liposomes as immunological adjuvants and vaccine carriers

ELSEVIER Journal of Controlled Release 41 (1996) 49-56

journal of controlled

release

Liposomes as immunological adjuvants and vaccine carriers

Gregory Gregoriadis*, Ihsan Gursel, Mayda Gursel, Brenda McCormack Centre for Drug Delivery Research, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC IN lAX,

UK

Received 5 October 1995; revised 17 January 1996; accepted 24 January 1996

Abstract

Work by numerous laboratories in the last two decades has shown that liposomes promote humoral and cell-mediated immunity to a large variety of bacterial, protozoan, viral and tumour cell antigens. This immunoadjuvant action of liposomes depends on their structural characteristics which control vesicle fate in vivo including the mode of antigen interaction with antigen-presenting cells. Liposomal adjuvanticity is further promoted by receptor mediated targeting to macrophages or the presence of co-adjuvants including cytokines. The immunoadjuvant action of liposomes is supplemented by their ability to act as a carrier for co-entrapped B and T-cell epitopes, thus eliminating the need for a carrier protein. A technique has been developed recently for the entrapment of live microbial vaccines into giant liposomes under conditions which retain their viability. Such liposomes (containing microbial vaccines and other soluble antigens or cytokines if required) could be used as carriers of vaccines in cases where there is a need to prevent interaction of vaccines with maternal antibodies or preformed antibodies to vaccine impurities.

Keywords: Liposomes; Antigens; Immune response; Immunological adjuvant; Vaccines; Interleukins

1. Introduction

A large number of structurally unrelated agents have been known for several decades to potentiate immune responses to weak vaccine antigens. Among these, best known are aluminium hydroxide (alum), saponins, Freund 's complete and incomplete adjuvants, and more recently, l ipopolysaccharide (LPS), muramyl dipeptide (MDP) derivatives, pluronic block co-polymers and l iposomes (see Refs. [1-4] for reviews on individual adjuvants). Most of these adjuvants, however, are toxic. Therefore, concerted efforts are being made to develop safe and effective adjuvants to meet the challenges of new-generation

Corresponding author.

vaccines [1-4] . These should consist of inexpensive, non-toxic and non-immunogenic materials, should be stable on storage, biodegradable and preferably promote both humoral and cell mediated immunity. In certain cases, they should also act synergistically together with other adjuvants (e.g. cytokines). Lipo- somes, also known for their potential and actual uses in targeted drug delivery [5], appear to satisfy many of these criteria [2,6].

2. Methodology for the incorporation of antigens and other agents into liposomes

Effective use of l iposomes in vaccines is depen- dent on simple and reproducible techniques for the

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50 G. Gregoriadis et al. / Journal of Controlled Release 41 (1996) 49 56

incorporation of antigens into vesicles which should ideally, exhibit a narrow size distribution. In this respect, there has been considerable progress recently [7] and a wide spectrum of l iposome-based drugs can now be produced by related biotechnology and cosmetics companies. Some of these formulations are currently being tested clinically [8-1 l] and a few are already licensed [11]. Techniques producing liposomes, however, can be limited in terms of being applicable only to certain drugs (e.g. those of low molecular weight) and others require the use of detergents, sonication or organic solvents [7]. On the other hand, a procedure developed over 10 years ago in the author 's laboratory [12-14] does not require such conditions. The procedure, producing mul- t i lamellar vesicles [15] and described elsewhere in detail [13,14], is simple, amenable to scale-up and involves dehydration of water-loaded small unilamellar vesicles (SUV) in the presence of free drugs, fol lowed by rehydration. Dehydration-rehydration vesicles (DRV) are known [12-16] to entrap up to 80% or more of drugs, cyclodextrin inclusion com- plexes (e.g. hydroxypropyl-/R-cyclodextrin complex with dehydroepiandrosterone, a drug presently used as a co-adjuvant for co-entrapped antigen; B. McCormack and G. Gregoriadis, in preparation), plasmid DNA, or antigens present (e.g. Table 1). Further, they can be freeze-dried in the presence of a

cryoprotectant, retaining most of their contents on reconstitution with saline [17]. DRV can also be microfluidized to smaller vesicles (mean diameter of around 100 nm) which retain much of the originally entrapped solute [18]. Dehydration-rehydration technology has recently led to the formation of giant vesicles (up to several microns in diameter) entrapping live microorganisms (e.g. B a c i l l u s sub t i l i s ) with an up to 60 -70% efficiency (Refs. [19,20] and I. Gursel and G. Gregoriadis, unpublished data) under conditions which preserve their viabili ty (see later). In short, a combination of DRV and microfluidization technologies are capable of generating well-char- acterized l iposomes composed of a variety of lipids and containing one or more antigens, cytokines, microbes and other agents for a wide range of uses as adjuvants or vaccine carriers.

3. Immunopotentiation with liposomes

Initial observations [21] of the immunological adjuvant properties of liposomes, have been confirmed and extended to include a wide range of antigens from bacteria, protozoa, viruses, tumours, spermatozoa, venoms and other sources [2,6,22]. It is now widely accepted that association (e.g. in form of antigen entrapped within the liposomal aqueous

Table 1 Entrapment of solutes in dehydration-rehydration vesicles

Solute Amount used Amount of phospholipid Entrapment (% of used)

Tetanus toxoid 1.00 mg 16 40-82 BSA 2.00 mg 16 40-43 RIVE 0.05 mg 16 29-31 A/Sichuan 0.05 mg 16 38-45 HBsAg 0.20 mg 16 31-33 Poliovirus 3-VP2 peptide 0.22 mg 16 62-68 Poliovirus I-VP2 peptide 0.22 mg 16 74-82 Leishmania major antigens (LV 39 mixed isolate) 0.20 mg 16 34-43 Interleukin-2 2.5 X 104-2.5 × 105 units 16 49-70 lnterleukin- 12 0.35-40.00 #g 16 20-28 Interleukin-15 2.5 × 104 2.5 × 105 units 16 32-45 PGL2 plasmid DNA 0.01-0.10 mg 32 ~ 40 55

32 b 45 63 32 ~ 40-92

HP-/3-CD/DHEA complex 0.5-5.0 mg (DHEA) 32 21-37% ~

~,, b and ~denote neutral, negatively charged and positively charged DRV, respectively; for futher details see ref. [13]. dDRV were composed of equimolar DSPC and cholesterol; egg phosphatidylcholine was used in all other preparations; HP/3-CD/DHEA denotes inclusion complex made of dehydroxyepiandrosterone and hydroxylpropyl-fl-cyclodextrin.

G. Gregoriadis et al. / Journal of Controlled Release 41 (1996) 49 -56 5!

phase, electrostatically adsorbed onto the bilayer surface or, as with membrane-soluble viral subunits, hydrophobically inserted into the lipid phase) of liposomes and antigen (rather than a non-interacting mixture of these) is a general prerequisite for adjuvanticity to occur [23,24]. Moreover, the diversity of liposomal formulations found to enhance immunogenicity [1-4,6,22] suggests that, generally, liposomal adjuvanticity is independent of specific vesicle features (e.g. composition, size, surface charge) or mode of immunization in terms of antigen choice or route and frequency of injection. Indeed, approaches to the formulation of liposome-associated antigens and protocols for immunization have been largely empirical. Yet, liposome-induced adjuvanticity was observed for nearly all antigens studied [1-4,6,22]. Nonetheless, it is likely that, for optimal function, liposomal characteristics may have to be tailored for individual antigens.

As shown already [25], liposomal adjuvanticity (in terms of humoral immunity; HI) occurs during primary immunization, and is observed with most IgG subclasses (e.g. IgG, IgG2a, IgG2b , IgG3) with no apparent shift observed in subclass responses when compared to responses obtained with the free antigen. It has also been found [26], that liposomes increase the proportion of IgG2a/2b to IgG 1 antibody levels. The mechanism of liposome-induced HI appears to be related to the fate of liposomes in vivo. It is likely, for instance, that antibody production is partly promoted as a result of liposomes acting as a depot supplying antigen to antigen presenting cells (APC) such as macrophages [23,27-29] at rates favouring its efficient processing by the cells and presentation, and partly through liposome migration to the lymph nodes [27]. Induction of cell-mediated immunity (CMI) is another important feature of liposomal adjuvanticity [6,22,32]. Evidence to this effect includes positive delayed-type by hyper- sensitivity (DTH) reactions [30], data from lymph node lymphocyte proliferation tests [31] and induction of cytotoxic T lymphocytes [32,33]. Liposome- induced CMI is unlikely, however, to be the result of the antigen-depot mechanism as adjuvants such as oil emulsions and alum acting similarly, induce pre- dominantly HI. A more plausible explanation for CMI is that antigens are presented by liposomes in a hydrophobic microenvironment in a way similar to

that exhibited by antigens conjugated to a lipidic moiety. Such antigens induce DTH in proportion to the lipid's hydrophobicity [34]. Events leading to CMI by liposomes may also be favoured by efficient vesicle localization in the regional lymph nodes [27,35]. Recent studies on liposomal adjuvanticity at the subcellular level suggest that, following the localization of vesicles in liposomes, degraded liposomal antigen is recycled to endosomes and presented to T cells in association with the major histocompatibility complex (MHC) class II molecules [36]. It has also been suggested that liposomes engender class I processing of the entrapped antigens in vitro. Apparently, this can only occur by employ- ing vesicles composed of lipids that render them unstable in the acidic milieu of the endosomes [33]: such (pH-sensitive) liposomes fuse with the endo- somal membrane and release their antigen in the cytosol where the antigen is processed and presented in the context of MHC class I molecules [33,36]. It appears, however, that the usefulness of pH-sensitive liposomes in this respect is limited to in vitro systems only. Thus, experiments with pH-sensitive and conventional (pH-insensitive) liposomes in vivo have shown [33] that both types induce antigen specific CD8+ CTL. As predicted by this author [37], although much of the liposomal antigen is carried to the macrophages [6,22,38] and processed in the lysosomes via the lysosomotric pathway, some of it is expected to escape into the cytoplasm. It has also been suggested [33] that a portion of the liposomal antigen gains access to dendritic cells which are responsible for promoting recognition in the specific CTL precursors. It is feasible [39] that dendritic cells acquire some of the antigen in vivo to retain it more efficiently than macrophages or, because of the increased density of class I molecules on their surface, such cells may require much less antigen for efficient presentation.

4. Strategies for the optimization of liposomal adjuvanticity

Approaches to further improve the immunoadjuvant action of liposomes include receptor-mediated targeting to macrophages [40], the use of a variety of co-adjuvants [6,22], the modification of the vesicles'

52 G. Gregoriadis et al. / Journal of Controlled Release 41 (1996) 49 -56

structural characteristics [6,41] and the use of cytokines [42-47]. For instance, liposomes coated with a mannose-terminating ligand promoted greater IgG responses in mice against entrapped tetanus toxoid than did ligand-free liposomes [39], presumably because of improved targeting of vesicles to the macrophages which are known to express mannose receptors on their surface. Administration of antigens in liposomes together with interleukin-2 (IL-2) has also proved to be an effective way to augment immune responses to a variety of antigens. These include tetanus toxoid [42,43], bacterial polysaccharide [44], influenza A virus [45], inactivated influenza virus [48] and HSV-recombinant glycoprotein D [47] vaccines. Recent evidence indicates a similar co-adjuvant activity for interleukins 12 and 15 (M. Gursel and G. Gregoriadis, in preparation).

It is likely that, on the basis of data on liposome- mediated HI and CMI [2,6,21], liposomal adjuvanticity reflects the vesicular structure of the system and, as already mentioned, its lipidic nature rather that the system's lipid composition or structural characteristics. Structural characteristics are known [5], however, to control the behaviour of liposomes in vivo and must therefore play a decisive role both qualitatively and quantitatively in the expression of immunoadjuvant activity. There are, for instance, numerous studies on the extent to which bilayer fluidity [25,41,48,49], number of bilayers [23], vesicle size [50], surface charge [51], lipid to antigen mass ratio [25,41,52] and mode of antigen localization within liposomes [23,25,47,52,53], influence adjuvanticity (for a detailed discussion see references [2,6]).

5. Liposomes as carriers of oligopeptide antigens

Liposomes are known to induce IgG responses to small peptides containing helper T-cell (Th-cell) epitopes [50,54], but they fail to do so for peptides containing B-cell epitopes alone [55]. The latter become immunogenic and promote immunological memory on coupling to Th-cell epitope-containing (carrier) proteins [56]. This approach is, however,

associated with disadvantages, including immune responses to the carrier proteins, masking of epitopes, antigenic competition [54] and the require- ment of a potent adjuvant such as complete Freund's adjuvant [57]. Studies of peptide immunogenicity also indicate that B-cell peptides may become immunogenic through co-polymerization [58] and covalent coupling [59] or co-linear synthesis [60] to a Th-cell peptide sequence. Recently, it has been suggested [61] that use of a mixture of B- and Th-cell peptides may be sufficient for the elicitation of a helper effect.

The possibility of a T-cell epitope providing help for a B-cell epitope when both epitopes are entrapped in the same liposomes has recently been investigated [31] as a means to present the epitopes to the immune system in the absence of covalent bonding. Epitopes chosen were a peptide from the S region (S peptide) and a peptide from the pre-S 1 region (pre-Sl peptide) of the HBsAg. Under appropriate conditions, these peptides are known to induce antibodies binding to relevant sites on the HBsAg [62]. Moreover, the S peptide was chosen from a region which contains an H-2 s Th-cell epitope while the pre-S~ peptide was taken from a site adjacent to an H-2 ~ Th-cell epitope [62] but has been specifically designed to exclude that epitope. Immunization experiments (SJL (H-2 ~) mice) with the two peptides showed [31] that the Th-cell epitope could provide help for the B-cell epitope only when the peptides were co-entrapped in the same liposomes (Table 2). Furthermore, this helper effect was found to correlate with the ability of the S peptide (co-entrapped with the pre-S peptide) to stimulate T-cell proliferation in vitro [31]. Interestingly, anti-pre-S~ peptide sera were able to interact with the full recombinant HBsAg + pre-S large protein (400 amino acids) preparation (Table 2). In other experiments [31] a degree of help, albeit lower, for pre-Sj peptide was also observed in mice treated with the two peptides co-emulsified with incomplete Freund's adjuvant. These data suggested that liposomes can not only act as an immunological adjuvant for peptides, they could also serve as a carrier for Th- and B-cell epitopes thus dispensing with the need for covalent coupling to a carrier protein and associated prob- lems.

G. Gregoriadis et al. / Journal of Controlled Release 41 (1996) 49-56 53

Table 2 Immune responses (IgGl) in SJL (H-2 ~) mice immunized with liposomal HBsAg peptides

Peptide used for immunization Antibody response a

A n t i - S Anti-pre-S ~ Anti-rHBsAg b Anti-rHBsAgC{'} ~

Liposomal S

Liposomal pre-S Liposomal (S + pre-S~ (co-entrapped)

Liposomal S + liposomal pre-S ~ (separately entrapped)

12 800 ND 12 800 12 800 12 800

102 400 51 200 204 800 102 400 409 600 204 800 409 600 409 600 ND <50d(6) ND 51 200 1600 25 600 51 200 1600 25 600

102 400 3200 25 600 102 400 3200 25 600 102 400 12 800 204 800

3200 <50d(5) ND 51 200

204 800 204 800

800 800

1600 1600 3200

<50d(6) 25 600

102 400 102 400 102 400 102 400

3200 6400

25 600 51 200

SJL (H-2~)mice were injected intramuscularly with 20 #g peptide on day 0 and day 56. They were bled 2 weeks later and sera (50-fold diluted) tested by ELISA. Sera from mice immunized with free S or pre-S ~ or a mixture of free S and pre-S ~ gave values of < 50 when tested for anti-S~, anti-pre-S, anti-pre-S~ or anti-rHBsAg (S region) or anti-rHBsAg (full-length) IgGl (from ref. [31] with permission). ND, not determined Values are from individual animals and denote dilutions required for readings to reach values of 0.2 or less. h226 amino acids (S region only). ~Full length (includes S and pre-S regions). dAll animals tested (number in parentheses) gave readings of 0.2 or less.

6. Microbial vaccines: a role for liposomes

Most studies on the immunological adjuvant properties of liposomes [6,22,53] have been carried out with vesicles of submicron average diameter produced by a variety of techniques [7] and capable of incorporating efficiently peptides and proteins but not larger, microbial vaccines. These include attenuated or killed viruses and bacteria, for example measles, polio virus, Bordetel la pertussis, Bacille Calmette-Gu~rin and Salmonel la typhi [63]. Al- though most microbial vaccines are highly immunogenic, there are instances where their administration in sufficiently large liposomes may be required. In the case of multiple vaccines, for instance, consisting of a mixture of soluble and particulate antigens or vaccine formulations also containing cytokines, presentation of all materials (co-entrapped in the same liposomes) together to antigen presenting cells, may be advantageous in terms of improving immunogenicity. Furthermore, it may be that entrapment of

microbial vaccines into appropriate (large) liposomes will prevent or, at least, curtail interaction of such vaccines with relevant antibodies, for example maternal antibodies or preformed antibodies to vaccine impurities. Work with antigen-containing liposomes has already shown [64] that such interactions are indeed avoided in vivo.

We have already shown [19,20] that 'empty' giant liposomes (average diameter up to about 9 /zm) freeze-dried in the presence of killed or live Bacil lus

subtilis spores or killed Bacille Calmette-Gu6rin (BCG) (in the presence or absence of a soluble antigen such as tetanus toxoid) and subsequently reconstituted in physiological saline, generate vesicles of similar size entrapping up to 35% of the microorganisms and up to 16% of the toxoid when present (Table 3). The presence of spores within the vesicles (3 -20 spores per vesicle, depending on the phospholipid composition [20]) was confirmed by confocal fluorescence microscopy using spores labelled with fluorescein isothiocyanate [19]. More-

54 G. Gregoriadis et al. / Journal of Controlled Release 41 (1996) 49-56

Table 3 Entrapment of B. subtilis spores and tetanus toxoid into giant liposomes

Giant vesicles B. subtilis

Killed Live

Tetanus toxoid

(PC)A 31.6 --_ 24.2(12) 0.0(4) (PC)B 26.7 + 12.1(7) 8.42 + 2.6(4) (DSPC)A 0.0(4) (DSPC)B 21.3+ 8.9(6) 11.1 + 1.9(4) (PC)A 22.5 + _ 9.8(6) 4.1 +- 3.8(4) (PC)B 34.6 _+ 5.2(6) 16.5 -+ 11.0(4) (DSPC)A 20.1 _+ 12.1 +- 12.4(6) 3.3 -+ 3.1(4) (DSPC)B 26.8 _+ 4.4(6) 12.4 -+ 9.8(4) (PC)B '~ 9.0 + 5.4(3) 3.5 +- 3.1(3) (DSPC)B" 10.2 _+ 3.6(3) 16.5 + 6.0(3)

~2~I-labelled B. subtilis and tetanus toxoid were entrapped separately or together (in this case toxoid was unlabelled) in giant vesicles made of egg phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC) by Methods A or B (see ref. [20]). Results based on radioactivity measurements or the fluorescamine method for the toxoid co-entrapped with spores are expressed as % -+ S.D. of material used for entrapment. In one experiment, entrapment of ~2~I-labelled BCG in PC giant vesicles was 27.8% (Method B). Numbers in parentheses denote number of preparations. "Denotes preparation with co-entrapped B. subtilis and tetanus toxoid (from ref. [20] with permission).

over , the m i l d n e s s of the p rocedure a l l owed the

p r e se rva t i on o f spore v iab i l i ty af ter e n t r a p m e n t [20].

Add i t iona l e x p e r i m e n t s [19] r evea l ed tha t g iant

l i posomes were able to quan t i t a t ive ly re ta in the i r

spore or t e tanus toxo id con ten t s in the p r e sence of

(mouse) b l o o d p l a s m a at 37°C for up to 24 h.

Fur ther , f o l l owing the i r i n t r a m u s c u l a r in j ec t ion into

mice , g ian t l i posomes were c lea red f rom the site o f

in jec t ion toge the r wi th the i r e n t r a p p e d vacc i ne con-

tents.

7. Conclusions

T h e s t ruc tura l versa t i l i ty of l iposomes , the i r abi l i ty

to inco rpora t e a wide var ie ty of an t igens regard less

of s ize and so lubi l i ty and a f avourab l e b iod i s t r i bu t ion

profi le h a v e r e n d e r e d the s y s t e m an e f fec t ive m e a n s

for the i m m u n o - p o t e n t i a t i o n and de l ive ry of pep t ides

and mic rob ia l vacc ines a lone or in c o n j u n c t i o n wi th

o the r c o - a d j u v a n t s such as cy tok ines . T he p r o m i s e of

the sys tem as a vacc i ne car r ie r has recen t ly b e e n

subs t an t i a t ed wi th the first l i p o s o m e b a s e d vacc i ne

(aga ins t hepat i t i s A) ( E p a x a l - B e r n a ) l i censed for use

in h u m a n s [65] and by the e n c o u r a g i n g resul ts o f

phase I and phase II c l in ical tr ials wi th a var ie ty o f

o ther l i posoma l vacc ines (e.g. t r iva len t inf luenza ,

hepat i t i s B, d iph ther ia , and te tanus toxo id vacc ines )

[11].

Acknowledgments

I am gra tefu l to Mrs . C o n c h a Per r ing for exce l l en t

secre tar ia l ass is tance .

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