Recent advances in veterinary vaccine adjuvants

Recent advances in veterinary vaccine adjuvants

International Journal for Parasitology 33 (2003) 469–478 www.parasitology-online.com Invited review Recent advances in veterinary vaccine adjuvants ...

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International Journal for Parasitology 33 (2003) 469–478 www.parasitology-online.com

Invited review

Recent advances in veterinary vaccine adjuvants Manmohan Singh*, Derek T. O’Hagan Chiron Vaccines Research, Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608, USA Received 30 August 2002; received in revised form 9 January 2003; accepted 14 January 2003

Abstract Next generation veterinary vaccines are going to mainly comprise of either subunit or inactivated bacteria/viruses. These vaccines would require optimal adjuvants and delivery systems to accord long-term protection from infectious diseases in animals. There is an urgent need for the development of new and improved veterinary and human vaccine adjuvants. Adjuvants can be broadly divided into two classes, based on their principal mechanisms of action: vaccine delivery systems and ‘immunostimulatory adjuvants’. Vaccine delivery systems are generally particulate e.g. emulsions, microparticles, ISCOMS and liposomes, and mainly function to target associated antigens into antigen presenting cells (APC). In contrast, immunostimulatory adjuvants are predominantly derived from pathogens and often represent pathogen associated molecular patterns, e.g. LPS, MPL and CpG DNA, which activate cells of the innate immune system. Recent progress in innate immunity is beginning to yield insight into the initiation of immune responses and the ways in which immunostimulatory adjuvants might enhance this process in animals and humans alike. q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Veterinary vaccine adjuvants; Immunostimulators; Vaccine delivery systems; Microparticles; Emulsions

1. Introduction Widespread vaccination in animals still remains the most successful method to prevent losses in farm animals from infectious diseases (Aucouturier et al., 2001). Conventional veterinary vaccines have mainly consisted of live attenuated pathogens, whole inactivated organisms or inactivated bacterial toxins (Chang et al., 1998). Generally, these approaches have been successful for vaccine development due to the induction of antibodies, which neutralise viruses or bacterial toxins, inhibit binding of microorganisms to cells or promote their uptake by phagocytes. Although attenuated forms of the pathogen are used as veterinary vaccines, however, concerns about these occasionally reverting to the virulent form still exist. Employing killed organisms or parts thereof, is an alternative for these vaccines but they provide lesser degree of protection than attenuated forms. Also, non-living vaccines have generally proven ineffective at inducing potent cell-mediated immunity (CMI), particularly of the Th1 type. T helper cells can be classified into Th2 and Th1 subtypes, mainly based on their production of cytokines in mice, Th1 responses are * Corresponding author. Tel.: þ 1-510-923-7877; fax: þ1-510-923-2586. E-mail address: [email protected] (M. Singh).

characterised by the production of g interferon (IFN). In addition, although live vaccines can induce cytotoxic T lymphocytes (CTL), live attenuated vaccines may cause disease in immunosuppressed animals and some pathogens are difficult to grow in culture, making the development of inactivated vaccines impossible (Bowerstock and Martin, 1999). As a result of these limitations, several new approaches to veterinary vaccine development have emerged, which may have significant advantages over more traditional approaches. These approaches include recombinant protein subunits and plasmid DNA (Rankin et al., 2002). While these new approaches may offer some advantages, a general problem is that these vaccines may not be cost effective for veterinary use and are often poorly immunogenic (Bahenmann and Mesquita, 1987; Loehr et al., 2001). Traditional vaccines often contain many components that can elicit additional T cell help or function as adjuvants, e.g. bacterial DNA or LPS in whole cell vaccines. However, these components have been eliminated from new generation vaccines, which, therefore, need potent adjuvants. In the very recent past, there has been great interest in DNA vaccines for veterinary applications (Loehr et al., 2001), since they appear to offer significant potential for the induction of potent CTL and mucosal responses.

0020-7519/03/$30.00 q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0020-7519(03)00053-5

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Immunological adjuvants were originally described by Ramon (1924) as “substances used in combination with a specific antigen that produced a more robust immune response than the antigen alone”. This broad definition encompasses a very wide range of materials (Vogel and Powell, 1995). However, despite extensive evaluation of a large number of candidates over many years, the main adjuvant currently approved for human use by the US Food and Drug Administration are aluminium based mineral salts (generically called alum). Alum has a good safety record, but comparative studies in humans and animals show that it is a weak adjuvant for antibody induction to recombinant protein vaccines and induces a Th2, rather than a Th1 response (Gupta, 1998). A key issue in adjuvant development is toxicity, since safety concerns have restricted the development of many adjuvants since Freund’s adjuvant and alum were first introduced more than 50 years ago (Bahenmann and Mesquita, 1987; Edelman, 1997). Many experimental adjuvants have advanced to animal trials and some have demonstrated high potency, but most have proven too toxic for routine use. Some of the main issues that might determine the use of these compounds for veterinary applications are injection site reactogenicity, elimination or biodegradation of the adjuvant and duration of retention at site of injection. For standard prophylactic immunisation in healthy animals, only adjuvants that induce minimal local and systemic adverse effects will prove acceptable. Additional practical issues that are important for adjuvant development include stability, ease of manufacture, cost and applicability to a wide range of vaccines. Examples of different classes of adjuvants that are being evaluated for vaccines against infectious diseases in humans and animals are shown in Table 1.

2. Role of adjuvants in veterinary vaccine development Adjuvants can be used to improve the immune response to vaccine antigens in several different ways, including: (1) increasing the immunogenicity of weak antigens; (2) enhancing the speed and duration of the immune response; (3) modulating antibody avidity, specificity, isotype or

subclass distribution; (4) stimulating CTL; (5) promoting the induction of mucosal immunity; (6) enhancing immune responses in immunologically immature or senescent individuals; (7) decreasing the dose of antigen in the vaccine to reduce costs or (8) helping to overcome antigen competition in combination vaccines. The mechanisms of action of most adjuvants still remain only poorly understood, since immunisation often activates a complex cascade of responses and the primary effect of the adjuvant is often difficult to clearly discern. However, if one accepts the geographical concept of immune reactivity, in which antigens that do not reach the local lymph nodes do not induce responses (Zinkernagel et al., 1997), it becomes easier to propose mechanistic interpretations for some adjuvants, particularly those based on a ‘delivery’ mechanism. If antigens, which do not reach lymph nodes, do not induce responses, then, any adjuvant, which enhances delivery of antigen into the cells that traffic to the lymph node, may enhance the response. A subset of dendritic cells are thought to be the key cells which circulate in peripheral tissues and act as ‘sentinels’, being responsible for the uptake of antigens and their transfer to lymph nodes, where they are then presented to T cells. Circulating immature DCs are efficient for antigen uptake, while mature dendritic cells are efficient at antigen presentation to T cells. Hence, promoting antigen uptake into dendritic cells, trafficking to lymph nodes and dendritic cells maturation are thought to be key components to the generation of potent immune responses. Dendritic cells are thought to be the most effective antigen presenting cells (APC), although macrophages can also function in this role. The dominant paradigm in immunology for several decades was that the immune system evolved to discriminate self from non-self (Bretscher and Cohn, 1970). This hypothesis resulted in significant progress in understanding the clonal recognition of antigenic epitopes mediated by B and T lymphocytes. However, the self/non-self framework offers little insight into why some non-self antigens are found to be poorly immunogenic. In the last decade, alternative models of immunity have been established, which emphasise the selective pressures on the host to induce a pro-inflammatory innate immune response following exposure to pathogen associated molecular patterns

Table 1 Selective list of different classes of adjuvants which have been evaluated for enhancing immune responses to vaccines in animals Mineral salts Immunostimulatory adjuvants

Lipid particles Particulate adjuvants Mucosal adjuvants

Aluminium hydroxide, aluminium phosphate, calcium phosphate Cytokines, e.g. IL-2, IL-12, GM-CSF, saponins, (e.g. QS21), MDP derivatives, bacterial DNA (CpG oligos), LPS, MPL and synthetic derivatives, lipopeptides Emulsions, e.g. Freund’s (CFA and IFA), ISA 25, 51, 206, SAF, MF59, liposomes, virosomes, ISCOMS, cochleates PLG microparticles, poloxamer particles, virus-like particles Heat-labile enterotoxin (LT), cholera toxin (CT), mutant toxins, e.g. LTK63 and LTR72, microparticles, polymerised liposomes, chitosan

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(Janeway, 1989; Medzhitov and Janeway, 1997) and tissue damage (Matzinger, 1994, 1998; Shi et al., 2000). These responses are not antigen-specific and are mediated by the innate immune system, which is the first line of immune defence and is highly conserved throughout many species. Pathogen associated molecular patterns are perceived as ‘danger signals’ following binding to toll-like receptors on phagocytic APC and induce the release of pro-inflammatory cytokines, which stimulate and focus the adaptive immune response (Fearon, 1997; Fearon and Locksley, 1996). Traditional veterinary vaccines such as inactivated pathogens and attenuated viral vaccines often contain most of the features of real pathogens and, therefore, are sufficiently potent to induce protective immune responses. In contrast, recombinant vaccines, which may be used in the future, are highly purified, lack many of the features of the original pathogen and do not evoke strong immune responses. Hence, it can be argued that the role of adjuvants for recombinant vaccines is to ensure that the vaccine resembles infection closely enough to initiate a potent immune response (Janeway, 1989; Fearon, 1997). In addition, the innate immune system directs the balance of humoural and CMI (Fearon and Locksley, 1996), and adjuvants can control the type of acquired immune response induced (Yip et al., 1999). Adjuvants can be divided into different broad groups based on their principal modes of action, depending on whether or not they have direct immunostimulatory effects on APC or function as antigen delivery systems. However, any classification of adjuvants is difficult and many examples resist easy definitions.

3. Immunostimulatory adjuvants Monophosphoryl lipid A is derived from LPS of Salmonella minnesota, a gram negative bacteria and, therefore, is classified as a pathogen associated molecular pattern. Like lipo polysaccharide, monophosphoryl lipid A is thought to interact with TLR4 on APC, resulting in the release of pro-inflammatory cytokines. In a number of preclinical studies, monophosphoryl lipid A has been shown to induce the synthesis and release of IL-2 and IFN-g, which promote the generation of Th1 responses (Gustafson and Rhodes, 1992; Ulrich and Myers, 1995). Monophosphoryl lipid A has been formulated into emulsions to enhance its potency (Ulrich, 2000). Structure – function studies of MPL allowed identification of a new generation of synthetic adjuvants based on aminoalkyl glucosamine phosphate compounds (Johnson et al., 1999), the lead candidate (RC-529), which is currently being evaluated in trials. In addition, several synthetic mimetics of monophosphoryl lipid A are available from alternative sources, which are yet to be evaluated in clinical trials (Hawkins et al., 2002). In the last few years, a whole new class of adjuvant active compounds have been identified, following the demonstration that bacterial DNA, but not vertebrate DNA, had

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direct immunostimulatory effects on immune cells in vitro (Rankin et al., 2002; Messina et al., 1991; Tokunaga et al., 1984). The immunostimulatory effect was due to the presence of unmethylated CpG dinucleotides (Krieg et al., 1995), which are under-represented and methylated in vertebrate DNA. Unmethylated CpG in the context of selective flanking sequences are thought to be recognised by cells of the innate immune system to allow discrimination of pathogen-derived DNA from self-DNA (Bird, 1987). It has recently been shown that responses to CpG DNA are mediated by binding to TLR9 (Hemmi et al., 2000). Previously, it was reported that CpG are taken up by nonspecific endocytosis and that endosomal maturation is necessary for the cell activation and the release of proinflammatory cytokines (Sparwasser et al., 1998). The Th1 adjuvant effect of CpG appears to be maximised by their conjugation to protein antigens (Klinman et al., 1999) or their formulation with delivery systems (Fig. 1) (Singh et al., 2001b). Although, CpG have mainly been evaluated in rodent models, recent papers have described sequences that are active in non-human primates (Hartmann et al., 2000) and farm animals (Rankin et al., 2002). A third group of immunostimulatory adjuvants are the triterpenoid glycosides or saponins, derived from the bark of a Chilean tree, Quillaja saponaria (Quil A). Saponins appear to function mainly through the induction of cytokines. Saponins have been widely used as adjuvants for many years and have been included in several veterinary vaccines. QS21, which is a highly purified fraction from Quil A, has been shown to be a potent adjuvant for Th1 cytokines (IL-2 and IFN-g) and antibodies of the IgG2a isotype, which indicates a Th1 response in mice (Kensil, 1996). Saponins have been shown to intercalate into cell

Fig. 1. Antibody responses following two intramuscular immunisations 4 weeks apart in mice with CpG adjuvant adsorbed to cationic polylactideco-glycolide microparticles co-administered with HIV-1 env gp120 recombinant protein adsorbed onto anionic polylactide-co-glycolide microparticles. For comparison, we evaluated polylactide-co-glycolide with gp120 adsorbed and CpG with gp120. In addition, the responses induced were compared with gp120 in MF59. Geometric mean titres ^ s.e. represented for each group.

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membranes, through interaction with structurally similar cholesterol, forming ‘holes’ or pores (Glaueri et al., 1962). It is currently unknown if the adjuvant effect of saponins is related to pore formation, which may allow antigens to gain access to the endogenous pathway of antigen presentation, promoting a CTL response (Sjolander et al., 2001).

4. Particulate antigen delivery systems The use of particulate adjuvants or antigen delivery systems, as alternatives to immunostimulatory adjuvants, has been evaluated by several groups. Particulate adjuvants (e.g. emulsions, microparticles, ISCOMS, liposomes, virosomes and virus-like particles) have comparable dimensions to the pathogens, which the immune system evolved to combat. Immunostimulatory adjuvants may also be included in particulate delivery systems to enhance the level of response or focus the response through a desired pathway, e.g. Th1. In addition, formulating potent immunostimulatory adjuvants into delivery systems may limit adverse events, through restricting the systemic circulation of the adjuvant. 4.1. Lipid particles as adjuvants Complete Freund’s adjuvant is a potent, but toxic water in mineral oil adjuvant, which may contain killed mycobacteria (Rankin et al., 2002; Lindblad, 2000). Incomplete Freund’s adjuvant (IFA) is an emulsion without the killed mycobacterium. IFA has found use in farm animal vaccination due to its strong adjuvant effect (Aucouturier et al., 2001; Rankin et al., 2002). Some of the veterinary vaccines that have used IFA include foot-and-mouth disease, equine influenza virus, hog cholera, rabies, parainfluenza, Newcastle disease and infectious canine hepatitis (Chang et al., 1998). Several water-in-oil (w/o) and oil-inwater (o/w) emulsions with or without mineral oils have found mass applications in vaccination against foot-andmouth disease and Newcastle diseases in farm animals (Bahenmann and Mesquita, 1987; Barnett et al., 1996). Vaccination for the foot-and-mouth disease in animals has been extensively carried out in two mineral oil emulsions from Seppice Montanide ISA 206 and ISA 25 (Barnett et al., 1996). Emulsified vaccines based on mineral oils like Drakeol and Marcol also induce high levels of immunity in cattle and pigs (Cunliffe and Graves, 1963). A potent o/w adjuvant, the Syntex adjuvant formulation was developed using a biodegradable-oil (squalane) in the 1980s, as a replacement for Freund’s adjuvants. However, Syntex adjuvant formulation contained a bacterial cell wall based synthetic adjuvant, threonyl muramyl dipeptide and a nonionic surfactant, poloxamer L121 and proved too toxic for widespread use in humans (Edelman, 1997). Therefore, a squalene o/w emulsion was developed (MF59) without the presence of additional immunostimulatory adjuvants, which

proved to be a potent adjuvant with an acceptable safety profile (Ott et al., 1995). Because of its safety and efficacy, MF59 may have applications for veterinary use. In many studies, emulsions have also been used as delivery systems for immunostimulatory adjuvants, including monophosphoryl lipid A and QS21. This approach allows immunostimulatory adjuvants to be targeted for enhanced uptake by APC. An o/w emulsion containing monophosphoryl lipid A and QS21 induced protection in a mouse model of malaria that was comparable or better than the levels of protection induced with the vaccine in Freund’s complete adjuvant (Ling et al., 1997). An alternative emulsion based approach involves the use of the Montanide series of adjuvants (ISA 25, 51, 206, etc), which can be formulated as w/o, o/w or w/o in water emulsions (Lawrence et al., 1997; Aucouturier et al., 2000). The water in mineral oil (Drakeol) adjuvant (ISA-51) has been evaluated as an vaccine adjuvants in animals (Bowerstock and Martin, 1999). Liposomes are phospholipid vesicles which have been evaluated both as adjuvants and as delivery systems for antigens and adjuvants (Alving, 1992; Gregoriadis, 1990). Liposomes have been commonly used in complex formulations, often including monophosphoryl lipid A, which makes it difficult to determine the contribution of the liposome to the overall adjuvant effect. Immunopotentiating reconstituted influenza virosomes are unilamellar liposomes comprising mainly phosphatidylcholine, with influenza haemagglutinin intercalated into the membrane. The use of viral membrane proteins in the formation of virosomes offers the opportunity to exploit the targeting and fusogenic properties of the native viral membrane proteins, perhaps resulting in effective delivery of entrapped antigens into the cytosol for CTL induction (Bungener et al., 2000). An alternative approach to vaccine delivery which may have some advantages over traditional liposomes has been described using ‘archaeosomes’, which are vesicles prepared from the polar lipids of Archaeobacteria (Krishnan et al., 2000). In some studies, archaeosomes have been shown to be more potent than liposomes (Krishnan et al., 2000; Conlan et al., 2001). Cationic lipid vesicles have also been described recently, which comprise cationic cholesterol derivatives with or without neutral phospholipids (Guy et al., 2001). The immunostimulatory fractions from Q. saponaria (Quil A) have been incorporated into lipid particles comprising cholesterol, phospholipids and cell membrane antigens, which are called ISCOMs (Barr et al., 1998). The principal advantage of the preparation of ISCOMS is to allow a reduction in the dose of the haemolytic Quil A adjuvant and to target the formulation directly to APC. In addition, within the ISCOM structure, the Quil A is bound to cholesterol and is not free to interact with cell membranes. Therefore, the haemolytic activity of the saponins is significantly reduced (Barr et al., 1998; Soltysik et al., 1995). It is well established that ISCOMS induce cytokine

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production in a range of mouse strains and a recent study has indicated that the induction of IL-12 is key to the adjuvant effect of ISCOMS (Smith et al., 1999). In previous studies, strong IFN-g responses were also described (Emery et al., 1990). ISCOM formulations have also evaluated in various animal models and a licensed ISCOM based vaccine is used to protect horses from equine influenza in Sweden (Sjolander et al., 2001). An alternative approach involving lipid vesicles has also been described involving non-ionic surfactant vesicle or ‘niosomes’, which have induced potent responses in small animal models (Brewer et al., 1998). In addition, it has been suggested that an important component of the adjuvant effect of synthetic lipopeptide antigens is their ability to aggregate into particulate structures (Tsunoda et al., 1999), although interaction with toll-like receptors is also important. In addition, we have shown that the potency of lipopeptides can be enhanced by their formulation into particulate delivery systems (Nixon et al., 1996).

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microparticles can be used as delivery systems for adjuvant active molecules, including CpG DNA (Singh et al., 2001b). Similar anionic microparticles can also be used for delivery of adsorbed proteins and are effective for CTL induction in mice (Kazzaz et al., 2000). Polymers which self-assemble into particulates (poloxamers) (Newman et al., 1998) or soluble polymers (polyphosphazenes) (Payne et al., 1998) may also be used as adjuvants, but the safety and tolerability of these approaches remains to be further evaluated. For example, recombinant Ty VLPs from Saccharomyces cerevisiae carrying a string of up to 15 CTL epitopes from Plasmodium species have been shown to prime protective CTL responses in mice following a single immunisation (Gilbert et al., 1997). In addition, Ty VLPs have also been shown to induce CTL activity in macaques against co-expressed SIV p27 (Klavinskis et al., 1996).

5. Alternative routes of immunisation 4.2. Microparticles as adjuvants Antigen uptake by APC is enhanced by association of antigen with polymeric microparticles or by the use of polymers or proteins which self-assemble into particles. The biodegradable and biocompatible polyesters, the polylactide-co-glycolides are the primary candidates for the development of microparticles as adjuvants, since they have been used in humans and animals for many years as suture material and as controlled release drug delivery systems (Okada and Toguchi, 1995; Putney and Purke, 1998). The adjuvant effect achieved through the encapsulation of antigens into polylactide-co-glycolide microparticles was first demonstrated by several groups in the early 1990s (Eldridge et al., 1991; O’Hagan et al., 1991a,b, 1993). In contrast to alum, polylactide-co-glycolide microparticles have been shown to be effective for the induction of CTL responses in rodents (Nixon et al., 1995; Maloy et al., 1994; Moore et al., 1995). The adjuvant effect of microparticles appears to be largely a consequence of their uptake into APC. Microparticles also appear to have significant potential as an adjuvant for DNA vaccines (Hedley et al., 1998; Singh et al., 2000). We have recently described a novel approach in which cationic microparticles with adsorbed plasmids were used to dramatically enhance the potency of DNA vaccines in rodents and primates (Singh et al., 2000). Importantly, the cationic microparticles enhanced responses in a range of animal models, including non-human primates (Table 2). They efficiently adsorbed DNA and delivered several plasmids simultaneously on the same formulation, at a range of different loading levels (Briones et al., 2001; O’Hagan et al., 2001) The microparticles appeared to be effective as a consequence of efficient delivery of the adsorbed plasmids into dendritic cells, the most important APC for presentation of antigen to naive T cells (Denis-Mize et al., 2000). In addition, cationic

Although most vaccines have traditionally been administered by intramuscular or subcutaneous injection, mucosal administration of vaccines offers a number of important advantages, including easier administration, reduced adverse effects and the potential for frequent boosting in farm animals. In addition, local immunisation induces mucosal immunity at the sites where many pathogens initially establish infection of hosts. In general, systemic immunisation has failed to induce mucosal IgA antibody responses. Oral immunisation would be particularly advantageous in isolated communities and farms, where access to veterinary health care is difficult. Moreover, mucosal immunisation would avoid the potential problem of infection due to the re-use of needles. The most attractive route for mucosal immunisation is oral, due to the ease and acceptability of administration through this route. However, due to the presence of acidity in the stomach, an extensive range of digestive enzymes in the intestine and a protective coating of mucus which limits access to the mucosal epithelium, oral immunisation has proven extremely difficult with non-living antigens. However, novel delivery systems and adjuvants may be used to significantly enhance the responses following oral immunisation. 5.1. Mucosal immunisation with microparticles Both systemic and mucosal responses against albumin were generated in cattle using alginate microparticles administered orally and intranasally (Bowerstock and Martin, 1999; Rebelatto et al., 2001). In mice, oral immunisation with polylactide-co-glycolide microparticles has been shown to induce potent mucosal and systemic immunity to entrapped antigens (Challacombe et al., 1992, 1997; Eldridge et al., 1990; O’Hagan, 1994). In addition,

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Table 2 Levels of enhancement of antibody responses achieved with cationic PLG/DNA microparticles in comparison to naked DNA (HIV-1 gag) following two intramuscular immunisations 4 weeks apart in various animal models Species

Mice Guinea pigs Rabbits Rhesus macaques

DNA dose (mg)

1 100 250 500

Fold increase over naked DNA Naked DNA

PLG/CTAB/DNA

22 868 644 19

7,664 12,882 8,778 10,220

mucosal immunisation with microparticles induced protection against challenge with Bordetella pertussis (Cahill et al., 1995; Jones et al., 1996; Shahin et al., 1995; Conway et al., 2001), Chlamydia trachomatis (WhittumHudson et al., 1996) and Salmonella typhimurium (Allaoui-Attarki et al., 1997). The ability of microparticles to perform as effective adjuvants following mucosal administration is largely a consequence of their uptake into the specialised mucosal associated lymphoid tissue (O’Hagan, 1996). While microparticles have significant potential for mucosal delivery of vaccines, their potency may be improved by their use in combination with additional adjuvants (Bowerstock and Martin, 1999). Accumulated experimental evidence suggests that simple encapsulation of vaccines into microparticles is unlikely to result in the successful development of oral vaccines and improvements in the current technology are clearly needed (Brayden, 2001). 5.2. Adjuvants for mucosal immunisation The most potent mucosal adjuvants currently available are the bacterial toxins from Vibrio cholerae and Escherichia coli, cholera toxin and heat-labile enterotoxin, respectively. However, since cholera toxin and heat-labile enterotoxin are the causes of cholera and travellers diarrhoea, they are generally considered too toxic for use in humans. Therefore, they have been genetically manipulated to reduce toxicity (Dickinson and Clements, 1995; Douce et al., 1995, 1997). Single amino acid substitutions in the enzymatic A subunit of heat-labile enterotoxin allowed the development of mutant toxins that retained potent adjuvant activity, but showed negligible or dramatically reduced toxicity (Di Tommaso et al., 1996; Giannelli et al., 1997; Giuliani et al., 1998). Heat-labile enterotoxin mutants have been used by the oral route to induce protective immunity in mice against Helicobacter pylori challenge (Marchetti et al., 1998). In addition, heat-labile enterotoxin mutants have been shown to be potent oral adjuvants for influenza vaccine (Barackman et al., 2001) and model antigens (Douce et al., 1999). Nevertheless, due to the significant challenges associated with oral immunisation, various alternative routes of

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immunisation have been evaluated with heat-labile enterotoxin mutants, including nasal, intravaginal and intrarectal. Of these, intranasal immunisation offers the most promise, both due to the potent responses induced by this route and due to the easy access and simple administration devices, which already exist. On many occasions, the ability of heat-labile enterotoxin mutants to induce potent antibody responses following intranasal immunisation has been demonstrated (Rappuoli et al., 1999). In recent studies, heat-labile enterotoxin mutants have shown protection against challenge with B. pertussis (Ryan et al., 1999), Streptococcus pneumoniae (Jakobsen et al., 1999) and herpes simplex virus (O’Hagan et al., 1999) following intranasal immunisation and the induction of potent CTL responses (Simmons et al., 1999; Neidleman et al., 2000). In addition, we recently showed that the potency of heat-labile enterotoxin mutants may be enhanced by their formulation into a novel bioadhesive microsphere delivery system (Fig. 2) (Singh et al., 2001a). Although the mechanisms of action of cholera toxin and heat-labile enterotoxin remain to be fully defined, it appears that there are important contributions to the adjuvant effect

Fig. 2. Following two intranasal immunisations 4 weeks apart in mice, enhanced serum antibody responses were obtained with influenza vaccine (HA) and mucosal adjuvant LTK63 in combination with bioadhesive HYAFF microspheres (HA þ LTK63 þ HYAFF). For comparison, mice were also immunised with antigen alone (HA), antigen and microspheres (HA þ HYAFF) or antigen plus adjuvant (HA þ LTK63). Geometric mean titres ^ s.e. represented for each group.

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from the B subunit binding domain, the presence of an intact A subunit, which interacts with regulatory proteins inside cells and also the enzymatic activity of the A1 subunit (Rappuoli et al., 1999). Recent studies have indicated that potent mucosal adjuvants such as cholera toxin may also allow vaccination following topical application to the skin (Glenn et al., 1998) and that this approach may be applicable to animals and humans (Glenn et al., 2000). In addition, epidermal immunisation may be achieved using needle-free devices, which use helium gas to deposit powdered vaccine into the epidermis (Chen et al., 2000). An alternative approach to the development of mucosal adjuvants involves the use of plant lectins (Lavelle et al., 2001). Furthermore, oral immunisation may also be achieved through the ingestion of transgenic plants expressing antigens and adjuvants (Tacket et al., 1998; Richter et al., 2000).

6. Future developments in vaccine adjuvants Several recent issues have served to highlight the urgent need for the development of new and improved vaccines for veterinary applications. These problems have included: (1) emergence of new diseases, (2) re-emergence of ‘old’ infections and (3) continuing spread of antibiotic resistant bacteria. In this review, we have suggested that the adjuvants to be used in these vaccines may have to closely mimic an infection and/or induce localised tissue damage to elicit protective immunity in animals. This may be achieved through the use of particulate delivery systems, which have similar dimensions to pathogens and are able to target antigens to macrophages and dendritic cells. If this hypothesis is correct, it suggests that a delicate balance must be maintained between the desired initiation of immune responses and avoidance of the problems potentially associated with a robust response, e.g. local tissue damage and systemic cytokine release. Further developments in the delivery of adjuvants may be achieved through the identification of specific receptors on APC, which might be extra- or intracellular. If intracellular, then a means to promote uptake of the delivery system by the relevant cells may also be required for optimal efficacy. Future developments in adjuvants will most likely also be driven by the economics of immunisation. For veterinary applications particularly, a significantly lower cost per animal would be necessary in comparison to human vaccines. So far, the adjuvants utilised extensively in the veterinary field have been either mineral oil emulsions or aluminium hydroxide with additional compounds for immunopotentiation. However, further developments in novel adjuvants will likely be driven by a better understanding of the mechanism of action of currently available veterinary vaccine adjuvants and this is an area of research that requires additional work.

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Acknowledgements We would like to acknowledge the contributions of our colleagues in Chiron Corporation to the ideas contained in this review, particularly, Rino Rappuoli. We would also like to thank all the members of the Vaccine Delivery Group at Chiron.

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