Adjuvants—a classification and review of their modes of action

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Eisevier Pn: S0264410X(96)00183-1

ELSEVIER

Vaccine, Vol. 15, No. 3, pp. 246-256, 1997 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-41 ox/97 $17+0.00

Review Adjuvants-a classification and review of their modes of action John C. Cox and Alan R. Coulter Since early this century, various substances have been added to vaccines and certain formulations have been devised in an attempt to render vaccines more eflective. Despite a plethora of options, only aluminium salts have gained acceptance as human vaccine adjuvants and even veterinary vaccines are largely dependent upon the use of aluminium salts. Currently, many new vaccines are under development and there is a desire to simplzfy vaccination schedules both by increasing the number of components per vaccine and decreasing the number of doses required for a vaccine course. New, more eflective adjuvants will be required to achieve this. 0 1997 Elsevier Science Ltd. Keywords: adjuvants; adjuvant mode of action; adjuvant classification; adjuvant combinations

Adjuvants (from the Latin, adjuvarezto help) have been used to improve vaccine efficacy from the early 1920~‘~~.While the number of substances with adjuvant activity and the literature describing their use has expanded enormously3-5, their mode of action has remained largely mysterious and empirical. The purpose of this review is to consider in detail the ways in which an adjuvant can act and to attempt a classification of adjuvants based on their mode of action. The end benefit can be threefold. Firstly, if the pathogenesis of a disease is known, then an adjuvant which can generate a protective immune response can be selected for vaccine formulation. Alternatively, if the pathogenesis and immunology are not well understood, then adjuvants which can generate a range of different immune responses can be rationally selected for study. Thirdly, this knowledge can be used to combine various adjuvants in order to enhance different effects as desired. This review will discuss a range of both substances and processes which, when added to or performed upon a vaccine, will increase its immunogenicity. This approach combines both adjuvant and vehicle as defined by Allison and Byar@ and permits such well-known adjuvants as water-in-oil (w/o) emulsions (e.g. Freund’s incomplete adjuvant)’ and the various microencapsulation technologies to be included within the broad heading of adjuvants. The reader is referred to recent reviews3p5 for more detailed information on particular adjuvants and to a review by Morein et ~1.~for a current view on mucosal immunity and vaccination of infants in the presence of maternal antibodies.

Immunology Research, CSL Ltd, 45 Poplar Road, Parkville, Vie. 3052, Australia. (Received 24 May 1996; revised 30 July 1996; accepted 7 August 1996)

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MODES OF ACTION OF ADJUVANTS Adjuvants may act in one or more of five ways (see Table 1).

Immunomodulation This refers to the ability of many adjuvants to modify the cytokine network. In general, only immunomodulatory adjuvants will exert an adjuvant effect when presented at a separate time or site to the immunogen. Immunomodulation may result in a general upregulation of the entire immune system, but most commonly results in upregulation of certain cytokines and a concomitant down regulation of others. Two major subsets of CD4+ T cells, viz Thl and Th2 have been well described for mouse9,” and man” and their existence is postulated for other animal species12. Thl responses typically induce complement fixing antibody and strong delayed-type hypersensitivity (DTH) reactions and are associated with y-IFN, IL-2 and IL-12 whilst Th2 responses result in high circulating and secretory antibody levels, frequently IgE and the cytokines IL-4, IL-5, IL-6 and possibly IL-lo. Thl and Th2 responses are mutually inhibitory”. Selection of the appropriate immunomodulatory adjuvant will not only lead to an enhanced immune response but will also determine the isotype of IgG, which other immunoglobulins and how much CD4+directed, cell mediated immunity (CMI) is generated. The immune response never swings totally in one direction or the other. The most notable swings are produced by aluminium salts >90% Th2l 3 and bacterial endotoxins and derivatives (lipid A, monophosphoryl lipid A) which induce a predominantly Thl type responsei4. Presentation This refers to the ability of an adjuvant to preserve the conformational integrity of an antigen and to present

Adjuvants: J.C. Cox and A. R. Coulter Table 1

Modes of adjuvant

action Benefit

Action

Adjuvant

1. lmmunomodulation

Generally small molecules or proteins which modify the cytokine network Generally amphipathic molecules or complexes which interact with immunogen in its native conformation . Particles which can bind or enclose immunogen and which can fuse with or disrupt cell membranes . w/o emulsions for direct attachment of peptide to cell surface MHC-1 . Particulate adjuvants which bind immunogen. Adjuvants which saturate Kupffer cells . Carbohydrate adjuvants which target lectin receptors on macrophages and DCs . w/o emulsion for short term . Microspheres or nanospheres for long term

2. Presentation 3. CTL Induction

4. Targeting

5. Depot generation

type

immune effector cells. This will occur when an adjuvant is able to interact with an antigen in such a way that conformational epitopes are more effectively maintained. The main benefits are an improved in vivo activity and an increased shelf life. Three major sets of interactions are required to achieve an effective antibody response. The first interaction is with professional antigen presenting cells (APC), typically dendritic cells (DC) and Langerhans cells (LC), and possibly macrophages, although their role is still in disputet5. Antigen is taken up by rece tormediated endocytosis, or fluid-phase pinocytosisl 6?and the resultant endosome fuses with a lysosome to form an endolysosome. About this stage, external signals, probably dominated by GM-CSF’7 directs the DC to a regional draining lymph node or the spleen and initiates antigen processing and presentation. Antigen is processed into small peptides which then meet with major histocompatibility class II molecules (MHC-II) which have already been assembled in the endoplasmic reticulum (ER) and are processed through the Golgi and trans-Golgi reticulum. The resultant complex is then transported to the surface of the APC where the peptide is displayed in association with MHC-II18. The APC will also secrete IL-l, the amount of which will be determined by the degree of upregulation of the APC. This upregulation may be one of the functions of an immunomodulatory adjuvant. Local concentrations of IL-l will attract CD4’ cells to the APC, and those with T-cell receptors (TCR) complementary to the peptideMHC-II complex will undergo clonal expansion. It is probable that the Thl/Th2 switch is determined at this stage. If this is so, then an immunomodulatory adjuvant must be present at this time and place in an effective concentration to be useful in this role. The second interaction involves antigen and B cell, and recognition is primarily between surface immunoglobulin” and antigen. Bound antigen is internalized by Ig receptor-mediated endocytosis, digested in an endolysosome and the resultant peptides again expressed on the B cell surface in association with MHC-II. By this time, T cell subsets capable of recognizing this complex have already undergone clonal expansion and are available to help and direct the B cell to clonally expand into plasma cells which actively excrete immunoglobulin of the same specificity as the Ig on the surface of the initial B cell. Cytokine exchange will also determine the antibody isotype. this to appropriate

Upregulation of immune response. Selection of Thl or Th2 Increased neutralizing antibody response. Greater duration of response Cytosolic processing of protein yielding correct class 1 restricted peptides Simple process if promiscuous peptide known Efficient use of adjuvant and immunogen As above. May also determine type of response if targeting selective Efficiency Potential for single-dose vaccine

The third interaction is partly speculative, though there is increasing supportive evidence*‘.*’ that follicular dendritic cells (FDC) can provide a long-term reservoir of native antigen which is essential both for effective affinity maturation of the immune response and for persistence of biologically relevant antibody production. It is argued*’ that these responses depend upon availability of antigen in native conformation. Thus antigen presentation confers three major benefits; firstly it will maximize the amount of conformationally-relevant (i.e. neutralizing) antibody, secondly it will influence the affinity of the antibody and finally it can influence the duration of the immune response. Induction of CDS+ cytotoxic T-lymphocyte (CTL) responses The induction of CTL responses generally requires that antigen be processed within the cell cytosol (the endogenous pathway) where peptides, generally 9-mers, become incorporated within the closed-end groove of the MHC class 1 molecule and are then expressed on the cell surface. This processing contrasts with the exogenous pathway described in the previous section. Current evidence suggests that turnover of cellular proteins occurs in a 26s multi-enzyme complex. The proteolytic component of this complex is the proteasome, a highly conserved 20s structure comprising 24-28 subunits22-25. The majority of proteins which pass through this complex exit as peptides which are further processed by exopeptidases to amino acids. However, a small proportion are selectively transported to the ER by specialized transporter proteins (TAP1 and TAP2) where they are incorporated into the groove of the formin? MHC-1 and passed via the Golgi to the cell surface ‘. There is increasing evidence that a low molecular mass protein (LMP) is a specialized proteasome where two (or more) of the subunits are MHC encoded26. The presence of these MHC encoded subunits within a proteasome may modify the proteolytic cleavage towards peptides which are MHC-compatible. Production of LMP is upregulated by y-IFNZ6 and it is tempting to speculate that this is a mechanism whereby the proportion of peptide capable of insertion into MHC-1 can be increased in response to a cytokine warning signal. For an adjuvant to be useful for CTL induction, it must facilitate incorporation or persistence of

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Adjuvanfs: J.C. Cox and A. R. Coulfer appropriate peptide into MHC-1. The most effective way to achieve this is for the adjuvant to interact in some way with cell membranes so that antigen associated with the adjuvant is deposited within the cytosol in a form suitable for normal processing in the proteasome. This may occur by fusion with the external membrane or by endocytosis/pinocytosis followed by endosome membrane fusion or rupture (endosomal escape). Incorporation of an immunomodulator within this adjuvant formulation, especially one which induces y-IFN production, could be expected to increase relevant MHC-1-peptide expression. Although most cells express MHC-1, the most effective target cell for CTL induction is an APC and most probably a DC2’. An alternative mechanism for CTL induction is by direct attachment of peptide to empty externallyexposed MHC-12s. This has been achieved with a w/o formulation where the aqueous phase contains peptide and a universally recognized protein e.g. tetanus toxoid29. It is postulated that the w/o emulsion creates a depot which will attract DCs and at the same time protect the peptide from proteolysis. The tetanus toxoid will cause the DCs to migrate to lymphoid tissue and, whilst presenting tetanus-derived peptides, T cells will be attracted, some of which will be CD8 cells which recognize the original CTL peptide. Targeting This defines the ability of an adjuvant to deliver an immunogen to immune effector cells, generally via APCs. Although little data exists, it is likely that the vast majority of vaccine delivered is lost either by serum protease degradation or by first-pass removal in the liver. This form of adjuvant activity may not modify the type of immune response but rather will affect the amount of immunogen required to achieve a given effect i.e. the efficiency of generation of the immune response. However, if targeting can be selective for macrophages rather than DC, or the converse, the type of immune response may be substantially modified as shown in recent studiesI where depletion of macrophages led to a strong Th2 shift in response. There are several ways in which an adjuvant can achieve this effect. The most common is to interact with antigen in such a way as to form multimolecular aggregates. These aggregates will encourage uptake by macrophages and DC and, if an immunomodulatory adjuvant is included, will ensure that antigen and immunomodulator are delivered to the same APC. Adjuvants that have this property are termed “particulate adjuvants” and will be discussed in more detail. In specific applications, particles of 1-1Opm can be delivered orally to optimize uptake by Peyer’s patches”. Alternatively, delivery to macrophages3’ and DCS~~ can be increased when the adjuvant has sugar moieties (e.g. saponins), or other cell-surface-receptor recognizing molecules (e.g. recognition of GM-l ganglioside by LTB, CTB)33, or where immunogen can be attached to a mannose polymer (e.g. mannan, acemannan)34 or other carbohydrate. A third option is for an adjuvant to saturate Kupffer cells in the liver so that antigen, preferably not connected to adjuvant, can be preferentially taken up by APC. Such a mechanism of action has been postulated for many of the various carbohydrate adjuvants35.

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Depot generation This can be achieved as a short term or long-term depot, the latter giving either a continuous or pulsed release. Short-term depots are typified by aluminium salts and w/o emulsions, where antigen is trapped at the injection site and therefore cannot be lost by liver clearance. Excision of the injection site 8-10 days after dosing has little if any effect on magnitude or duration of response36 suggesting antigen has either been removed or walled-off by that stage. Long term depots are best achieved using synthetic polymers such as polylactide coglycolide (PLG) to produce microspheres which degrade to yield a pulsed delivery. These microspheres are preferably of a size >lO pm so that they must remain at the injection site until biodegradation permits removal of their contents (immunogen and preferably adjuvant) by APC. Release times from 1 to 6 months can be achieved with reasonable precision3’,“‘.

CLASSIFICATION

OF ADJUVANTS

There are a number of different criteria which can be used to group adjuvants to permit their rational comparison. This review which allocates adjuvants into two broad groups: particulate and non-particulate, is based on a previous review3 but with certain changes as the mode of action of certain adjuvants have become better understood. Descriptions of adjuvants will be brief and readers are referred to selected references and the earlier review for further detail. Particulate adjnvants (see Table 2 for summary) Adjuvants which exist as microscopic particles and owe at least some of their adjuvant activity to this property. Generally the benefits of particulate adjuvants are only fully realised when immunogen is able to be incorporated into or at least associated with the particle.

These are an insoluble, gel-like precipitate of aluminium hydroxide, aluminium phos hate or alum with a particle size from 100 to 1000 nm Y‘. Immunogen is bound by electrostatic interaction to pre-formed gel or during gel formation in situ41342. They have been widely used in human and veterinary vaccines since 1930 and have an excellent safety record. They induce strong Th2 responses, good targeting (if the immunogen is adsorbed), result in a moderate depot effect but induce minimal CTL or CM1 induction. Strong IgE responses’3*43,44 are frequently reported. Aluminium salts are inexpensive, safe, and simple to formulate38. Water-in-oil emulsions36~45 These are microdroplets of water, stabilized by surfactant (typically mannide mono-oleate) in a continuous oil phase (typically mineral oil, squalene or squalane). Freunds incomplete adjuvant (FIA) has been used for human and veterinary vaccines46*47 but is now largely discredited (perhaps unjustly) due to a low incidence of site reactivity. Emulsions based on metabolizable oils have a superior safety profile29.

Adjuvants: J.C. Cox and A. R. Coulter

Table2

Characteristics

of particulate

adjuvants

Adjuvant

lmmunomoduiation

Targeting

Presentation

Aluminium salts WI0 emulsions O/w emulsions ISCOMs@ Liposomes

Strong Weak Weak Strong _

+ + +++ ++

-

Th2, IgE Thl and Th2 Thl and Th2 Thl and Th2

_ +++ ++++ +++

CTL Induction

Depot

- or +++b

+ST= +++ST

++++ ++

_ _

Microparticles 40 pm >lO pm Calcium salts Proteasomes/virosomes Stearyl tyrosine

@Win Algammulin

++++ +++LT” +ST

+ 4-k

Mod Thl and Th2 Mod Thl Mod Thl and Th2

“ST, short term (5 2 week); bGood CTL response for externally

+++ +ST

+

+ST

applied peptide only; %T, long term (weeks to months)

They are poorly immunomodulatory (in absence of local irritant effect), provide good short term depots, are inexpensive, relatively simple to formulate and induce good antibody responses especially for hydrophilic immunogens. W/o emulsions provide an excellent formulation into which soluble immunomodulators can be incorporated. Emulsions can be unstable. Oil-in-water (o/w) emulsions48*49 These are microdroplets of oil (typically squalene or squalane, size about 200 nm), stabilized by surfactants (typically Tween 80 and or Span 85) in a continuous water phase and are under development as human vaccine adjuvants50*5’, frequently in association with soluble immunomodulators [e.g. muramyl dipeptide (MDP) derivatives or block copolymers]. O/w emulsions result in excellent antigen presentation and moderate targeting. They are inexpensive, safe5’ and excellent basic formulations into which lipophilic immunomodulators can be incorporated; in addition they are highly suited for amphipathic molecules where presentation is important. It is important to incorporate immunogen into the oil phase. Immune stimulating complexes (ISCOM@ adjuvant-Iscotec AB)53, ISCOM matrix is an open cage-like structure about 40 nm dia resulting from the interaction of saponins with cholesterol and phospholipid. Immunogenic ISCOMs are ISCOMs into which protein or other immunogenic molecules have been incorporated. They are used for veterinary vaccines and are being studied in various candidate vaccines in man in conjunction with defined saponin fractions55. ISCOMs induce strong Thl and Th2 responses, good targeting and presentation, and excellent CTL responses. They are inexpensive, safe in animal studies and simple to formulate. It is important to incorporate immunogen into ISCOM for an effective CTL response56.

Single or multilamellar bilayer membrane vesicles varying in size from 20 nm to 3 pm comprising cholesterol and phospholipid. The immunogen may be

membrane-bound (lipophilic and amphipathic molecules) or within the inter-membrane spaces (hydrophilic molecules). Liposomes have the potential to give good targeting, CTL 9 and presentation and provide a safe, basic formulation into which hydrophilic and lipophilic immunomodulators can be incorporated. However, they are difficult to formulate and to incorporate immunogen, and need soluble immunomodulators to be effective in most situations.

Nano- and microparticles These are small solid particles in the range 10-1000 nm (nanoparticles) and l-100 pm (microparticles) formed from a range of biocompatible and biodegradable polymers most typically cyanoacrylates and the PLG copolymers. They can act as a long-term depot (weeks to many months), give excellent targeting if ~5 pm dia and offer the best option for single-dose multi-release vaccines. Incorporation of immuno modulatory adjuvants into particles3’ increases their effectiveness. Preparation is difficult, and certain issues related to manufacture and control remain to be resolved.

Other particulate adjuvants A number of other particulate adjuvants warrant mention although they have received less attention to date. o(a) Calcium salts62. Similar to aluminium salts but lacking immunomodulatory activity. Approved for human use. o(b) Proteosomes63. Multimeric aggregates of bacterial (e.g. Neisseria meningitidis) transmembrane proteins into which structure amphipathic immunogens can be incorporated. While they do not have immunomodulatory activity, they provide good presentation and targeting of immunogens. CTL induction is minimal. Anamnestic responses to the carrier protein can reduce their effectiveness for repeated use. o(c) Virosomes64 As for proteosomes but with virus-derived transmembrane proteins e.g. influenza haemagglutinin.

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Adjuwanfs: J.C. Cox and AR. Coulter Table 3 Characteristics

of principal non-particulate

adjuvants

Adjuvant

lmmunomodulation

MDP-hydrophilic MDP-Lipophilic Non-ionic block cooolvmers Saponins ’ 1 Lipid A (MPL) Cytokines Carbohydrate polymers Derivatized polysaccharides

Strong Th2 Strong Thl ? Strong Thl , Th2 Strong Thl Various Mod Thl. IL-l induction ?

aFor self-aggregating

copolymers,

Targeting

CTL

Comments

+

Use in w/o emulsions Use in o/w emulsions Use in w/o or o/w emulsions Form ISCOMs. Use with liposomes, MPL Use with o/w emulsions, liposomes, saponins Use preferably with some particulate adjuvant Preferably conjugate?

- or +++a -

+++ _

+++ 4-H

-

_ -

e.g. CRL 1005

e(d) Stearyl tyrosine65. This adjuvant forms a stable complex with hydrophilic proteins to act as a mediumterm depot and stimulates Thl responses. Safety profile is reportedly good. o(e) y-Inulin 6. Inulin in the y form exists as small (l-2 pm) ovoid particles which are immunomodulatory, inducing a Thl response possibly by modification of the alternative complement pathway. a-inulin is acceptable for human injection. o(f) Algammulin67. A combination of aluminium hydroxide and y-inulin with the combined properties. Has been in phase 1 clinical trial. Non-particulate adjuvants (see Table 3 for summary) These are adjuvants where activity does not depend unon anv narticulate or multimeric nature. They are generally- immunomodulators though some improve with a targeting. Most benefit from association particulate adjuvant. Muramyl dipeptide (MDP) and derivatives@‘69 N-acetyl muramyl-L-alanyl-D-isoglutamine is the component of a peptidoglycan adjuvant-active extracted from Mycobacteria. Reportedly less toxic derivatives include threonyl MDP, murabutide, N-acetylglucosaminyl-MDP (GMDP), murametide and nor-MDP. Lipophilic derivatives include MTP-PE. MDP and derivatives are potent IL-l inducers; hydrophilic derivatives tend to stimulate Th2, whereas lipophilic derivatives tend to stimulate Th17’. They are best used in association with particulate adjuvants, especially w/o emulsions, o/w emulsions and liposomes but have been associated with adverse reactions in clinical trial”.

Non-ionic block copolymers72973 These polymers most typically comprise a region of hydrophobic polyoxypropylene (POP) flanked by regions of polyoxyethylene (POE). Molecular weights range from 2500 to 12500 with POE comprising 520% of this total. Different polymers are used as additives to the oil phase of o/w or w/o emulsions. Their primary action is to enhance presentation of amphipathic molecules, although they may have immunomodulatory activity. Non-ionic block copolymers are not biodegradable74 and have been implicated in dose-site reactivity75. Recent observations with hi@er molecular weight block copolymers e.g. CRL 1005 suggest that these adju-

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vants may be more appropriately considered as particulate adjuvants where targeting to APCs is a primary mode of action.

These are complex mixtures of triterpenoids, average Mw 2000, extracted from the bark of the Quillaiu saponaria tree. Saponin is a crude extract, Quil A and Spikoside are partially purified whilst QS21 (Stimulon) and ISCOPREP@ 703 are defined entities or mixtures. Other sources of saponins have been described79. Saponins induce strong Thl and Th2 responses and moderate CTL responses to some proteins probably as a result of forming mixed protein-saponin micelles. Quil A is a widely used veterinary adjuvant and defined saponins are proposed as human adjuvants. They are inexpensive, simple to formulate and generally safe. Lipid AsoYs Lipid A is a disaccharide of glucosamine with two phosphate groups and five or six fatty acid chains (generally Cl2 to C,, in length). Removal of the 1’ phosphate group leaves 4’ monophosphoryl lipid A (MPL) which is a proposed human vaccine adjuvant either alone or in association with o/w emulsions, liposomes, QS21 or CWS (cell wall skeleton). Lipid A and MPL induce strong Thl responses.

Cytokines are generally glycoproteins of molecular weight around 20 kDa which are proposed as human and veterinary vaccine additives. They have various actions, e.g. IL-l (T and B cell maturation), y-IFN (Thl upregulation, enhanced MHC expression), IL-2 (Thl upregulation), IL-4 (Th2 upregulation) and GM-CSF (co-migratory signal for DCs). IL-12 has recently been shown to induce strong Thl shifts and may have potential as an adjuvant in human vaccines83,84. Cytokines are expensive, species-specific, and in addition there are concerns regarding stability, toxicity and potential autoimmunity. If these issues can be overcome, they may become important components of some prophylactic and most therapeutic vaccines. Carbohydrate polymers~~85*86 These are polymers of mannose (e.g. mannan) and Jl-3 glucose (e.g. glucan, acemannan, lentinan) which have been proposed as human vaccine adjuvants either

Adjuvanfs: J.C. Cox and A. R. Coulter Table 4

Further non-particulate adjuvants

Adjuvant

Action

Comments

Reference

AvridineDDA

Unacceptable toxicity

48,104,105,106,107

CWS(cell wall skeleton) DHEA,(dehydroepi- androsterone) Vitamin D3 TDM(Trehalose dimycolate)

Thl induction Presentation (in liposome or o/w emulsion) Thl induction? Thl induction Th2, secretory IgA induction? Thl induction

99 108,109,110 108,109,110 111

P,CSS Poly I:CPoly ICLC Poly A:U

Targeting. Potent CTL induction Both Thl ($FN) and Th2 (IL-4) induction Th2 induction (IL-6)

Use with MPL in o/w emulsions Administration difficult Administration difficult Administration difficult. Toxicity unacceptable Potentially toxic Poly I:C toxic

mixed with87 or conjugated to immunogen. They can stimulate and target macrophages (via mannose receptors), DCs (via DEC 205 receptor)32 and upregulate Thl responses. Derivatized polysaccharides88’89 These are generally high molecular weight sulphated dextrans, or diethylaminoethyl (DEAE) dextran and have been used as veterinary (and are proposed as human) vaccine adjuvant, the former in one case in association with w/o emulsions”. Their action is complex, possibly by saturation of Kupffer cells35, possibly by T or B-cell mitogenicity. Bacterial toxins91V92 These are complex proteins e.g. cholera toxin (CT), CTB pentamer, Escherichia coli labile toxin (LT), LTB, which are potent mucosal adjuvants in some animal models. LT substitution mutants93’94 have been proposed as possible human adjuvants. The adjuvant action of bacterial toxins is not understood and may involve molecular targeting via the GM 1 ganglioside receptor or stimulation of endogenous adenylate cyclase activityg5. Other non-particulate adjuvants Table 4 selects several additional adjuvants which merit mention either because of past prominence or current promise. ADJUVANT

COMBINATIONS

To this point we have considered a number of basic adjuvant formulations in terms of their modes of adjuvant action (Table 1). In summary, immunomodulators influence both the magnitude of the immune response, the Thl/Th2 balance of that response and hence the isotype of antibody produced and the extent of DTH. Presentation is important when neutralizing antibody is a major requirement and may influence affinity and duration of the response. Adjuvants capable of cytosolic delivery are the best option if CTL responses are desired. Targeting increases the efficiency of delivery of antigen to APCs and becomes important when antigen cost is high. Short-term depots similarly increase efficiency whilst long-term depots offer the opportunity for singledose multi-release vaccines. The purpose of adjuvant combinations is to combine various adjuvant components to achieve the desired mix of immunological responses. The best-known adjuvant

112,113 114,115,116 116,117,118

combination is Freund’s complete adjuvant (FCA)7 which combines the immunomodulatory properties of tuberculosis (essentially TDM and Mycobacterium MDP) along with the short-term depot effect of w/o emulsions, This adjuvant generates very strong Thl and Th2 responses and is especially suited to hydrophilic immunogens. The Ciba-Geigy adjuvant formulation96 is a modification of FCA which uses a metabolizable oil (squalene) and nor-MDP. It has been used successfully in clinical trial. Despite the success of w/o formulations as a basis for adpant combinations (especially FCA and TiterMax@)’ they do not normally induce CTL responses and require multiple doses for effective immunization i.e. long-term depots are not established. Many combined adjuvants based on o/w emulsions have been described. The best known are the Syntex adjuvant formulation9* (SAF) which contains the nonionic block co olymer L121 and threonyl MDP; the Ribi DETOX 8 adjuvant99 which contains MPL and CWS (cell wall skeleton) and the Chiron MF59 adjuvant5’ which contains MTP-PE (muramyl tripeptidephosphatidylethanolamine), a lipophilic MDP derivative. These adjuvant formulations are of major value for amphipathic immunogens; for hydrophilic immunogens, the o/w component of the formulation will confer minimal benefits and a w/o based formulation would be preferable. Liposomes offer a versatile formulation into which various immunomodulatory molecules can be incorporated. Examples include MPL”‘, lipophilic MDP”’ and Quil A’02. Although hydrophilic molecules can be incorporated within a liposome, the efficiency is generally low and liposome formulations are most suited for amphipathic immunogens. One other interesting combination is the mixture of MPL and QS21’03. Selection of the “best” adjuvant combination requires some knowledge of the chemical nature of the protective immunogen(s) and some idea of the nature of the immune response which is likely to be protective. However, even where knowledge of both these issues is minimal, rational selection of a small number of basic formulations and additives should permit selection of an effective adjuvant system. It is hoped that this review will help in this rational selection.

ACKNOWLEDGEMENTS We thank Rodney Harris for maintenance of the literature data base and Professors Ian Gust and Graham Mitchell for valuable comments and criticism.

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