Adjuvants and their modes of action

ELSEVIER

Livestock Production Science42 (1995) 153-162

Adjuvants and their modes of action J. Alexander *, J.M. Brewer Department

ofImmunology, University of Strathclyde, Todd Centre, 31 Taylor Street, Glasgow G4 ONR, Scotland, UK

Abstract The trend towards the use of peptides and subunit proteins in modem vaccine design has necessitated the use of immunological adjuvants to achieve effective immunity. Aluminium hydroxide, a component of the diphtheria, tetanus and hepatitis B vaccines, was first described as an adjuvant over 60 years ago and is the only adjuvant currently approved for use in humans. It is also a common component of many veterinary vaccines. While this adjuvant is effective at enhancing antibody titres to antigens, the effectiveness of aluminium hydroxide is limited due to its inability to promote cell mediated immunity. Freund’s Complete Adjuvant (FCA) has been used experimentally and does stimulate cellular immunity, but is unsuitable for human and veterinary use as it promotes, amongst other toxic side effects, local inflammation and granuloma formation at the site of injection. Thus, in recent years there has been a great deal of interest in developing novel, cheap, effective and safe adjuvants which stimulate cellular, as well as humoral immunity to be. used with medical and veterinary vaccines. In addition, the recent unravelling of numerous immunological pathways has facilitated the rational development of new adjuvants and allowed a better understanding of the modes of action of traditional adjuvants. Keywords: Adjuvant; TH 1; TH2; Cytokine; Targeting

1. Background Many of the currently available veterinary vaccines rely on the use of attenuated or inactivated pathogens. While in some cases this approach has proved successful, there is greater scope for vaccine design using modern biochemical or recombinant techniques which have made feasible the bulk production of purified, defined antigen from almost any pathogen source (Zanetti et al., 1987; Milich, 1989). The advantages of this approach are many fold, for example, the vaccine need only contain the antigens necessary for induction of immunity, thus avoiding the inclusion of counterprotective or even pathology inducing antigens. Furthermore, the storage and delivery of the vaccine may be * Corresponding author. 0301-6226/95/$09.50

0 1995 Elsevier Science B.V. All rights reserved

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made easier due to the relative stability of sub-unit, recombinant and synthetic vaccines (Zanetti et al., 1987; Milich, 1989). Thus, these techniques have been utilised to produce vaccines against diseases such as Aujeszky’s disease (pseudorabies; Geskypur, Rhone Merieux), feline leukemia (Leukocell; Norden Labs.), enteric colibacillosis in cattle and pigs (Nobivac LTK88 and Nobivac LT-K99; Intervet), and a multitude of other vaccines currently under development. Despite the advantages of this approach to vaccine design, one of the problems with isolated antigens is their low immunogenicity when compared with the same antigens as part of the whole pathogen (Bomford, 1989). This problem can be overcome by genetic attenuation of the infectious agent (e.g., Omnivac-PRV pseudorabies vaccine; BioLogics) or incorporating pathogen

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genes directly into attenuated carriers/vectors such as Vaccinia or Salmonella. However, this approach introduces several difficulties such as carrier induced suppression and potential virulence of the vector (Bomford, 1989). An alternative would be to deliver the antigen formulated with an immunological adjuvant. In the broadest sense, an immunological adjuvant can be described as a substance that acts non specifically to enhance the response to an antigen. Currently the only adjuvants approved for use in humans are the Alum gels (Bomford, 1989). These and mineral oils are also the mainstay of veterinary vaccines (Veterinary Formulary, 1994). Unfortunately, while these adjuvants are good stimulators of antibody production, they are generally poor at promoting a cell mediated response (Bomford, 1980). A good cellular immune response is, however, essential for the successful control of a large number of diseases, particularly those caused by parasites and viruses (Bomford, 1989). Freund’s Complete Adjuvant (FCA), a water in oil emulsion containing killed mycobacteria, is ideal in this respect, generating a wide spectrum of immunological responses to antigens (Bomford, 1980). However, FCA is unsuitable even for veterinary vaccines as it is highly toxic, can cause chronic inflammation and induce autoimmune complications (Edelman, 1980; Warren and Chedid, 1988). In recent years therefore, a great deal of research effort has been directed towards developing new adjuvants which have the potency but do not have the adverse effects of FCA. As a result numerous new alternative adjuvants have been studied and described for both human and veterinary use and have been the subjects of many detailed reviews (Edelman, 1980; Warren and Chedid, 1988; Advances in Veterinary Science and Comparative Medicine (Vol. 35), 1990; Woodard, 1990; Research in Immunology (44th Forum), 1992). These have been developed using a number of different approaches and produce their effects by a number of different routes or mechanisms. These are described below. 1.2. Adjuvant classification Perhaps the most logical classification of adjuvants was that proposed by Woodard ( 1990) who suggested that adjuvants be divided into three main groups, surfactant adjuvants, vesicle adjuvants and water soluble

adjuvants on the basis of their physical characteristics. We have therefore comprehensively classified existing and developmental adjuvants according to this scheme (Table 1). The surfactant adjuvant classification includes aluminium hydroxide, which as well as being used clinically, is extensively used in veterinary vaccines particularly with toxoids such as tetanus, clostridia and pasteurellosis (Veterinary Formulary, 1994). Another surfactant adjuvant which has been used in veterinary vaccines is saponin (e.g., with Alhydrogel in some feline leukemia vaccines) and a purified saponin, Quil A is currently used in a Respiratory Syncytial Virus vaccine for calves (Veterinary Formulary, 1994). While Quil A is still a very complex mixture of triterpine glycosides, attempts have been made to purify active components from the mixture. One extract, QS2 1, retains adjuvanticity but has relatively low toxicity compared with Quil A (Kensil et al., 1991). When Quil A is mixed with cholesterol, it spontaneously forms micelles which can incorporate proteins and lipids in an adjuvant active structure known as an ISCOM (Morein et al., 1984). ISCOMs have been extensively used in a number of experimental vaccines, particularly viral vaccines (reviewed Claassen and Osterhaus, 1992) and an equine influenza vaccine using ISCOMs as adjuvant is currently available (Veterinary Formulary, 1994). ISCOMs have also been shown to potentiate antigen specific systemic and mucosal immune responses when administered orally (Mowat et al., 1991)) an attractive route of administration especially where infection via the mucosal surfaces occurs. Of the other vesicle adjuvants, oil adjuvanted preparations are the most extensively used in veterinary vaccines (e.g., Equine herpes vaccine, Leptospirosis vaccine, porcine Escherichia coli vaccine and porcine parvovirus vaccine; Veterinary Formulary, 1994). Experimentally, the adjuvant activity of water in oil emulsions (e.g., Freund’s incomplete adjuvant (FIA) ) can be enhanced by the addition of killed mycobacteria in the oil phase (FCA) . As described above, this adjuvant formulation is far too toxic for veterinary use and alternative water in oil emulsions have been tested in veterinary vaccines (e.g., Specol; Boersma et al., 1992). Another approach has been to formulate antigen in oil in water emulsions which allow more rapid dissemination of the oil phase and thus reduce inflammation

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Table 1 Classification of adjuvants on the basis of physical characteristics with examples A.

Surfactant adjuvants (I) Inorganic surfactants. e.g., aluminium hydroxide (Glenny et al., 1926). aluminium salts (Edelman, 1980). beryllium (Behbehani et al., 1985) (II) Natural organic surfactants (and derivatives). e.g., trehalose dimycolate (TDM; Retzinger et al., 1981), LPS (Lipid A; Johnson et al., 1956), saponon (Quil A; QS-21; Kensil et al. 1991). retinoic acid (Goettsch et al., 1992). Vitamin E (Afzal et al., 1984) (III) Synthetic organic surfactants. _ cationic aliphatic nitrogenous bases, e.g., dimethyl dioctadecyl ammonium bromide (DDA; Gall, 1966; Woodard, 1989). _ non ionic surfactants, e.g., block polymer surfactants (Pluronics@ (Hunter et al., 1981) , Tetronics” (Hunter and Bennett, 1984) etc.), sorbitan trioleate (Span 85@‘; Woodard, 1989), polyethylene-glycolglycerol ricinoleate (Cremophor EL; Austrup et al., 1991). fatty acid esters (Bomford, 1981). - lipopeptides (Bessler et al., 1985) and lipid conjugated proteins (Hunter, 1973). - Sulpholipopolysacchatides ( SLPs; Hilgers et al., 1985) Vesicle adjuvants (I) Water in oil emulsions. e.g., Freund’s complete/incomplete adjuvant (FCA/FIA; Edelman, 1980; Warren Chedid, 1986) (II) Oil in water emulsions. e.g., RIB1 adjuvant system (RAS; Masihi et al., 1986), syntex adjuvant formulation (SAF; Allison and Byars, 1986), lipovant (Reynolds et al., 1980), intmlipid (Siddiqui et al., 1981). (III) Liposomes. (Allison and Gregoriadis, 1974; Gregoriadis, 1990). (IV) Immunostimulating complexes. (ISCOM’s; Momin et al., 1984; Mowatt et al., 1991). (V) Other particulate systems. e.g., non-ionic surfactant vesicles (NISV; Brewer & Alexander, 1992). nanopatticles (Steineker et al., 1991). microparticles (Singh et al., 1991; G’Hagan et al., 1991). proteosomes (Lowell et al., 1988). virus-like particles (Griffiths et al., 1991), gamma inulin (Leslie et al., 1990), bentonite (Gallily and Garvey, 1968). Dextran Sulphates (Diamantstein et al., 1971) , diethylamincethyl (DEAE) dextran (Joo and Emod, 1988), Solid matrix antibody-antigen (SMAA) complexes (Randall and Young, 1989), Carbopols’” (Gualandi et al., 1988)

C.

Water soluble adjuvants & derivatives (I) Muramyl dipeptides. (Leclerc and Vogel, 1986). (II) Cholera toxins. (Lycke et al., 1989) (III) Cytokines. e.g., interleukin 1 (IL- 1; Staruch and Wood, 1983; McCullough et al., 1991) , interleukin 2 (IL-2; McCullough et al., 1991; Good et al., 1988a). interferon y (IFNy; Heath and Playfair, 1990), interleukin 12 (IL-12; Afonsoet al. 1994)

at the site of injection compared with water in oil emulsions (Woodard, 1989). The adjuvant activity of these formulations may be enhanced by the incorporation of alternatives to killed mycobacteria such as Trehalose dimycolate (TDM; Retzinger et al., 1981), mummy1 dipeptide (MDP) and analogues (e.g., SAF; Allison and Byars, 1986) or Lipopolysaccharide (LPS) derivatives (e.g., Lipid A; Masihi et al., 1986). An oil in water adjuvant formulation containing monophosphoryl lipid A and cell wall skeleton (CWS) has

recently been applied to a potential brucellosis vaccine, the inclusion of CWS reducing the amount of oil necessary in the adjuvant from 50% to 1% (Masihi et al., 1983). The adjuvant activity of oil in water emulsions may also be increased by the incorporation of synthetic non-ionic surfactants (e.g., block copolymers) at the oil/water interface (Hunter et al., 1981; Hunter and Bennett, 1984), although non ionic surfactants have been shown to form adjuvant active vesicles dispensing with the requirement for an oil phase (NISV; Brewer

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and Alexander, 1992). The capacity of phospholipids to form bilayered vesicles or liposomes has been more extensively studied (Bangham and Horne, 1964; Gruner, 1987; New, 1990) and the capacity of these structures to act as adjuvants was first described in 1974 by Allison and Gregoriadis. Liposomes have been used in a variety of experimental vaccines (reviewed Gregoriadis, 1990) and can be formulated with other adjuvants such as Lipid A (Alving, 1987) or MDP derivatives (Dukor and Schumann, 1987). Recently, poly( lactide-co-glycolide) microspheres have been evaluated as adjuvants (Singh et al., 1991) and have also been shown to be effective adjuvants when administered orally (O’Hagan et al., 1993).

Vesicular antlger

2. Mechanisms of action Traditional explanations of adjuvant action were of necessity simplistic due to our limited understanding of the initiation and regulation of the immune response. However, recent advances in our understanding of immunology allow not only more detailed analysis of how traditional adjuvants may have worked, but how to design new adjuvants to best attain the appropriate response (Fig. 1). 2. I. Depot hypothesis This is the earliest proposed mechanism for adjuvant action (Glenny et al., 193 1). It suggests that repository adjuvants such as the water in oil emulsions (Herbert, 1968) or aluminium salts (Glenny et al., 1931) enhance antibody responses by the slow and even release of small quantities of antigen over a long period of time. This may produce a secondary immune response to the antigen after a single injection. Liposomes and possibly other vesicle systems also form short lived depots which probably play a limited role in their adjuvant activity (Shek and Sabiston, 1982). The limitation of this hypothesis in its simplest form can be demonstrated by the excision of the antigen depot which does not affect the overall antibody response (Freund and Lipton, 1955; Herbert, 1968). This may be due to the partial dissemination of the depot and formation of secondary depots at draining lymph nodes after injection (Warren and Chedid, 1988).

Cytotdxic T cell response

Cell mediated immunity. 6 cell help for :lgG2a

Hunioral Immunity. B cell help for :-

IgGI, /gf

Fig. 1. Schematic diagram of the probable major regulatory pathways determining the evolution of the immune response in the mouse. Macrophage (M~J) driven activation of Thl cells (Gajewski et al., 1991) and CD8+ T cells (DeBrick et al., 1991) probably due to the effects of IL-12 (Scott,1993). results in the production of IFl’Iy which promotes the further activation of Thl cells (Gajewski and Fitch, 1989) and antagonises the proliferation of Th2 cells ( MOSmann et al., 1991) B cell (B) activation of Th2 cells (Gajewski et al., 1991) which may be enhanced by IL-1 from multiple sources (Greenbaum et al., 1988). results in IL-4 production which further upregulates Th2 activation (Swain et al., 1990) and IL-10 production. IL-10 acts to inhibit IFNy production by Thl cells (Fiotentino et al., 1989) and CD8 + T cells (Mosmann et al., 1991). Similar, though not necessarily identical pathways undoubtedly operate in other mammals, for example, the dichotomy in CD4 + T cells has been identified in humans (Del Prete et al., 1991) .

2.2. Changes in cell trafJicking The antigen depot also provides a focus for interaction between cells of the immune system and the adjuvant/antigen preparation. The main cell types involved in this reaction are those of the macrophage/monocyte lineage. Most adjuvants also induce increased and pro-

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longed circulation of lymphocytes through local lymph nodes, an effect known as lymphocyte trapping (Edelman, 1980). Theoretically, this would enhance the probability of effective interaction between antigen, antigen presenting cells and immunocompetent cells such as lymphocytes. Many adjuvants activate complement which will result in enhanced cell recruitment. 2.3. Surface activity With the exception of the water soluble compounds, one of the consistent features of the adjuvants described in Table 1 is surface activity or the possession of a surface interface. This theme was first described by Gall ( 1966) in studies of the aliphatic nitrogenous bases. More recent work with the block polymer surfactants prepared in oil in water emulsions reinforce the close association between surface and adjuvanticity (Hunter et al., 1981; Hunter and Bennett, 1984). This relatively hydrophobic surface concentrates antigen and host proteins (opsonins) and effectively displays them to cells of the immune system (Hunter et al., 1981; Hunter and Bennett, 1984). Many vaccine antigens are isolated from cell membranes (e.g., malaria circumsporozoite (CS) protein; Good et al., 1988) and possess a transmembrane region of hydrophobic amino acids. In these circumstances the conformation of the antigen is lost, a feature essential for the production of high affinity antibodies to the native protein. Studies of myelin basic protein, an encephalogenic protein, have shown that certain peptide specific antibodies could only recognise the intact protein when it was bound to the surface of a liposome (Boggs et al., 1985). This indicates that inserting the antigen into a model membrane alters its conformation significantly. Water in oil emulsions such as Freund’s incomplete/complete are also known to denature antigen allowing recognition of internal antigen determinants (Kenney et al., 1989). 2.4. Antigen targeting Association of antigen with large particular structures (e.g., vesicular adjuvants such as liposomes) is known to increase the delivery of antigen to antigen presenting cells (Gregoriadis, 1990). Indeed, large carrier proteins such as Bovine serum albumin (BSA) or Keyhole limpet haemocyanin (KLH) have been shown

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to function as delivery systems in addition to their more conventional role in providing T cell help for smaller immunogenic peptides (Francis et al., 1985). In this fashion, the amount of antigen presented to immunocompetent cells is increased, effectively increasing the antigenic dose (Beatty et al., 1981; Shek and Lukovitch, 1982). An adjuvant effect is also seen when monoclonal antibodies are employed to target conjugated antigens to class II MHC molecules on antigen presenting cells, further demonstrating the important role efficient delivery of antigen plays in adjuvanticity (Carayanniotis and Barber, 1990). There is also a wealth of evidence that certainly liposomes influence which antigen presenting cell processes associated antigen (Dal Monte and Szoka, 1989a,b), in this case the macrophage. This is of major significance as current evidence would indicate that presentation of antigen via macrophages preferentially stimulates THl responses, delayed type hypersensitivity and IgG2a production and B cell presentation stimulates TH2 expansion, IgCl and IgE production (Fig. 1; Gajewski et al., 1991; Abbas et al., 1991; Brewer et al., 1994). A large number of recent studies using liposomes and ISCOMS also implicate macrophages as the most important cell in processing foreign antigen for class I MHC presentation and a cytotoxic CD8 + response (Fig. 1; Heeg et al., 1991; Harding et al., 1991; Zhou et al., 1992; Reddy et al., 1992; Lopes and Chain, 1992). 2.5. Effects on MHC class II expression One of the requirements for activation of CD4 restricted T helper cells is presentation of antigen derived peptides in the context of the MHC class II surface glycoprotein (Unanue, 1984). Certain antigen presenting cells constitutively express high levels of class II (Allison and Byars, 1986), whereas others require extrinsic factors to modulate surface class II (Beller et al., 1980). Such factors include the T cell derived cytokine IFN-y (Steeg et al., 1982), certain bacteria and bacterially derived components (BCG; Ezekowitz et al., 1981; Monophosphoryl lipid A; Tomai and Johnson, 1989; LPS: Ziegler et al., 1984) and adjuvants such as FCA (Behbehani et al., 1985)) Beryllium (Behbehani et al., 1985) block copolymers (L81; Howerton et al., 1990), and ISCOMS (Campbell and Peerbaye, 1992). Therefore, certain adjuvants

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may function by upregulation of MHC class II expression thus increasing antigen presentation to T cells. 2.6. The costimulatory role of adjuvants In addition to their function in antigen presentation (Unanue, 1984)) antigen presenting cells also have an important costimulatory role in activating T cells (Weaver and Unanue, 1990). This activity, unlike T cell receptor recognition of the class II/antigen complex, is non specific for antigen. The exact mediators involved are not completely defined, but it is now clear from several studies that IL- 1 is an essential co-stimulator for the proliferation of the CD4+ TH2 subset associated with help for antibody production (Greenbaum et al., 1988; Weaver et al., 1988) and IL-12 is involved in the generation of the CD4+ THl subset associated with the generation of cell mediated immunity (Fig. 1; reviewed Scott, 1993). IL-l exists in both the soluble form or in a membrane form found associated with the surface of the antigen presenting cell (Kurt-Jones et al., 1985); both have costimulatory activity (Greenbaum et al., 1988; Weaver et al., 1988). This mechanism of T cell activation would also help to explain the adjuvant activity of exogenous IL- 1 administered with antigen (Staruch and Wood, 1983; McCullough et al., 1991). Given that after administration of alum and antigen in vivo, anti IL-l antibodies were able to inhibit in vitro proliferation of T cells (Grun and Maurer, 1989) and the ability of LPS to stimulate IL- 1 expression in vitro (Kurt-Jones et al., 1986)) it seems reasonable to suggest that other adjuvants may also be able to function in this role. Furthermore, the ability of certain adjuvants to select for cell mediated as opposed to humoral immunity (Bomford, 1980; Grun and Maurer, 1989) strongly suggests that these adjuvants may induce the production of costimulatory molecules responsible for the expansion of THl cells. A more critical aspect of this mechanism related to antigen presentation in the absence of costimulatory signals. In this situation T helper cells enter a state of unresponsiveness or tolerance due to an inability to synthesise IL-2 which is both antigen and MHC specific (Mueller et al., 1989). The implications in effective vaccination are obvious. It is not surprising, therefore, that many groups are examining the adjuvant activity of recombinant cytokines, in other words the actual costimulatory molecules themselves (see Table 1, and

Blecha and Charley, 1990). Recent results using IL-12 as an adjuvant against Leishmania major are particularly impressive ( Afonso et al., 1994)) although other cytokines such as IL-4 or IL-5 may prove beneficial depending on which type of immune response is appropriate.

3. Conclusions There is an increasing trend away from classical attenuated or killed whole pathogen vaccines towards developing chemically defined preparations. This is primarily for safety reasons but ultimately subunit, recombinant and synthetic vaccines will be less costly, more stable, with fewer storage and delivery problems and amenable to high standards of quality control. These defined antigens will need to incorporate adjuvants if immunogenicity is to be maximised. Many of the adjuvants currently in widespread use are not ideal because they either induce an inappropriate immune response or they have potentially severe contra-indications. Nevertheless, as the secrets of the immune response are unravelled and the mechanisms of adjuvant action are dissected and understood, new safe designer adjuvants will become available. Selective use of recombinant cytokines probably point towards some of the exciting ways forward and early studies with veterinary vaccine have been encouraging. However, as the in vivo half-life of these substances is extremely short, slow release delivery systems will probably have to be utilised. Targeting vaccines in an appropriate vehicle to the correct antigen presenting cell to induce the desired immune response, is an alternative option awaiting exploitation. Furthermore, if such a vehicle also carried the appropriate costimulatory cytokine( s) , a maximal response could be anticipated. Alternatively, inhibiting cytokines could be incorporated to switch off inappropriate immune responses. Irrespectively, exciting new developments in vaccine technology are to be anticipated in the near future.

Acknowledgements J.M. Brewer is funded by Proteus Molecular Design Ltd.

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References

Abbas, A.K., Williams, M.E., Burstein, H.J., Chang, T., Bossu, P. and Lichtman, A.H., 1991. Activation and functions of CD4+ T cell subsets. lmmunol. Rev., 123: 5-22. Advances in Veterinary Science and Comparative Medicine (Volume 35). 1990. In: Blecha, F. and Charley, B. (Eds.), lmmunomodulation in Domestic Food Animals. Academic Press Ltd.,

London. Afonso, L.C.C., Scharton, T.M., Vieira, L.Q., Wysocka, M., Trinchieri, G. and Scott, P., 1994. The adjuvant effect of interleukin12 in a vaccine against Leishmiania major. Science, 263: 23523-l. Afzal, M., Tengerdy, R.P., Ellis, R.P., Kimberling, C.V. and Morris, C.J., 1984. Protection of rams against epididymitis by a B. ouirvitamin E adjuvant vaccine. Vet. lmmunol. lmmunopathol., 7: 293-304. Allison, A.C. and Gregoriadis, G., 1974. Liposomes as immunological adjuvants. Nature, 252: 252. Allison, A.C. and Byars, N.E., 1986. An adjuvant formulation that selectively elicits the formation of antibodies of protective isotypes and of cell-mediated immunity. J. lmmunol. Methods, 95: 157-186. Alving, C.R., 1987. Liposomes as carriers for vaccines. In: Ostro, M.J. (Ed.), Liposomes: from Biophysics to Therapeutics. Marcel Dekker, New York, pp. 195-218. Austrup, F., Kucharzik, T. and Kolsch, E., 1991. Cremophor EL as an adjuvant affecting immunoglobulin class switch in the immune response to the thymus independent antigen (u( I- > 3) dextran B 1355s. Immunology, 73: 508-509. Bangham, A.D. and Home, R.W., 1964. Negative staining of phospholipids and their structural modification by surface active agents as observed in the electron microscope. J. Mol. Biol., 8: 660668. Beatty, J.D., Beatty, B. and Paraskevas, F., 1981. Liposome stimulation of anti-BSA antibody production in mice. J. Surg. Res., 31: 259-267. Behbehani, K., Beller, D.I. and Unanue, E.R., 1985. The effects of beryllium and other adjuvants on la expression on macrophages. J. Immunol., 134: 2047-2049. Beller, D.I., Keily, J. and Unanue, E.R., 1980. Regulation of macrophage populations. I. Preferential induction of la-rich peritoeal exudates by immunologic stimuli. J. lmmunol., 124: 1426-1432. Bessler, W.G., Cox, M., Lex, A., Suhr, B., Weismuller, K-H. and Jung, G., 1985. Synthetic lipopeptide analogs of bacterial lipoprotein are potent polyclonal activators for murine B lymphocytes. J. lmmunol., 135: 1900-1905. Boersma, W.J.A., Bogaerts, W.J.C., Bianchi, A.T.J. and Classen, E., 1992. Adjuvant properties of stable water-in-oil emulsions: Evaluation of the experience with Specol. Res. Immunol., 143: 503512. Boggs. J.M., Hashim, G.A., Day, E.G. and Moscarello, M.A., 1985. Lipid induced recognition of a conformational determinant (residues 65 to 83) in myelin basic protein. J. lmmunol., 133: 26172622.

159

Bomford, B., 1980. The comparative selectivity of adjuvants for humoral and cell mediated immunity. Clin. Exp. lmmunol., 39: 435441. Bomford, R., 1981. The adjuvant activity of fatty acid esters. The role of acyl chain length and degree of saturation. Immunology, 44: 187-192. Bomford, R., 1989. Adjuvants for anti-parasite vaccines. Parasitol. Today, 5: 4146. Brewer, J.M. and Alexander, J., 1992. The adjuvant activity of nonionic surfactant vesicles (niosomes) on the BALB/c humoral response to bovine serum albumin. Immunology, 75: 570-575. Brewer, J.M., Richmond, J. and Alexander, J., 1995. The demonstration of an essential role for macrophages in the in vivo generation of lgG2a antibodies. Clin. Exp. lmmunol., in press. Campbell, J.B. and Peerbaye, Y.A., 1992. Saponin. Res. lmmunol. 143: 526-530. Carayanniotis, G. and Barber, B.H., 1990. Characterisation of the adjuvant free serological response to protein antigens coupled to antibodies specific for class II MHC determinants. Vaccine, 8: 137-144. Claassen, I. and Osterhaus, A., 1992. The ISCOM structure as an immune enhancing moiety: experience with viral systems. Res. lmmunol., 143: 531-541. DalMonte, P.R. and Szoka, F.C., 1989a. Effect of liposome encapsulation on antigen presentation in vitro: Comparison of presentation by peritoneal macrophages and B cell tumors, J. lmmunol., 142: 1437-1443. DalMonte, P.R. and Szoka, F.C., 1989b. Antigen presentation by B cells and macrophages of cytochrome c and its antigenic fragment when conjugated to the surface of liposomes. Vaccine, 7: 401-408. DeBrick, J.E., Campbell, P.A. and Staerz, V.D., 1991. Macrophages as accessory cells for class I MHC restricted immune responses. J. lmmunol., 147: 2846-2851. Del Prete, G.F., De Carli, M., Mastromauro, C., Biagiotti, R., Macchia, D., Falangiani, P., Ricci, M. and Romagnani, S., 1991. Purified protein derivative of Mycobacterium tuberculosis and excretory-secretory antigen(s) of Toxocara canis expand in vitro human T cells with stable and opposite (type 1 T helper or type 2 T helper) profile of cytokine production. J. Clin. Invest., 88: 346-350. Diamantstein, T., Wagner, B., Beyse, I., Odenwald, M.V. and Schultz, G., 1971. Stimulation of antibody formation by polyanions. V. relationship between enhancement of sheep red blood cell uptake by the spleen and adjuvant action of dextran sulphate. Eur. J. Immunol., 1: 429433. Dukor, P. and Schumann, G., 1987. Modulation of non-specific resistance by MTP.PE. In: Madje, J.A. (Ed.), International Symposium on Immunological Adjuvants and Modulators of Non Specific Resistance to Microbial Infections. June 1986. Alan R. Liss Inc., New York. pp. 255-265. Edelman, R., 1980. Vaccine adjuvants. Rev, lnf. Dis., 2: 370-383. Ezekowitz, R.A.B., Austyn, J., Stahl, P.D. and Gordon, S., 1981. Surface propeties of Bacillus calmefte-guerin activated mouse macrophages. Reduced expression of mannose specific endocytosis, Fc receptors and antigen F4/80 accompanies the induction of la. J. Exp. Med., 154: 60-76.

160

J. Alexander. J.M. Brewer/Livestock Production Science 42 (1995) 153-162

Fiorentino, D.F., Bond, M.W. and Mosmann, T.R., 1989. TWO types of helper T cell. IV. Th2 secrete a factor that inhibits cytokine production by Thl clones. I. Exp. Med., 170: 2081-2095. Francis, M.J., Fry, C.M., Rowlands, D.J., Brown, F., Bittle, J.L., Houghten, R.A. and Lemer, R.A., 1985. Immunological priming with synthetic peptides of foot and mouth disease virus. J. Gen. Viral., 66: 2347-2354. Freund, J. and Lipton, M.M., 1955. Experimental allergic encephalomyelitis after the excision of the injection site of antigen-adjuvant emulsion. J. Immunol., 75: 454459. Gall, D., 1966. The adjuvant activity of aliphatic nitrogenous bases. Immunology, 11: 369-386. Gajewski, T.F., Joyce, J. and Fitch, F.W., 1989. Antiproliferative effect of IFNy in immune regulation. III. Differential selection of Thl and Th2 murine helper T lymphocyte clones using mcombinant IL-2 and recombinant IFNy. J. Immunol., 143: 15-22. Gajewski, T.F., Pinnas, M., Wong, T. and Fitch, F.W., 1991. Murine Thl and Th2 clones proliferate in response to distinct antigen presenting cell populations. J. Immunol., 146: 1750-1758. Gallily, R. and Garvey, J., 1968. Primary stimulation of rats and mice with haemocyanin in solution and adsorbed onto bentonite. J. Immunol., 101: 924-929. Glenny, A.T., Pope, C.G., Waddington, H. and Wallace, U., 1926. Immunological notes. XXIII. The antigenic value of toxoid precipitated by potassium alum. J. Pathol., 29: 38-39. Glenny, A.T., Buttle, G.A.H. and Stevens, M.F., 1931. Rate of disappearance of diptheria toxoid injected into rabbits and guinea pigs: toxoid precipitated withalum. J. Pathol. Bacterial., 34: 267215. Goettsch, W., Hatori, Y. and Sharma, R.P.. 1992. Adjuvant activity of all-trcms-retinoic acid in C57BL/6 mice. hit. J. Immunopharmacol., 14: 143-150. Good, M.F., Miller, L.H., Kumar, S., Quakyi, IA., Keister, D., Adams, J.H., Moss, B., Berzofsky, J.A. and Carter, R., 1988. Limited immunological recognition of critical malaria vaccine candidate antigens. Science, 242: 574-577. Greenbaum. L.A., Horowitz, J.B., Woods, A., Pasqualini, T., Reich,E-P. and Bottomly, K., 1988. Autocrine growth of CD4+ T cells: Differential effects of IL-1 on helper and inflammatory T cells. 3. lmmunol., 140: 1555-1560. Gregoriadis, G., 1990. Immunological adjuvants: A role for liposomes. Immunol. Today, 11: 89-97. Griffith& J.C., Berrie, E.L., Holdsworth, L.N., Moore, J.P., Harris, S.J., Senior, J.M., Kingsman, SM., Kingsman, A.J. and Adams, S.E., 1991. Induction of high titer neutralising antibodies using hybrid human deficiency virus V3-Ty viruslike particles in a clinically relevant adjuvant. J. Viral.. 65: 450-456. Grun, J.L. and Maurer, P.H., 1989. Different T helper cell subsets elicited in mice utilising two different adjuvant vehicles: The role of endogenous IL- 1 in proliferative responses. Cell. Immunol. , 121: 134-145. Gmner. SM., 1987. Materials properties of liposomal bilayers. In: Ostro, M.J. (Ed.), Liposomes: from Biophysics to Therapeutics. Marcel Dekker, New York, pp. l-38. Gualandi, G.L.. Losio, N.M., Muratori, G. and Foni, E., 1988. The ability by different preparations of porcine parvovirus to enhance

humoral immunity in swine and guinea pigs. Microbiologica, 11: 363-369. Harding, C.V., Collins,D.S., Kanagawa, 0. and Unanue,E.R., 1991. Liposome encapsulated antigens engender lysosomal processing for class II presentation and cytosolic processing for class I presentation. J. Immunol., 147: 286&2863. Heath, A.W. and Playfair, J.H.L., 1990. Conjugation of interferon gamma to antigen enhances its adjuvanticity. Immunology, 7 1: 454-456. Heeg, K., Kuon, W. and Wagner, H., 1991. Vaccination of class 1 restricted murine CDS+ cytotoxic lymphocytes towards soluble antigens: ISCOM-OVA complexes enter the class I MHC restricted antigen pathway and allow sensitisation against the immunodominant peptide. Eur. J. Immunol., 21: 1523-1527. Herbert, W.J., 1968. The mode of action of mineral oil emulsion adjuvants on antibody production in mice. Immunology, 14: 301318. Hilgers, L.A.T., Snippe, H., Jansze, M. and Williers, J.M.N., 1985. Combination of two synthetic adjuvants: synergistic effects of a surfactant and a polyanion on the humoral immune response. Cell. Immunol., 92: 203-209. Howerton, D.A., Hunter, R.L., Ziegler, H.K. and Check, I.J., 1990. Induction of macrophage Iaexprcssion in vivo by synthetic block polymer, L81. J. Immunol., 144: 1578-1584. Hunter, R.L., 1973. Selective stimulation of delayed type hypersensitvity by a lipid conjugated protein antigen which is localized in thymus dependent lymphoid tissue. J. Immunol., 110: 183190. Hunter, R.L., Strickland, F. and Kezdy, F., 1981. The adjuvant activity of nonionic block polymer surfactants. I. The role of the hydrophile-lipophile balance. J. Immunol., 127: 1244-1250. Hunter, R.L. and Bennett, B., 1984. The adjuvant effect of nonionic block polymer surfactants. 2. Antibody formation and inflammation related to the structure of triblock and octablock copolymers. J. Immunol., 133: 3167-3175. Johnson, A.J., Gaines, S. and Landy, M., 1956. Studies on the 0 antigen of Salmonella typhosa 5. Enhancement of the antibody response to protein antigens by the purified lipopolysaccharide. J. Exp. Med., 103: 225-246. Joo, I. and Emod, J., 1988. Adjuvants effect of DEAE-dextran on cholera vaccines. Vaccine, 6: 233-237. Kenney, J.S., Hughes, B.H., Masada, M.P. and Allison, A.C., 1989. Influence of adjuvants on the quantity, affinity, isotype and epitope specificity of murine antibodies. J. Immunol. Methods, 121: 157-166. Kensil, C.R., Patel, U., Lennick, M. and Marciani, D., 1991. Separation and characterisation of saponins with adjuvant activity from Quilaja saponaria Molina cortex. J. Immunol., 146: 431437. Kurt-Jones, E.A., Belier, D.I., Mizel, S.B. and Unanue, E.R., 1985. Identification of a membrane associated IL-l in macrophages. Proc. Natl. Acad. Sci. USA, 82: 1204-1208. Kurt-Jones, E.A., Virgin, H.W. and Unanue, E.R., 1986. In vivo and in vitro expression of macrophage membrane IL-I in response to soluble and particulate stimuli. J. Immunol., 137: 10-14. Leclerc, C. and Vogel, F., 1986. Synthetic immunomodulators and synthetic vaccines. CRC. Crit. Rev. Ther. Drug Carrier Systems, 2: 353-406.

J. Alexander, J.M. Brewer/Livestock Production Science 42 (1995) 153-162

Leslie, D.E., Nicholson, S., Dimitrakakis, M., Johnston, N. and Gust, I.D., 1990. Humoral responses in mice using gamma inulin preparations as adjuvants for hepatitis B vaccines. Immunol. Cell Biol., 68: 107-I 12. Lopes, L.M. and Chain, B.M., 1992. Liposome mediated delivery stimulates a class I restricted cytotoxic T cell response to soluble antigen. Eur. J. Immunol., 22: 287-290. Lowell, G.H., Ballou, W.R., Smith, L.F., Wirtz, R.A., Zollinger, W.D. and Hockmeyer, W.T., 1988. Proteosome lipopeptide vaccines: Enhancement of immunogenicity for malaria CS peptides. Science, 240: 800-802. Lycke, N., Bromander, A.K., Ekman, L., Karlsson, U. and Holmgren, J., 1989. Cellular basis of immunomodulation by cholera toxin in vitro with possible association to the adjuvant activity in vivo. J. Immunol., 142: 20-27. Masihi, K.N., Lange, W., Brehmer, W. and Ribi, E., 1983. Effects of mycobacterial fractions and muramyl dipeptide on the resistance of mice to aerogenic influenza virus infection. Int. J. Immunopharmacol., 5: 403-410. Masihi, K.N., Lange, W., Brehmer, W. and Ribi, E., 1986. Immunobiological activites of nontoxic lipid A: Enhancement of nonspecific resistance in combination wiht trehalose dimycolate against viral infection and adjuvant effects. Int. J. Immunopharmacol., 8: 339-345. McCullough, K.C., Pullen, L. and Parkinson, D., 1991. The immune response against foot and mouth disease virus: influence of the T lymphocyte growth factors IL-l and IL-2 on the murine humoral response in vivo. Immunol. Lett., 31: 41-46. Milich, D.R., 1989. Synthetic T and B cell recognition sites: implication for vaccine development. In: Dixon, F.J. (Ed.), Advances in Immunology, Vol. 45. Academic Press, San Diego, CA, pp. 195-282. Morein, B., Sundquist, B., Hoglund, S., Dalsgaard, K. and Osterhaus, A., 1984. ISCOM, a novel structure for antigenic presentation of membrane proteins from enveloped viruses, Nature, 308: 457459. Mosmann, T.R.. Schmacher, J.H., Street, N.F., Budd, R., O’Garra, A., Fong, T.A.T., Bond, M.W., Moore, K.W.M., Sher, A. and Fiorentino, D.F., 1991. Diversity of cytokine synthesis and function of mouse CD4 + T cells. Immunol. Rev., 123: 209. Mowat, A.M., Donachie, A.M., Reid, G. and Jarrett, O., 1991. Immune stimulating complexes containing Quil A and protein antigen prime class I MHC restricted T cells and are immunogenic by the oral route. Immunology, 72: 3 17-322. Mueller, D.L., Jenkins, M.K. and Schwartz, R.H., 1989. Clonal expansion versus clonal inactivation: A costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Annu. Rev. Immunol., 7: 481-51 I. New, R.R.C., 1990. Liposomes: a Practical Approach. IRL Press, Oxford. O’Hagan, D.T., McGee, J.P., Holmgren, J., Mowat, A.MCI., Donachic, A.M., Mills, K.H.G., Gaisford, W., Rahman, D. and Challacombe, S.J., 1993. Biodegradable microparticles for oral immunisation. Vaccine, 11: 149-154. Randall, R.E. and Young, D.F., 1989. Immunisation against multiple viruses by using solid-matrix-antibody-antigen complexes. J. Virol., 63: 1808-1810.

161

Reddy, R., Zhou, F., Nair, S., Huang, L. and Rouse, B.T., 1992. In vivo cytotoxic T lymphocyte induction with soluble proteins administered in Iiposomes. J. Immunol., 148: 1585-1589. Research in Immunology (44th Forum). 1992. Characteristics and practical use of new-generation adjuvants as an acceptable alternative to Freund’s complete adjuvant. In: Claassen, E. and Boersma, W.J. A. (Eds. ) Institut Pasteur/Elsevier, Paris. Retzinger, G.S., Meredith, S.C., Takayama, K., Hunter, R.L. and Kezdy, F.J., 1981. The role of surface in the biological activities of trehalose 6,6’-dimycolate: Surface properties and development of a model system. J. Biol. Chem., 256: 8208-8216. Reynolds, J.A., Harrington, D.G., Crabbs, C.L. Peters, C.J. and DiLuzio, N.R., 1980. Adjuvant activity of a novel metabolisable lipid emulsion with inactivated viral vaccines. Infect. Immunol., 28: 937-943. Scott, P., 1993. IL-12: Initiation cytokine for cell-mediated immunity. Science, 260: 496-497. Shek, P.N. and Lukovitch, S., 1982. The role of macrophages in promoting the antibody response mediated by Iiposome associated protein antigens. Immunol. Lett., 5: 305-309. Shek, P.N. and Sabiston, B.H., 1982. Immune responses mediated by liposome associated protein antigens. I. Potentiation of the plaque forming response. Immunology. 45: 349-356. Siddiqui, W.A., Kan, S., Kramer, K., Case, S. and Palmer, K., I98 1. Use of a synthetic adjuvant in an effective vaccination of monkeys against malaria. Nature, 289: 64-66 Singh, M., Singh, A and Talwar, G.P., 1991. Controlled delivery of diptheria toxoid using biodegradable poly( o.L-lactide) microcapsules. Pharm. Res., 8: 958-961. Staruch, M.J. and Wood, D.D., 1983. The adjuvanticity of interleukin 1 in vitro. J. Immunol., 130: 2191-2194. Steeg, P.R., Moore, R.N., Johnson, H.M. and Oppenheim, J.J., 1982. Regulation of murine macrophage Iaexpression by a lymphokine with immune interferon activity. J. Exp. Med., 156: 1780-1793. Steineker, F., Kreuter, J. and Lower, J., 1991. High antibody titres in mice with polymethylmethacrylate nanoparticles as adjuvant for HIV vaccines. AIDS, 5: 431435. Swain, S.L., Weinberg, A.D., English, M. and Huston, G., 1990. IL4 directs the development of Th2-like helper effecters. J. Immunol., 145: 3796-3806. Tomai, M.A. and Johnson, A.G., 1989. T cell and interferon-gamma involvement in the adjuvant action of a detoxified endotoxin. J. Biol. Resp. Mod., 8: 625-3. Unanue, E.R., 1984. Antigen presenting function of the macrophage. Annu. Rev. Immunol., 2: 3951128. Veterinary Formulary, 1994. SecondEdition Pharmaceutical Press, London.

In: Debuf, Y.M. (Ed.).

Warren, H.S. and Chedid, L.A., 1988. Future prospects for vaccine adjuvants. CRC Crit. Rev. Immunol., 8: 83-99. Weaver, C.T., Hawrylowicz, C.M. and Unanue, E.R., 1988. T helper cell susbsets require the expression of distinct costimulatory signals by antigen presenting cells. Proc. Natl. Acad. Sci. USA, 85: 8181-8185. Weaver, CT. and Unanue, E.R., 1990. The costimulatory function of antigen presenting cells. Immunol. Today, I I: 49-55. Woodard, L.F., 1989. Adjuvant activity of water insoluble surfactams. Lab. Animal Sci., 39: 222-225.

162

J. Alexander, J.M. Brewer/Livestock

Woodard, L.F., 1990. Surface chemistry and classification of vaccine adjuvants and vehicles. In: Mizraki, A. (Ed.), Bacterial Vactines. Alan R. Liss, Inc., New York, pp. 281-306. Zanetti, M., Sercarz, E. and Salk, J., 1987. The immunology of new generation vaccines. Immunol. Today, 8: 18-25.

Production Science 42 (1995) 153-162

Zhou, F., Rouse, B.T. and Huang, L., 1992. Induction of cytotoxic T lymphocytes in vivo with protein antigen entrapped in membranous vehicles. J. Immunol., 149: 1599-1604. Ziegler, H.K., Staffileno, L.K. and Wentworth, P., 1984. Modulation of macrophage Iaexpression by LPS. I. Expression of Ia in vitro. J. Immunol., 133: 1825-1835.