Recent advances in veterinary vaccine adjuvants

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 immun-

ity (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

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 gener-

ation 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

International Journal for Parasitology 33 (2003) 469–478

www.parasitology-online.com

* Corresponding author. Tel.: þ1-510-923-7877; fax: þ1-510-923-2586.

E-mail address: [email protected] (M. Singh).

http://www.elsevier.com/locate/PARA

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 deter-

mine the use of these compounds for veterinary applications

are injection site reactogenicity, elimination or biodegrada-

tion 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’ mechan-

ism. 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 effec-

tive 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 discrimi-

nate 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 follow-

ing 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 Aluminium hydroxide, aluminium phosphate, calcium phosphate

Immunostimulatory adjuvants 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

Lipid particles Emulsions, e.g. Freund’s (CFA and IFA), ISA 25, 51, 206, SAF, MF59,

liposomes, virosomes, ISCOMS, cochleates

Particulate adjuvants PLG microparticles, poloxamer particles, virus-like particles

Mucosal adjuvants Heat-labile enterotoxin (LT), cholera toxin (CT), mutant toxins, e.g.

LTK63 and LTR72, microparticles, polymerised liposomes, chitosan

M. Singh, D.T. O’Hagan / International Journal for Parasitology 33 (2003) 469–478470

(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 pre-

clinical 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 demon-

stration that bacterial DNA, but not vertebrate DNA, had

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 non-

specific endocytosis and that endosomal maturation is

necessary for the cell activation and the release of pro-

inflammatory 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 polylactide-

co-glycolide microparticles co-administered with HIV-1 env gp120 recom-

binant protein adsorbed onto anionic polylactide-co-glycolide micro-

particles. 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.

M. Singh, D.T. O’Hagan / International Journal for Parasitology 33 (2003) 469–478 471

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, viro-

somes 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 immuno-

stimulatory 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 myco-

bacteria (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, para-

influenza, Newcastle disease and infectious canine hepatitis

(Chang et al., 1998). Several water-in-oil (w/o) and oil-in-

water (o/w) emulsions with or without mineral oils have

found mass applications in vaccination against foot-and-

mouth 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 non-

ionic 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, includ-

ing 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 formu-

lations, 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 pre-

pared 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 choles-

terol derivatives with or without neutral phospholipids (Guy

et al., 2001).

The immunostimulatory fractions from Q. saponaria

(Quil A) have been incorporated into lipid particles com-

prising cholesterol, phospholipids and cell membrane anti-

gens, 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


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 import-

ant. In addition, we have shown that the potency of

lipopeptides can be enhanced by their formulation into

particulate delivery systems (Nixon et al., 1996).

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 encapsula-

tion 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 micro-

particles 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

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 (polox-

amers) (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

Although most vaccines have traditionally been admin-

istered 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 advan-

tageous 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. How-

ever, 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,


mucosal immunisation with microparticles induced pro-

tection against challenge with Bordetella pertussis

(Cahill et al., 1995; Jones et al., 1996; Shahin et al., 1995;

Conway et al., 2001), Chlamydia trachomatis (Whittum-

Hudson 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 Escheri-

chia 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 manipu-

lated 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

immunisation have been evaluated with heat-labile entero-

toxin 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

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 DNA dose (mg) Fold increase over naked DNA

Naked DNA PLG/CTAB/DNA

Mice 1 22 7,664 .300

Guinea pigs 100 868 12,882 .15

Rabbits 250 644 8,778 .12

Rhesus macaques 500 19 10,220 .2000

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.


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 immunis-

ation 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 adju-

vants 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 poten-

tially associated with a robust response, e.g. local tissue

damage and systemic cytokine release. Further develop-

ments 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 under-

standing of the mechanism of action of currently available

veterinary vaccine adjuvants and this is an area of research

that requires additional work.

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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Recent advances in veterinary vaccine adjuvants