Vaccine 22 (2004) 2391–2395

ISCOMATRIXTM adjuvant: a potent inducer of humoraland cellular immune responses�

M.J. Pearse∗, D. DraneCSL Limited, 45 Poplar Road, Parkville, Vic., Australia

Available online 7 April 2004

Abstract

ISCOMATRIXTM adjuvant is capable of inducing broad and potent humoral and cellular immune responses. The components are welldefined and the manufacturing process is simple and robust. Many vaccines containing the ISCOMATRIXTM adjuvant have been testedin a range of animal models, including human and non-human primates. Strong antibody and T cell responses have been induced in thesestudies[1]. The antibody response is often achieved with lesser amounts of antigen than other adjuvant systems and the maximal responseshave also been reached more quickly. Both CD4+ and CD8+ T cell responses are induced with the cytotoxic T lymphocyte responses beingvery long lived. Additionally, ISCOMATRIXTM adjuvant can be used in vaccines for induction of mucosal immune responses. This reviewprovides an overview of the immune responses that can be elicited using ISCOMATRIXTM vaccines and the current state of knowledgeregarding the mechanism of action of this adjuvant.© 2004 Elsevier Ltd. All rights reserved.

Keywords: Saponin; Adjuvant; ISCOMATRIX; ISCOM; Cellular immune responses; Humoral immune responses

1. Introduction

The typical cage-like structures of ISCOMATRIXTM ad-juvant (Fig. 1) comprise saponin, cholesterol and phospho-lipid molecules which are held together by hydrophobicforces[2].

Saponins have been shown to have potent adjuvantactivity and have been used in animal vaccines as anadjuvant for many years[3]. The saponin used in themanufacture of ISCOMATRIXTM adjuvant for clinicaluse is called ISCOPREPTM saponin which is a purifiedsaponin fraction from the bark of theQuillaija saponariatree. The ISCOPREPTM saponin is prepared by a pro-prietary process that is robust and highly reproducible.The complexing of saponin with cholesterol and phospho-lipid to form the ISCOMATRIXTM adjuvant is beneficialfor a number of reasons. These include a reduction inhaemolytic activity, increase in saponin stability and in-creased capacity to associate with a broad range of anti-gens.

� ISCOMATRIXTM Trade Mark of ISCOTEC AB, a CSL LimitedCompany.

∗ Corresponding author. Tel.:+61-393-891-254.E-mail address: martin.pearse@csl.com.au (M.J. Pearse).

The ISCOMATRIXTM adjuvant has been extensivelycharacterised and the raw materials used are well defined,making the technology suitable for the development ofnovel vaccines for human use. The manufacturing processis simple and has been extensively optimised to producea robust process that is well controlled and scalable. TheISCOMATRIXTM adjuvant is extremely stable maintainingboth physical and biological characteristics for many yearswhen stored at 2–8◦C.

The original ISCOMTM vaccines were manufacturedby incorporation of antigens into the structure duringformation [4]. This limited the use of the technology toantigens with hydrophobic areas such as membrane pro-teins and often the manufacturing process was complexand difficult to control. CSL have developed a processfor the manufacture of ISCOMATRIXTM adjuvant, whichdoes not contain antigen, as well as methods which en-able the association of antigens to form ISCOMATRIXTM

vaccines. Although the manufacturing process and com-ponents differ from that used for ISCOMTM vaccines, theadjuvant properties remain the same. The processes devel-oped are simple and amenable to large scale manufacture,making ISCOMATRIXTM adjuvant more suitable to thedevelopment of novel human vaccines that can be regis-tered.

0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.vaccine.2003.12.031

2392 M.J. Pearse, D. Drane / Vaccine 22 (2004) 2391–2395

Fig. 1. Electron micrograph of optimised ISCOMATRIXTM adjuvant fol-lowing negative staining. ISCOMATRIXTM adjuvant particles are typi-cally rigid, hollow, spherical, cage-like particles approximately 40 nm indiameter.

2. Immune responses to ISCOMTM andISCOMATRIXTM vaccines

2.1. Antibody responses

ISCOMTM and ISCOMATRIXTM vaccines have beenshown to be potent inducers of antibody responses in alarge number of studies using a range of antigens in manyanimal species, including human and non-human primates.

Recent studies in guinea pigs, sheep and baboons haveshown the significant benefit of ISCOMATRIXTM adjuvantin terms of antigen dose reduction. In guinea pigs the HIVantigen MNgp120, combined with ISCOMATRIXTM adju-vant, induced neutralising antibody responses using 10–100fold less antigen than was required with aluminium hydrox-ide adjuvant (Fig. 2) (Boyle J. et al., manuscript in prepa-ration). In sheep[5] and baboons (unpublished data) given

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Fig. 2. Neutralising antibody responses, as determined by a HIV MNgp120 V3 specific EIA, in guinea pigs receiving either 0.3 or 3�gHIV MNgp120 with ISCOMATRIXTM adjuvant (IMX) or 30�g HIVMNgp120 with aluminium adjuvant (ALUM).

influenza vaccine antigens, serum HAI titres induced withan ISCOMATRIXTM adjuvanted vaccine were equivalentto or better than unadjuvanted vaccines that contained 10fold more antigen. Similar dose reduction observations withISCOMATRIXTM influenza vaccines have also been madein humans. Furthermore, the addition of ISCOMATRIXTM

adjuvant to influenza vaccine induced higher and more rapidantibody responses than the conventional influenza vaccine[6]. This accelerated induction of antibody responses couldbe advantageous in epidemic situations when a rapid re-sponse, particularly in unprotected individuals, is requiredto prevent disease or reduce morbidity.

Another important feature of ISCOMTM and ISCOMAT-RIXTM vaccines is the ability to induce antibody responsesin the presence of pre-existing antibody. Many pathogensare capable of eliciting antibody responses that do not elim-inate the disease, for example, HBV, HCV and HIV. A vac-cine adjuvant capable of stimulating immune responses inthe presence of these antibodies is critical if therapeutic vac-cination strategies are to be successful. ISCOMTM vaccineshave shown potential at overcoming the neutralising effectsof passively acquired antibody in neonatal mice, horses andmacaques[7–9]. In non-human primates and horses, respec-tively, active immunity was induced in the presence of ma-ternal antibody against measles virus and equine herpes 2virus with ISCOMTM vaccines, but not conventional killedvaccines. Furthermore, ISCOMTM vaccines can induce Th1immune responses, including primary T cell responses inneonates[7]. In contrast, conventional vaccines elicit mainlyTh2 responses in this age group. This bias tends to persistafter boosting in the adult, which in many cases leads to animmunologically irrelevant response.

2.2. Cellular immune responses

The induction of cellular immune responses, particularlyin humans has to date proven to be a significant hurdle forthe development of therapeutic vaccines. Delivery systemssuch as DNA and viral vectors have offered some hope buthave potential safety problems, and in the case of DNAgenerally elicit poor cellular responses, in particular CTLresponses. Additionally, viral vectors have the problem ofinducing neutralising antibodies to the vector which lim-its repeated use. Prime-boost combinations of DNA andlive viral vector delivery are currently being evaluated,and although results have been promising in animal mod-els they are yet to be proven in humans. By contrast, theISCOMATRIXTM adjuvant has proven to be a potent in-ducer of cellular immune responses in many animal speciesincluding human and non-human primates. Furthermore,ISCOMATRIXTM adjuvant is simple to manufacture atlarge scale, can be used repeatedly and does not have thesafety concerns of DNA or viral vectors.

Many studies have been performed in non-human pri-mates with both ISCOMTM and ISCOMATRIXTM vaccinesand in all cases strong cellular immune responses have been

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Fig. 3. Haemagglutination inhibition (HAI) responses to influenza (A/New Calendonia) following intranasal immunisations. Mice were vaccinated with1�g influenza HA as either an ISCOMTM vaccine (ISCOM), ISCOMATRIXTM vaccine (IMX) or unadjuvanted vaccine (No adj).

induced. These studies have included comparison with otheradjuvants and delivery systems, and ISCOMTM based vac-cines have generally been superior at induction of cellu-lar immune responses, in particular cytotoxic T lymphocyte(CTL) responses[10]. In a study performed with an HCVcore protein ISCOMATRIXTM vaccine in rhesus macaques,strong CD4+ and CD8+ T cell responses against a broadrange of epitopes were observed in 100 and 60% of vac-cinated animals, respectively. The CD8+ T cell responseswere confirmed in a CTL lysis assay. Furthermore, there wasevidence to indicate that the animals which did not generateHCV core-specific CD8+ T cell responses to the peptidesanalysed, could not do so because of MHC restriction[11].The CTL response induced by immunisation with the HCVcore protein ISCOMATRIXTM vaccine was extremely longlived, with strong responses still detected almost a year af-ter the final dose. In contrast, the CTL responses generatedfollowing vaccination with recombinant vaccinia virus ex-pressing HCV core protein were diminished at week 4 andnegligible by week 18 post-vaccination. However, it shouldbe noted that this study examined only a single dose as theinduction of neutralising antibodies prevented repeat dosingwith vaccinia.

In all clinical trials performed with ISCOMATRIXTM vac-cines, cellular immune responses where evaluated were de-tected. CD4+ and CD8+ T cell responses were detected in60–90% and 20–80% of vaccinated subjects, respectively[6,12] (Davis I. et al., Frazer I. et al. and Basser R. et al.,manuscripts in preparation). Further studies are ongoing toevaluate the clinical usefulness of these types of immuneresponses.

The induction of cellular immune responses, especiallyCTL, is considered to be particularly important for ther-apeutic cancer vaccines. To this end a number of studieshave been performed using prophylactic and therapeuticmurine tumour models. These studies have shown thatISCOMATRIXTM vaccines given prophylactically pro-vide 100% protection against tumour challenge. More

importantly, ISCOMATRIXTM vaccines were also effectivein a stringent therapeutic model with complete clearance oftumours in animals vaccinated up to 24 days post-tumourimplant (unpublished data).

2.3. Protection in animal models

As described above, ISCOMTM and ISCOMATRIXTM

vaccines are potent inducers of both humoral and cellularimmunity. Consistent with this, these vaccines have beenshown to elicit protective immunity in a variety of animalspecies against a range of pathogens including viruses, bac-teria, mycoplasma and parasites[1,13]. Of particular rele-vance to human vaccine development, ISCOMTM vaccinesformulated with HIV-1 gp120, HIV-1 multicomponent anti-gens (gp120, p24, V2 and V3 peptides), SIV antigens[14,15]and EBV gp340[16] have been shown to protect monkeysagainst viral challenge with these pathogens. Furthermore,in a study in macaques, an ISCOMTM influenza vaccine wasshown to be more immunogenic and induced enhanced pro-tective efficacy compared with non-adjuvanted conventionalinfluenza vaccine[17].

2.4. Mucosal immune responses

The use of ISCOMTM vaccines for nasal vaccination wasrecently reviewed by Hu et al.[18]. It has been shown in micethat influenza ISCOMTM vaccines induce CTL as well asstrong mucosal (IgG and IgA) and systemic (IgG) antibodyresponses when delivered intranasally[19]. The responsesgenerated were equivalent to those obtained with the strongmucosal adjuvant LT (E. coli heat-labile entero-toxin). Ani-mal studies have shown that similar immune responses weregenerated with influenza ISCOMTM and ISCOMATRIXTM

vaccines following intranasal delivery (Fig. 3). Systemic andmucosal antibody responses in other species including sheep[5] and baboons (unpublished observation) have also beenobserved following intranasal delivery, although they have

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generally not been as strong as those in the mouse. Thelikely explanation for this is inefficient delivery of vaccine tonasal immune effector sites in these species. Moreover, mu-cosal antibody responses have also been observed at distantmucosal sites (genital and intestinal tracts) after intranasaldelivery with ISCOMTM vaccines[5,13,19]. Intranasal de-livery of ISCOMATRIXTM based vaccines has the prospectto increase the efficacy of vaccines for the elderly, in whomthe systemic immune system is waning, but the mucosal im-mune system remains active[13]. Of particular relevance tothe development of vaccines to gut parasites, Carol et al.[20] demonstrated the induction of strong immune responsesin the gut toEchinococcus granulosus antigens followingintranasal delivery of an ISCOMTM vaccine.

3. Mechanism of action

The ISCOMATRIXTM adjuvant induces a balancedTh1/Th2 response and is a potent inducer of antibody aswell as CD4+ and CD8+ T cell responses. This broad im-mune response is dependent on the induction of multipleinnate and adaptive immune mediators, cellular processesand the interplay between these elements. The details ofthis complex process are yet to be fully elucidated, how-ever, some details are emerging. Clearly, cytokine inductionis an important component of ISCOMATRIXTM adjuvantactivity. Upregulation of pro-inflammatory IL-1 was thefirst cytokine response observed to be related to the adju-vant activity of ISCOMTM vaccines[21]. Since then manyother cytokines have been shown to be up-regulated in re-sponse to ISCOMTM and ISCOMATRIXTM vaccines, theseinclude IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 and IFN-�[22,23]. This broad range of cytokines is consistent withthe mixed Th1/Th2 responses observed with ISCOMTM andISCOMATRIXTM vaccines.

Recent studies using a sheep afferent lymph node can-nulation model have shown that ISCOMATRIXTM adjuvantalone, in the absence of antigen, results in effects at thelevel of the draining lymph node such as an increase in lym-phocyte recruitment and the expression of pro-inflammatorycytokines (IL-1, IL-6, IL-8 and IFN-�) [24]. The produc-tion of inflammatory mediators by immune effector cellsis now recognised as being critical for the induction ofprimary immune responses. Cellular recruitment and acti-vation are greatly reduced in IL-12 knock-out mice givenISCOMTM vaccines, suggesting that IL-12 is important foradjuvant activity[25]. Together these observations suggestthat ISCOMTM vaccines activate innate immune responses,with IL-12 playing a key role. ISCOMTM vaccines have alsobeen shown to induce upregulation of MHC class II andCD86 expression on APCs[26]. The depot effect, whereby,antigen is trapped at the site of administration, in order to at-tract antigen presenting cells (APCs) is considered to be animportant function of adjuvants[27]. However, unlike alu-minium and oil-based adjuvants, ISCOMATRIXTM based

vaccines are cleared rapidly from the site of injection to thedraining lymph nodes, although there is some evidence ofdose site effects such as cellular infiltration.

The saponin component of the ISCOMATRIXTM ad-juvant is critical for the immunomodulatory properties.Saponin per se has adjuvant potential; however, optimalCTL induction is achieved when the antigen and adjuvantare associated as an ISCOMTM or ISCOMATRIXTM vac-cine (unpublished data). The most likely explanation forthis is that ISCOMATRIXTM vaccines, because of theirparticulate nature and/or hydrophobic properties, are moreefficiently taken up by cells of the immune system suchas APCs. Consistent with this, ISCOMATRIXTM vaccinesare efficiently taken up in vitro by dendritic cells, andthe antigens processed and presented to CD8+ T cells(Maraskovsky E. et al., in press). Following cellular up-take, ISCOMATRIXTM adjuvant structures were found tobe approximately equally distributed between the cytosoland intracellular compartments[26], which in part explainstheir capacity to access both endosomal and proteosomalcompartments for MHC classes I and II antigen processing,respectively. Access to the class I pathway is critical to theability of ISCOMATRIXTM vaccines to induce strong CTLresponses. Although not completely understood, it is thoughtthat the membrane-disrupting properties of the saponincomponent of the ISCOMATRIXTM structure might aidendosomal escape, thereby, releasing antigen to the cytosol.

To date there is little evidence to suggest that ISCOMAT-RIXTM adjuvant binds to specific receptors and unlike otheractivators of innate immune responses such as CpGs, LPSand DNA they do not appear to activate Toll Like Receptors(TLRs) (unpublished observations). It, therefore, remainsunclear how ISCOMATRIXTM vaccines induce cellular ac-tivation and up-regulate cytokine expression.

4. Concluding remarks

ISCOMATRIXTM vaccines have now been tested withnumerous antigens in a variety of experimental animalsand humans and been shown to be safe and highly im-munogenic. The key features of ISCOMATRIXTM vaccinesfor humoral responses are increases in the magnitude,speed and longevity of the response, as well as the capac-ity for antigen dose reduction. These properties make theISCOMATRIXTM adjuvant ideal for vaccine applicationsthat require a rapid response such as pandemics or in situa-tions where antigen is limited or expensive to manufacture.Addition of the ISCOMATRIXTM adjuvant to existingvaccines could lower production costs or improve immuno-genicity, particularly in settings where responses are affectedby the presence of pre-existing or maternal antibodies.The key features of ISCOMATRIXTM vaccines for cellularimmune responses are the ability to induce strong CD4+and CD8+ T cell responses and long lived CTL responseswithout the need for different formulations for prime and

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boost. The latter property has obvious advantages for man-ufacture, cost, delivery and potentially ease of registration.ISCOMATRIXTM vaccines also demonstrate significantpotential as a mucosal adjuvant, particularly for intranasaladministration. Manufacture of the ISCOMATRIXTM ad-juvant is simple, reproducible and can be performed atlarge scale. Additionally the ISCOMATRIXTM adjuvant hasbeen well characterised. The properties and features of theISCOMATRIXTM adjuvant warrant further investigation inthe clinic for novel human vaccines where cellular and/orhumoral immune responses are required.

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