Introduction

Inulin structure

'Inulin' is a simple, inert polysaccharide consisting of a family of linear -D-(21) polyfructofuranosyl -D-glucoses, in which an unbranched chain of up to 100 fructose moieties is linked to a single terminal glucose. It has a relatively hydrophobic, polyoxyethylene-like backbone, and this unusual structure, as well as its non-ionized nature, allows recrystallization and easy preparation in a very pure state. Inulin-based adjuvants are therefore molecularly polydisperse (molecular weights up to 16 000), neutral polysaccharides of simple, known composition. Inulin is the storage carbohydrate of Compositae and is obtained in high molecular weight from dahlia tubers.

Although the molecular composition of inulin is well known, different determinations of its solubility have been reported. An early quantitative study suggested that two distinct forms of inulin existed (the first obtained by precipitation from water, the second by precipitation from ethanol), both of which were substantially soluble in water at 37°C¹. The form obtained by precipitation from water was referred to as alpha inulin (-IN), and the form obtained by precipitation from ethanol as beta inulin (-IN).

Gamma inulin

Cooper and Carter described a third polymorphic form of particulate inulin, designated as gamma inulin (-IN)². -IN is virtually insoluble in water at 37°C, but is soluble in concentrated solution (>50 mg/mL) at temperatures in the range 70-ndash;80°C. The three polymorphic forms in which inulin crystallises may be characterized by their different solubility rates in aqueous media: one is instantly soluble at 23°C (beta ₂₃⁰ inulin), another form is soluble at 37°C with a half-time of 8 min (alpha ₃₇⁸ inulin) and the other form is virtually insoluble at 37°C (-IN). All forms are interconvertible; the more soluble and unstable progressing on standing to less soluble and more stable forms, only reversible by complete solution followed by recrystallization, with the end product being the stable -IN. However, only higher molecular weight inulins can attain the gamma form.

Biological effects of inulin

Complement activation by inulin derivatives

Inulin activates and exhausts complement when incubated with human serum, and was one of the first substances used for this purpose. This property of inulin resulted in the first demonstration of the alternative complement pathway (ACP)³. Complement activation is a characteristic only of particulate inulin, as dissolved inulin is biologically inactive. -IN is a far more potent complement activator than -IN or -IN, and was shown to be more effective than killed Staphylococcus aureus Cowan's type I and zymosan². The effect is dose dependent, reaching its maximum level at 10-ndash;20 g/mL in human plasma.

Gamma inulin has been shown to activate complement in vivo in several animal species⁴. An i.p. dose of 50 g -IN in mice gave detectable serum ACP depletion in 2 h, and higher doses (200-ndash;500 g) gave more than 50% depletion in 30 min⁵. Serum ACP levels returned to normal after 16-ndash;24 h post-treatment. Similar results were obtained in rabbits and dogs using various administration routes⁴.

Effects of inulin derivatives on the immune response

The complement system plays a central role in the immune response, and complement activation is a primary defence line against foreign substances⁶. Receptors for complement activation products are present on leucocyte surfaces⁷ and are closely related to activation and modulation of the immune response, particularly B-cell differentiation and antibody production. Thus, both the innate and the adaptive immune responses are regulated by complement⁸.

As expected for a potent complement activator, -IN is a highly immunoactive agent. Injection of -IN i.p. results in an increased number of peritoneal neutrophils and lymphocytes⁴. Although there was no macrophage activation after -IN administration as measured by respiratory burst (chemiluminescence), macrophages were found to be primed by -IN to respond to a phorbol ester trigger. Incubation of human peripheral blood lymphocytes with -IN plus tetanus toxoid enhanced secretion of IL-2 sevenfold to 41-fold, compared to toxoid alone⁴. Further experiments directed to study the mechanisms of action of -IN demonstrated that it induced C3 deposition on the surface of macrophages, followed by enhanced T-cell specific activation⁹.

Uses of gamma inulin

Adjuvant activity of inulin derivatives

In keeping with its role as a potent immune activator, -IN is an effective vaccine adjuvant. It can boost both cell-mediated and humoral immunity, inducing the ideal immunological response for a vaccine adjuvant¹⁰. Co-injection of -IN and keyhole limpet haemocyanin (KLH) increased total KLH-specific IgG responses up to 28-fold compared to antigen alone, while IgG2a, IgG2b and IgG3 responses showed increases of more than 100-fold¹¹.

Gamma inulin induced a uniform specific IgG response against KLH in different mouse strains, which without adjuvant varied over a 61-fold range¹⁰. KLH-specific IgG responses were also enhanced by -IN in guinea pigs and rabbits.

When -IN was co-crystallised with aluminium hydroxide (alum) a hybrid particle called 'algammulin' was formed. This inulin derivative binds antigen and conserves the ability to activate complement¹². Algammulin enhanced the response to KLH up to 17-fold greater than alum¹². The levels of all immunoglobulins specific for KLH were increased, and an enhanced Th1 response (IgG2a, IgG2b and IgA) and reduced IgE response was found, when compared to alum.

Gamma inulin and algammulin similarly enhanced IgG responses to diphtheria toxoid antigen¹³. Adding -IN alone gave 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 very low antigen doses, and varying the alum : inulin ratio in algammulin from 1:0 through 1:10, 1:20 and 1:40 to 0:1 showed a consistent shift from Th2 to Th1 responses. Algammulin also gave a higher response than alum or Freund's complete adjuvant (FCA) to whole-cell meningococci¹⁴. -IN also enhanced the response to a Taenia ovis recombinant antigen¹⁵. Sheep were immunized with GST-45W antigen mixed with -IN, algammulin, alum, FCA or saline. -IN was as effective at inducing cell-mediated responses as was FCA, and elicited a humoral response comparable to alum. However, FCA or Freund's incomplete adjuvant (FIA) were the only adjuvants able to induce protection against challenge infection, suggesting that high antibody titres are needed to confer protection against T. ovis.

Use of gamma inulin in contraceptive vaccines

Gamma inulin was tested in combination with liposomes and vitamin E to develop a contraceptive vaccine. Mice were treated with -IN 3 days before being immunized with either sperm, sperm protein extracts or human epididymal gene product 2 (HE2) antigen combined with liposomes and vitamin E. Use of the three adjuvants together gave high antisperm antibodies and, in contrast to the use of FCA, did not cause discomfort, pain or distress to the animals¹⁶.

Use of gamma inulin to improve influenza vaccine responses

In initial studies with a live influenza virus lethal challenge model, -IN induced heterotypic protection when given with the virus immunogen. Such protection is considered to be mediated not by antibody but by cytotoxic T cells. Groups of Balb/c mice were injected with live or gamma-irradiated influenza virus A/JAP combined with -IN. Fifty percent of the animals primed with the -IN/virus mixture survived a lethal challenge with live A/WSN influenza virus 29 days after immunization, compared to 3.8% of mice primed with virus only¹¹.

Gamma inulin as an alternative adjuvant for hepatitis B vaccines

Current research is directed to develop more immunogenic vaccines against hepatitis B (HB), to be used in either a prophylactic or a therapeutic setting. In our earlier work, hepatitis B surface antigen (HBsAg) adsorbed on algammulin or -IN gave primary antibody responses in mice up to 5.6-fold greater than those using an equivalent dose of alum alone¹⁷.

Recently, we have compared formulations of -IN and algammulin plus HBsAg (1 g per mouse) with a reformulation of the currently available human HB vaccine containing alum (50 g per mouse) and HBsAg (1 g per mouse). Groups of 10 C57Bl/6 mice were immunized at days 0 and 30, and humoral responses were evaluated at day 44. Analysis of the IgG subtypes showed higher levels of IgG2a specific for HBsAg in animals immunized with the -IN formulation, consistent with an increased Th1 response. Titres of anti-HBsAg specific total IgG, IgG1 and IgG2b were similar in all groups (Figure 1).

Figure 1.

Gamma inulin (-IN) boosts the immune response to hepatitis B antigen. Groups of 10 C57Bl/6 mice were immunized at days 0 and 30 with hepatitis B surface antigen (HBsAg; 1 g per mouse) in combination with either -IN (500 g per mouse), algammulin (500 g inulin per mouse) or alum (50 g per mouse). HBsAg-specific IgG was measured 14 days after the second immunization. -IN induced significantly higher levels of IgG2a while all three adjuvants induced similar levels of total IgG and IgG1. , -IN + HBsAg; , algammulin + HBsAg; , alum + HBsAg.

Full figure and legend (12K)

The cellular response to HBsAg was also measured. Splenocytes from immunized mice were re-stimulated with HBsAg in vitro. Interestingly, despite the Th1 shift in the antibody isotype titres, the cytokine profile of splenocytes obtained from mice immunized with -IN, algammulin and alum formulations were comparable, with similar levels of HBsAg-stimulated IFN-, IL-10 and IL-6 production (Figure 2).

Figure 2.

Pattern of hepatitis B surface antigen (HBsAg)-induced cytokine responses after immunization with HBsAg plus gamma inulin (-IN), algammulin or alum. Splenocytes were isolated from mice immunized with HBsAg plus either -IN, algammulin or alum, and incubated for 72 h in the presence of HBsAg (0.5 g/mL) or PBS. The levels of IFN-, IL-10 and IL-6 in response to in vitro re-stimulation with HBsAg were equivalent for all three adjuvants. , -IN + HBsAg; , algammulin + HBsAg; , alum + HBsAg.

Full figure and legend (9K)

Gamma inulin and malaria vaccines

Peptide epitopes of a malarial merozoite surface antigen (MSA2) conjugated with diphtheria toxoid protein gave higher antibody responses to both peptide and native antigen when injected with algammulin compared with FCA or alum¹⁸. In a Plasmodium chabaudi challenge model, most animals survived, provided the malaria antigens were administered with an adjuvant, with algammulin being at least as effective as FCA^{19, 20}.

In recent work, we combined -IN with merozoite surface protein (MSP) 5 (kindly provided by R. Coppel, Monash University, Melbourne, Australia), an antigen from Plasmodium falciparum. Mice (C57Bl/6) were immunized at days 0 and 30 with MSP 5 (25 g per mouse) combined with either -IN, FCA or PBS. The titres of anti-MSP 5-specific IgG were measured in plasma at day 44. The combination containing -IN induced higher anti-MSP 5 IgG titres (up to fivefold) than PBS controls, with the total IgG, IgG1 and IgG2a titres being equivalent to those of FCA treated animals (Figure 3). The cellular immune response to MSP 5 antigens elicited in vitro by splenocyte re-stimulation showed that -IN was as effective as FCA in inducing Th1 (IFN-) and Th2 (IL-10) cytokines (Figure 4).

Figure 3.

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

Full figure and legend (16K)

Figure 4.

Pattern of merozoite surface protein (MSP) 5-induced cytokine responses after in vitro restimulation. Splenocytes were collected from mice treated with MSP 5 and -IN or Freund's complete adjuvant (FCA), and cultured for 72 h in MSP 5 (0.5 g/mL). The levels of MSP 5-induced IFN- or IL-10 were similar in -IN and FCA treated mice. , -IN + MSP 5; , FCA + MSP 5.

Full figure and legend (6K)

The potency of -IN and algammulin was also tested with MSP 4/5 (provided by R. Coppel, Monash University, Melbourne, 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 30 with MSP 4/5 (25 g per mouse) combined with either -IN, algammulin or FCA. Total IgG titres specific for MSP 4/5 were measured in serum samples collected at day 44. Mice immunized with MSP 4/5 in -IN or algammulin had comparable IgG responses to those obtained in FCA treated mice (Figure 5).

Figure 5.

Use of gamma inulin (-IN) in a Plasmodium yoelii merozoite surface protein (MSP) 4/5 vaccine. Groups of C57Bl/6 animals were injected at days 0 and 30 with MSP 4/5 (25 g per mouse) formulated with either -IN, algammulin or Freund's complete adjuvant (FCA) and serum collected at day 44. The levels of MSP-specific IgG, IgG1 and IgG2a were similar for each of the adjuvant groups, confirming that inulin-based adjuvants can equal the potency of FCA for malaria vaccines. , -IN + MSP 4/5; , algammulin + MSP 4/5; , FCA + MSP 4/5.

Full figure and legend (17K)

Inulin as an adjuvant in a human papilloma virus vaccine

The safety and immunogenicity of an algammulin-based human papilloma virus vaccine was tested in humans²¹. The E7 protein of HPV 16 conjugated to a glutathione-S-transferase was combined with a high dose of algammulin and administered subcutaneously to five patients diagnosed with primary or recurrent cervical cancer. Three of the five patients developed a small subcutaneous lump (2-ndash;3 mm) at the site of immunization that diminished in size with time. All subjects showed new or increased reactivity with E7 over the course of immunization. No constitutional symptoms relating to the immunization were reported. In separate in vitro tests, T cells from mice immunized with E7GST protein plus algammulin or -IN showed strongly enhanced cytotoxicity against target cells transfected with E7 protein^{22, 23}.

Anti-tumour effects of gamma inulin

Non-specific immunotherapy has been used successfully in several cancer models²⁴. Many of the compounds used as immune activators are also known to activate ACP molecules²⁵. The anticancer properties of -IN were first described in a murine model of melanoma (B16/C57Bl system)⁵. Administration of multiple doses of -IN prolonged the mean survival of animals injected with B16 melanoma cell lines by up to 58%. The antitumour activity of -IN was confirmed to be due to ACP activation, as depletion of C3 abrogated the protective effect. Complete tumour eradication and tumour-free survival was achieved in 27% of mice by -IN treatment followed by secondary inoculation with succinyl-concanavalin A⁴. These survivors were then resistant to re-challenge with the tumour. Intra-tumour injection with -IN was also shown to effectively induce regression of squamous cell carcinoma in sheep⁴. This effect was enhanced if combined with cyclophosphamide. Equine sarcoids also responded to intralesional treatment with -IN, where eight of 17 tumours completely regressed and five more were partially resolved. Several spontaneous malignancies in dogs responded well to -IN combined with other therapies.

Discussion

The success of vaccination in improving population health has encouraged researchers to try to develop new vaccines to prevent or treat pathogens for which vaccines are not yet available. Unfortunately, many vaccine antigen candidates have low immunogenicity, or do not produce a desirable immune response; for example, they invoke an ineffective Th2 rather than a protective Th1 response. At least some of these difficulties could be overcome by the development and use of better adjuvants. The problem to date has been that the more potent adjuvants are invariably associated with high local or systemic toxicity, thereby precluding their use particularly in a prophylactic setting.

Gamma inulin has the properties of an ideal adjuvant for prophylactic vaccines. It is obtained from a natural source, and the purification process does not involve the use of toxic substances¹⁰. In its soluble form it has no physiological effects other than an osmotic diuresis at very high doses, and the breakdown products in vivo are simple sugars. Purified -IN passes pyrogenicity and sterility tests, thereby confirming a safe and commercially viable purification method. -IN is not itself antigenic and has little or no side-effects when used at vaccine adjuvant doses. It has already been shown to be safe and non-toxic when tested in a small number of humans with only minor granuloma reactions being observed.

The adjuvant properties of -IN have been studied in many animal models, demonstrating its capacity to enhance humoral and cellular responses against a wide variety of antigens, including KLH, T. ovis surface antigen, diphtheria and tetanus toxoids, influenza virus, HBsAg and malaria surface antigens (Table 1). These studies have highlighted the particular ability of -IN to enhance Th1 responses without the concomitant toxicity normally seen with Th1 adjuvants. In summary, inulin-based adjuvants are safe and effective adjuvants suitable for use in a wide variety of pathogen and cancer vaccines.

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

Full table

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Acknowledgements

The contributing authors DGS and NP have interest in shares of Vaxine Pty Ltd. Vaxine has proprietary interests in the inulin-based adjuvant technology described in this paper. We thank K. Hewitt and S. Emmet for technical assistance. Work described in this paper was funded by grants to Vaxine from the Biotechnology Innovation Fund and the ACT Innovation Grants Scheme.