AC.‘KNUWL.EDGMENTS We are grateful to Ms. Lyne Hourhard and S$VIC Christian for thexr skillful techmcal asslstancc. work was supported by grants from the Natural Sciences and Engineering Council of Canada.
fins
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Ostro. M. J.. td.. 19x7.
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to Thcrapcutics.
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LIPOSOME
ADJUVANTICITY
413
2. Gregoriadis, G., Ed., “Liposomes as Drug Carriers. Recent Trends and Progress.” Wiley, Chichester Sussex, 1988. 3. Shahum, E., and ThCrien, H.-M., Immunology 65,3 15, 1988. 4. ThCrien, H.-M., and Shahum, E., Immunol. Lett. 22, 253, 1989. 5. Van Rooijen, N., and Su, D., In “Immunological Adjuvants and Vaccines” (G. Gregoriadis, Ed.), pp. 159-170. Plenum, New York, 1989. 6. Therien, H.-M., Lair, D., and Shahum, E., Vaccine& 558, 1990. 7. Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin, M. A., and Coffman, R. L., J. Immunol. 136, 2348, 1986. 8. Kurt-Jones. E. A., Hamberg, S., Ohara, J., Paul, W. E., and Abbas, A. K., J. Exp. Med. 166, 1774. 1987. 9. Snapper, C. M., and Paul, W. E., Science 236,944, 1987. 10. Vitetta, E. S., Bossie, A., Fernandez-Botran, R., Myers, C. D., Oliver, K. G., Sanders, V. M., and Stevens, T. L., Immunol. Rev. 99, 193, 1987. 1 I. Abbas. A. K., Amer. J. Pathol. 129, 6, 1987. 12. Finkelman, F. D., Holmes, J., Katona, I. M., Beckmann, M. P., Park, L. S., Schooley, K. E., Coffmann, R. L., Mosmann, T. R., and Paul, W. E., Annu. Rev. Immunol. 8, 303, 1990. 13. Grun, J. L., and Maurer, P. H., Cell. Immunol. 121, 134, 1989. 14. McKearn, J. P., Paslay, J. W., Slack, J., Baum, C., and Davie, J. M., Immunol. Rev. 64, 5, 1982. 15. Hwang, K. L. In “Liposomes. From Biophysics to Therapeutics.” (G. Gregoriadis, Ed.), pp. 109-156. Marcel Dekker Inc., New York, 1987. 16. Shahum, E., and Therien, H.-M., Cell. Immunol. 123, 36, 1989. 17. Leserman, L. D., Machy, P., and Barbet, J., In “Liposome Technology” (G. Gregoriadis, Ed.) pp. 2940. CRC Press, Boca Raton FL, 1984. 18. Salacinski, P. R. P., McLean, C., Sykes, J. E. C., Clement-Jones, V. V., and Lowry, P. J., Anal. Biochem. 117, 136, 1981.
19. 20. 2 I. 22. 23.
ThCrien, H.-M., and Shahum, E., Cell. Immunol. 116, 320, 1988. Fry, D. W., White, J. C., and Goldman, I. D., Anal. Biochem. 90, 809, 1978. Davis, D., and Gregoriadis, G., Immunology 61, 229, 1987. Vannier, W. E., and Snyder, S. L., Immunol. Lett. 19, 59, 1988. Walden, P.. Nagy, Z. A., and Klein, J., J. Mol. Cell Immunol. 2, 191. 1986.