Immunology and Cell Biology

(2004)

82

, 611–616 doi:10.1111/j.1440-1711.2004.01290.x

© 2004 Australasian Society for Immunology Inc.

Special Feature

Inulin-derived adjuvants efficiently promote both Th1 and Th2 immune responses

D I E G O G S I L V A , P E T E R D C O O P E R a n d N I K O L A I P E T R O V S K Y

Autoimmunity Research Unit, Australian National University Medical School, The Canberra Hospital, Canberra, Australian Capital Territory, Australia

Summary

There has been a recent resurgence of interest into new and improved vaccine adjuvants. This interesthas been stimulated by the need for new vaccines to combat problematic pathogens such as SARS and HIV, and tocounter potential bioterrorist attacks. A major bottleneck in vaccine development is the low immunogenicity ofpurified subunit or recombinant proteins, creating the need for safe human adjuvants with high potency. A majorproblem in the search for the ideal adjuvant is that adjuvants that promote cell-mediated (Th1) immunity (e.g.Freund’s complete adjuvant) generally have unacceptable local or systemic toxicity that precludes their use inhuman vaccines. There is a need for a safe, non-toxic adjuvant that is able to stimulate both cell-mediated andhumoral immunity. Inulin-derived adjuvants that principally stimulate the innate immune system through theirability to activate the alternative complement pathway have proven ability to induce both cellular and humoralimmunity. With their excellent tolerability, long shelf-life, low cost and easy manufacture, they offer great potentialfor use in a broad range of prophylactic and therapeutic vaccines. Based on successful animal studies in a broadrange of species, human trials are about to get underway to validate the use of inulin-based adjuvants in prophylacticvaccines against hepatitis B, malaria and other pathogens. If such trials are successful, then it is possible that inulin-derived adjuvants will one day replace alum as the adjuvant of choice in most human prophylactic vaccines.

Key words:

adjuvant, complement, immunity, inulin, Th1, Th2, vaccine.

Introduction

Inulin structure

‘Inulin’ is a simple, inert polysaccharide consisting of afamily of linear

β

-D-(2

→

1) polyfructofuranosyl

α

-

D

-glucoses,in which an unbranched chain of up to 100 fructose moietiesis linked to a single terminal glucose. It has a relativelyhydrophobic, polyoxyethylene-like backbone, and this unusualstructure, as well as its non-ionized nature, allows recrystalli-zation and easy preparation in a very pure state. Inulin-basedadjuvants are therefore molecularly polydisperse (molecularweights up to 16 000), neutral polysaccharides of simple,known composition. Inulin is the storage carbohydrate of

Compositae

and is obtained in high molecular weight fromdahlia tubers.

Although the molecular composition of inulin is wellknown, different determinations of its solubility have beenreported. An early quantitative study suggested that twodistinct forms of inulin existed (the first obtained by precipi-tation from water, the second by precipitation from ethanol),both of which were substantially soluble in water at 37

°

C.

1

The form obtained by precipitation from water was referred toas alpha inulin (

α

-IN), and the form obtained by precipitationfrom ethanol as beta inulin (

β

-IN).

Gamma inulin

Cooper and Carter described a third polymorphic form ofparticulate inulin, designated as gamma inulin (

γ

-IN).

2

γ

-IN isvirtually insoluble in water at 37

°

C, but is soluble in concen-trated solution (>50 mg/mL) at temperatures in the range70–80

°

C. The three polymorphic forms in which inulincrystallises may be characterized by their different solubilityrates in aqueous media: one is instantly soluble at 23

°

C (beta

230

inulin), another form is soluble at 37

°

C with a half-time of8 min (alpha

378

inulin) and the other form is virtually insolu-ble at 37

°

C (

γ

-IN). All forms are interconvertible; the moresoluble and unstable progressing on standing to less solubleand more stable forms, only reversible by complete solutionfollowed by recrystallization, with the end product being thestable

γ

-IN. However, only higher molecular weight inulinscan attain the gamma form.

Biological effects of inulin

Complement activation by inulin derivatives

Inulin activates and exhausts complement when incubatedwith human serum, and was one of the first substances usedfor this purpose. This property of inulin resulted in the firstdemonstration of the alternative complement pathway (ACP).

3

Complement activation is a characteristic only of particulateinulin, as dissolved inulin is biologically inactive.

γ

-IN is a farmore potent complement activator than

α

-IN or

β

-IN, andwas shown to be more effective than killed

Staphylococcusaureus

Cowan’s type I and zymosan.

2

The effect is dose

Correspondence: Professor Nikolai Petrovsky, AutoimmunityResearch Unit, ANU Medical School, Canberra Hospital, PO Box 11,Woden, ACT 2606, Australia. Email: nikolai.petrovsky@anu.edu.au

Received 19 May 2004; accepted 19 May 2004.

612

DG Silva

et al

.

dependent, reaching its maximum level at 10–20

µ

g/mL inhuman plasma.

Gamma inulin has been shown to activate complement

invivo

in several animal species.

4

An i.p. dose of 50

µ

g

γ

-IN inmice gave detectable serum ACP depletion in 2 h, and higherdoses (200–500

µ

g) gave more than 50% depletion in30 min.

5

Serum ACP levels returned to normal after 16–24 hpost-treatment. Similar results were obtained in rabbits anddogs using various administration routes.

4

Effects of inulin derivatives on the immune response

The complement system plays a central role in the immuneresponse, and complement activation is a primary defenceline against foreign substances.

6

Receptors for complementactivation products are present on leucocyte surfaces

7

and areclosely related to activation and modulation of the immuneresponse, particularly B-cell differentiation and antibody pro-duction. Thus, both the innate and the adaptive immuneresponses are regulated by complement.

8

As expected for a potent complement activator,

γ

-IN is ahighly immunoactive agent. Injection of

γ

-IN i.p. results in anincreased number of peritoneal neutrophils and lymphocytes.

4

Although there was no macrophage activation after

γ

-INadministration as measured by respiratory burst (chemilumi-nescence), macrophages were found to be primed by

γ

-IN torespond to a phorbol ester trigger. Incubation of humanperipheral blood lymphocytes with

γ

-IN plus tetanus toxoidenhanced secretion of IL-2 sevenfold to 41-fold, compared totoxoid alone.

4

Further experiments directed to study themechanisms of action of

γ

-IN demonstrated that it induced C3deposition on the surface of macrophages, followed byenhanced T-cell specific activation.

9

Uses of gamma inulin

Adjuvant activity of inulin derivatives

In keeping with its role as a potent immune activator,

γ

-IN isan effective vaccine adjuvant. It can boost both cell-mediatedand humoral immunity, inducing the ideal immunologicalresponse for a vaccine adjuvant.

10

Co-injection of

γ

-IN andkeyhole limpet haemocyanin (KLH) increased total KLH-specific IgG responses up to 28-fold compared to antigenalone, while IgG2a, IgG2b and IgG3 responses showedincreases of more than 100-fold.

11

Gamma inulin induced a uniform specific IgG responseagainst KLH in different mouse strains, which without adju-vant varied over a 61-fold range.

10

KLH-specific IgGresponses were also enhanced by

γ

-IN in guinea pigs andrabbits.

When

γ

-IN was co-crystallised with aluminium hydroxide(alum) a hybrid particle called ‘algammulin’ was formed.This inulin derivative binds antigen and conserves the abilityto activate complement.

12

Algammulin enhanced the responseto KLH up to 17-fold greater than alum.

12

The levels of allimmunoglobulins specific for KLH were increased, and anenhanced Th1 response (IgG2a, IgG2b and IgA) and reducedIgE response was found, when compared to alum.

Gamma inulin and algammulin similarly enhanced IgGresponses to diphtheria toxoid antigen.

13

Adding

γ

-IN alonegave similar responses to those for alum for IgG1, but

fourfold to 100-fold higher than alum for IgG2a and IgG2b.Enhancement by algammulin was particularly marked at verylow antigen doses, and varying the alum : inulin ratio inalgammulin from 1:0 through 1:10, 1:20 and 1:40 to 0:1showed a consistent shift from Th2 to Th1 responses. Algam-mulin also gave a higher response than alum or Freund’scomplete adjuvant (FCA) to whole-cell meningococci.

14

γ

-INalso enhanced the response to a

Taenia ovis

recombinantantigen.

15

Sheep were immunized with GST-45W antigenmixed with

γ

-IN, algammulin, alum, FCA or saline.

γ

-IN wasas effective at inducing cell-mediated responses as was FCA,and elicited a humoral response comparable to alum. How-ever, FCA or Freund’s incomplete adjuvant (FIA) were theonly adjuvants able to induce protection against challengeinfection, suggesting that high antibody titres are needed toconfer protection against

T. ovis

.

Use of gamma inulin in contraceptive vaccines

Gamma inulin was tested in combination with liposomes andvitamin E to develop a contraceptive vaccine. Mice weretreated with

γ

-IN 3 days before being immunized with eithersperm, sperm protein extracts or human epididymal geneproduct 2 (HE2) antigen combined with liposomes andvitamin E. Use of the three adjuvants together gave highantisperm antibodies and, in contrast to the use of FCA, didnot cause discomfort, pain or distress to the animals.

16

Use of gamma inulin to improve influenza vaccine responses

In initial studies with a live influenza virus lethal challengemodel,

γ

-IN induced heterotypic protection when given withthe virus immunogen. Such protection is considered to bemediated not by antibody but by cytotoxic T cells. Groups ofBalb/c mice were injected with live or gamma-irradiatedinfluenza virus A/JAP combined with

γ

-IN. Fifty percent ofthe animals primed with the

γ

-IN/virus mixture survived alethal challenge with live A/WSN influenza virus 29 daysafter immunization, compared to 3.8% of mice primed withvirus only.

11

Gamma inulin as an alternative adjuvant for hepatitis B vaccines

Current research is directed to develop more immunogenicvaccines against hepatitis B (HB), to be used in either aprophylactic or a therapeutic setting. In our earlier work,hepatitis B surface antigen (HBsAg) adsorbed on algammulinor

γ

-IN gave primary antibody responses in mice up to 5.6-foldgreater than those using an equivalent dose of alum alone.

17

Recently, we have compared formulations of

γ

-IN andalgammulin plus HBsAg (1

µ

g per mouse) with a reformula-tion of the currently available human HB vaccine containingalum (50

µ

g per mouse) and HBsAg (1

µ

g per mouse).Groups of 10 C57Bl/6 mice were immunized at days 0 and30, and humoral responses were evaluated at day 44. Analysisof the IgG subtypes showed higher levels of IgG2a specificfor HBsAg in animals immunized with the

γ

-IN formulation,consistent with an increased Th1 response. Titres of anti-HBsAg specific total IgG, IgG1 and IgG2b were similar in allgroups (Fig. 1).

Inulin-based adjuvants

613

The cellular response to HBsAg was also measured.Splenocytes from immunized mice were re-stimulated withHBsAg

in vitro

. Interestingly, despite the Th1 shift in theantibody isotype titres, the cytokine profile of splenocytesobtained from mice immunized with

γ

-IN, algammulin andalum formulations were comparable, with similar levels ofHBsAg-stimulated IFN-

γ

, IL-10 and IL-6 production (Fig. 2).

Gamma inulin and malaria vaccines

Peptide epitopes of a malarial merozoite surface antigen(MSA2) conjugated with diphtheria toxoid protein gavehigher antibody responses to both peptide and native antigenwhen injected with algammulin compared with FCA or alum.

18

In a

Plasmodium chabaudi

challenge model, most animalssurvived, provided the malaria antigens were administeredwith an adjuvant, with algammulin being at least as effectiveas FCA.

19,20

In recent work, we combined

γ

-IN with merozoite surfaceprotein (MSP) 5 (kindly provided by R. Coppel, Monash Uni-versity, Melbourne, Australia), an antigen from

Plasmodiumfalciparum

. Mice (C57Bl/6) were immunized at days 0 and30 with MSP 5 (25

µ

g per mouse) combined with either

γ

-IN,FCA or PBS. The titres of anti-MSP 5-specific IgG weremeasured in plasma at day 44. The combination containing

γ

-INinduced higher anti-MSP 5 IgG titres (up to fivefold) thanPBS controls, with the total IgG, IgG1 and IgG2a titres beingequivalent to those of FCA treated animals (Fig. 3). The cellularimmune response to MSP 5 antigens elicited

in vitro

by spleno-cyte re-stimulation showed that

γ

-IN was as effective as FCAin inducing Th1 (IFN-

γ

) and Th2 (IL-10) cytokines (Fig. 4).The potency of

γ

-IN and algammulin was also tested withMSP 4/5 (provided by R. Coppel, Monash University, Mel-bourne, Australia), an antigen obtained from the parasite

Plasmodium yoelii

that is used in a mouse model of malaria.Groups of C57bL/6 mice were immunized at days 0 and 30with MSP 4/5 (25

µ

g per mouse) combined with either

γ

-IN,algammulin or FCA. Total IgG titres specific for MSP 4/5were measured in serum samples collected at day 44. Miceimmunized with MSP 4/5 in

γ

-IN or algammulin had compa-rable IgG responses to those obtained in FCA treated mice(Fig. 5).

Figure 2

Pattern of hepatitis B surface antigen (HBsAg)-induced cytokine responses after immunization with HBsAg plusgamma inulin (

γ

-IN), algammulin or alum. Splenocytes wereisolated from mice immunized with HBsAg plus either

γ

-IN,algammulin or alum, and incubated for 72 h in the presence ofHBsAg (0.5

µ

g/mL) or PBS. The levels of IFN-

γ

, IL-10 and IL-6in response to

in vitro

re-stimulation with HBsAg were equivalentfor all three adjuvants.

�

,

γ

-IN + HBsAg; , algammulin +HBsAg;

�

, alum + HBsAg.

Figure 1

Gamma inulin (

γ

-IN) boosts the immune response tohepatitis B antigen. Groups of 10 C57Bl/6 mice were immunizedat days 0 and 30 with hepatitis B surface antigen (HBsAg; 1

µ

gper mouse) in combination with either

γ

-IN (500

µ

g per mouse),algammulin (500

µ

g inulin per mouse) or alum (50

µ

g permouse). HBsAg-specific IgG was measured 14 days after thesecond immunization.

γ

-IN induced significantly higher levels ofIgG2a while all three adjuvants induced similar levels of total IgGand IgG1.

�

,

γ

-IN + HBsAg; , algammulin + HBsAg;

�,alum + HBsAg.

Figure 3 Gamma inulin (γ-IN) is an effective malaria vaccineadjuvant. Mice (C57Bl/6) were immunized with Plasmodiumfalciparum merozoite surface protein (MSP) 5 (25 µg per mouse)plus either γ-IN (500 µg per mouse), Freund’s complete adjuvant(FCA) (v/v) or PBS. Fourteen days after the second immunization,γ-IN treated mice had equivalent titres of MSP 5-specific IgG,IgG1, IgG2a and IgG2b to FCA treated mice. �, γ-IN + MSP 5;

, FCA + MSP 5; �, PBS + MSP 5.

614 DG Silva et al.

Inulin as an adjuvant in a human papilloma virus vaccine

The safety and immunogenicity of an algammulin-basedhuman papilloma virus vaccine was tested in humans.21 TheE7 protein of HPV 16 conjugated to a glutathione-S-transferase was combined with a high dose of algammulinand administered subcutaneously to five patients diagnosedwith primary or recurrent cervical cancer. Three of the fivepatients developed a small subcutaneous lump (2–3 mm) atthe site of immunization that diminished in size with time. Allsubjects showed new or increased reactivity with E7 over thecourse of immunization. No constitutional symptoms relatingto the immunization were reported. In separate in vitro tests,T cells from mice immunized with E7GST protein plusalgammulin or γ-IN showed strongly enhanced cytotoxicityagainst target cells transfected with E7 protein.22,23

Anti-tumour effects of gamma inulin

Non-specific immunotherapy has been used successfully inseveral cancer models.24 Many of the compounds used asimmune activators are also known to activate ACP mole-cules.25 The anticancer properties of γ-IN were first describedin a murine model of melanoma (B16/C57Bl system).5

Administration of multiple doses of γ-IN prolonged the meansurvival of animals injected with B16 melanoma cell lines byup to 58%. The antitumour activity of γ-IN was confirmed tobe due to ACP activation, as depletion of C3 abrogated theprotective effect. Complete tumour eradication and tumour-free survival was achieved in 27% of mice by γ-IN treatmentfollowed by secondary inoculation with succinyl-concanavalinA.4 These survivors were then resistant to re-challenge withthe tumour. Intra-tumour injection with γ-IN was also shownto effectively induce regression of squamous cell carcinomain sheep.4 This effect was enhanced if combined with cyclo-phosphamide. Equine sarcoids also responded to intralesionaltreatment with γ-IN, where eight of 17 tumours completelyregressed and five more were partially resolved. Several

spontaneous malignancies in dogs responded well to γ-INcombined with other therapies.

Discussion

The success of vaccination in improving population healthhas encouraged researchers to try to develop new vaccines toprevent or treat pathogens for which vaccines are not yetavailable. Unfortunately, many vaccine antigen candidateshave low immunogenicity, or do not produce a desirableimmune response; for example, they invoke an ineffectiveTh2 rather than a protective Th1 response. At least some ofthese difficulties could be overcome by the development anduse of better adjuvants. The problem to date has been that themore potent adjuvants are invariably associated with highlocal or systemic toxicity, thereby precluding their use partic-ularly in a prophylactic setting.

Gamma inulin has the properties of an ideal adjuvant forprophylactic vaccines. It is obtained from a natural source,and the purification process does not involve the use of toxicsubstances.10 In its soluble form it has no physiological effectsother than an osmotic diuresis at very high doses, and thebreakdown products in vivo are simple sugars. Purified γ-INpasses pyrogenicity and sterility tests, thereby confirming asafe and commercially viable purification method. γ-IN is notitself antigenic and has little or no side-effects when used atvaccine adjuvant doses. It has already been shown to be safeand non-toxic when tested in a small number of humans withonly minor granuloma reactions being observed.

The adjuvant properties of γ-IN have been studied in manyanimal models, demonstrating its capacity to enhancehumoral and cellular responses against a wide variety of

Figure 4 Pattern of merozoite surface protein (MSP) 5-inducedcytokine responses after in vitro restimulation. Splenocytes werecollected from mice treated with MSP 5 and γ-IN or Freund’scomplete adjuvant (FCA), and cultured for 72 h in MSP 5 (0.5 µg/mL). The levels of MSP 5-induced IFN-γ or IL-10 were similar inγ-IN and FCA treated mice. �, γ-IN + MSP 5; �, FCA + MSP 5.

L)

Figure 5 Use of gamma inulin (γ-IN) in a Plasmodium yoeliimerozoite surface protein (MSP) 4/5 vaccine. Groups of C57Bl/6animals were injected at days 0 and 30 with MSP 4/5 (25 µg permouse) formulated with either γ-IN, algammulin or Freund’scomplete adjuvant (FCA) and serum collected at day 44. Thelevels of MSP-specific IgG, IgG1 and IgG2a were similar for eachof the adjuvant groups, confirming that inulin-based adjuvants canequal the potency of FCA for malaria vaccines. �, γ-IN + MSP4/5; , algammulin + MSP 4/5; �, FCA + MSP 4/5.

Inulin-based adjuvants 615

antigens, including KLH, T. ovis surface antigen, diphtheriaand tetanus toxoids, influenza virus, HBsAg and malariasurface antigens (Table 1). These studies have highlighted theparticular ability of γ-IN to enhance Th1 responses withoutthe concomitant toxicity normally seen with Th1 adjuvants. Insummary, inulin-based adjuvants are safe and effective adju-vants suitable for use in a wide variety of pathogen and cancervaccines.

Acknowledgements

The contributing authors DGS and NP have interest in sharesof Vaxine Pty Ltd. Vaxine has proprietary interests in theinulin-based adjuvant technology described in this paper. Wethank K. Hewitt and S. Emmet for technical assistance. Workdescribed in this paper was funded by grants to Vaxine fromthe Biotechnology Innovation Fund and the ACT InnovationGrants Scheme.

References

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2 Cooper PD, Carter M. Anticomplementary action of poly-morphic ‘solubility forms’ of particulate inulin. Mol. Immunol.1986; 23: 895–901.

3 Gotze O, Muller-Eberhard HJ. The C3-activator system: an alter-nate pathway of complement activation. J. Exp. Med. 1971; 134:90S–108S.

4 Cooper PD. Solid-phase activators of the alternative pathway ofcomplement and their use in vivo. In: Sim RB (ed.) Activatorsand Inhibitors of Complement. Dordrecht: Kluwer AcademicPublishers, 1993; 69–106.

5 Cooper PD, Carter M. The anti-melanoma activity of inulin inmice. Mol. Immunol. 1986; 23: 903–908.

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inflammation. In: Weigle WO (ed.) Advances in Immunopathol-ogy. Miami: Symp Specialists, 1981; 141–60.

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8 Carroll MC. The role of complement and complement receptorsin induction and regulation of immunity. Annu. Rev. Immunol.1998; 16: 545–68.

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10 Cooper PD. Vaccine adjuvants based on gamma inulin. Pharm.Biotechnol. 1995; 6: 559–80.

11 Cooper PD, Steele EJ. The adjuvanticity of gamma inulin. Immu-nol. Cell Biol. 1988; 66: 345–52.

12 Cooper PD, Steele EJ. Algammulin, a new vaccine adjuvantcomprising gamma inulin particles containing alum: preparationand in vitro properties. Vaccine 1991; 9: 351–7.

13 Gupta RK, Griffin P, Chang AC et al. The role of adjuvants anddelivery systems in modulation of immune response to vaccines.Adv. Exp. Med. Biol. 1996; 397: 105–13.

14 Gonzalez S, Nazabal C, Vina L et al. Influence of several adju-vants on the immune response against a recombinant meningo-coccal high molecular weight antigen. Dev. Biol. Stand. 1998;92: 269–76.

15 Deol HS, Palmer DG, Dunsmore T et al. The influence ofgamma inulin and Algammulin on the immune response in sheepto a recombinant antigen of Taenia ovis. Vaccine 1995; 13:429–33.

16 Fuentes P, Cooper PD, Barnadas R et al. Use of gamma-inulin/liposomes/Vitamin E adjuvant combination in contraceptivevaccines. Int. J. Pharm. 2003; 257: 85–95.

17 Cooper PD, Turner R, McGovern J. Algammulin (gamma inulin/alum hybrid adjuvant) has greater adjuvanticity than alum forhepatitis B surface antigen in mice. Immunol. Lett. 1991; 27:131–4.

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Table 1 Antigens and models used for testing inulin-derived adjuvants

Antigen Adjuvant Models Outcome

KLH10–12 γ-IN, algammulin Mice, guinea pigs, rabbits Comparable to FCACervical cancer vaccine(HPV E7 protein)21–23

γ-INAlgammulin

MiceHumans

Enhanced CTL responseNo adverse effects; higher Ab and Th responses

Hepatitis B vaccine (HBsAg)17 γ-IN, algammulin Mice More effective than alumInfluenza virus11

Haemagglutinin γ-IN, algammulin Mice Tenfold Ab increase; better protection; lung virus clearanceWhole virus γ-IN Mice Strong Th1 responseLive virus γ-IN Mice Enhanced survival

Carcinomas, sarcomas4 γ-IN Sheep, dogs, horses, monkeys

Complete resolution in more than 50%

Melanoma5 γ-IN Mice Increased survival time; cure in 30% with additional immune modulators

Contraceptive vaccine16 γ-IN/liposome/vitamin E complex

Mice Highly effective; low toxicity

Whole meningococcus14 Algammulin Mice Higher Ab responses than six other adjuvantsTetanus toxoid4 γ-IN Human T cells 6–40-fold increase in IL-2

γ-IN Mice IgG production equal to alumTaenia ovis15 γ-IN, algammulin Sheep Cell mediated response comparable to FCAMSP219–20 Algammulin Mice Higher Ab responses than FCADiphtheria toxoid13 γ-IN, algammulin Mice Better than alum with IgG1 and IgG2a up to 100-fold higher

FCA, Freund’s complete adjuvant; FHA, filamentous haemagglutinin; γ-IN, gamma inulin; HPV, human papilloma virus; KLH, keyhole limpethaemocyanin; MSP2, merozoite surface antigen 2.

616 DG Silva et al.

19 Jones GL, Spencer L, Lord R et al. High-titer antisera productionusing three adjuvants and peptide conjugates derived frommalarial surface antigen MSA-2. Pept. Res. 1991; 4: 138–41.

20 Saul A, Lord R, Jones GL et al. Protective immunization withinvariant peptides of the Plasmodium falciparum antigen MSA2.J. Immunol. 1992; 148: 208–211.

21 Frazer I, Tindle R, Fernando G et al. Safety and immunogenicityof HPV16 E7/Algammulin. In: Tindle RW (ed.) Vaccines forHuman Papillomavirus Infection and Anogenital Disease. NewYork: RG Landes, 1999; 91–104.

22 Fernando GJ, Stewart TJ, Tindle RW et al. Vaccine-inducedTh1-type responses are dominant over Th2-type responses in the

short term whereas pre-existing Th2 responses are dominant inthe longer term. Scand. J. Immunol. 1998; 47: 459–65.

23 Fernando GJ, Stewart TJ, Tindle RW et al. Th2-type CD4+ cellsneither enhance nor suppress antitumor CTL activity in a mousetumor model. J. Immunol. 1998; 161: 2421–7.

24 Huber CH, Wolfel T. Immunotherapy of cancer: from vision tostandard clinical practice. J. Cancer Res. Clin. Oncol. 2004; 130:367.

25 Cooper PD. Complement and cancer: activation of the alter-native pathway as a theoretical base for immunotherapy. Adv.Immun. Cancer Ther. 1985; 1: 125–66.