Immunoi Cell Bioi (1988)66, 345-352 The adjuvanticity of gamma inulin Peter D. Cooper* and Edward J. Steele*"*" * Division of Virology and Cellular Pathology^ John Curtin School of Medical Research. Australian National University, Canberra, ACT 2601. and "^ Department of Biology, University of Wollongong, Wollongong NSW 2500, Australia (Submitted 9 May 1988. Accepted for publication 8 July 1988.) Summary Gamma-inulin (g-IN) is a polymorph identified as the active component of inulin preparations that specifically activates the alternative pathway of complement (APC). The APC is central to many leucocyte functions, including B cell activation. We show here that g-IN, when formulated as a pure, endotoxin-free, fine suspension insoluble at 37'*C and given at 50-100 |ig per mouse, is a potent adjuvant for both humoral and cell-mediated responses to a variety of antigens. g-IN increased secondary IgG responses five- to 28-foid (f*<0 001), using as antigen phosphorylcholine coupied to keyhoie limpet haemocyanin; subclasses IgG 2a, 2b, and 3 were boosted several hundred-fold, IgG 1 iO-foId. IgM and IgA were increased four-to six-fold. Delayed hypersensitivity, by footpad swelling after secondary challenge with sheep red blood cells (SRBC), was increased more than two-fold (/'<0-00i) if g-IN was included with the primary SRBC, equivalent to increasing primary doses 10-fold. g-IN was equally active if given 5 days before the primary SRBC. Thus it is an immune stimulant rather than a depot or vehicle for antigen. Mice primed subcutaneously with 30-300 HA units of H2N2 influenza virus (strain A/JAP) and challenged intranasally with a lethal dose of H1NI virus (strain A/WSN) all died, but if g-IN was given with the primary antigen 50% ofthe mice survived (/*<0-001), a deduced but not proven boost to cytotoxic T ceii-mediated immunity. Unpubiished work has shown that g-IN has no adverse eifects at adjuvant-active doses. g-IN is thus a promising new vaccine adjuvant. It also has a potential for antitumour therapy, and is a specific reagent for expioring the roie of compiement in vivo. INTRODUCTION The alternative pathway of complement (APC) plays a central role in the immune response, being active in many leucocyte functions (1-4), in addition to its lytic activity in the humoral phase (5). In particular, the APC is important in B cell activation, probably in the germinal centre of follicular lymphoid tissue (6). However, its precise action in the development of immunity, especially cell-mediated immunity, remains unclear. It has been known for some time that inulin preparations activate the alternative but not the classical pathway of complement Abbreviations used in this paper: APC, alternative pathway of complement; BSA, bovine scrum albumin; DTH, delayed hypersensitivity; g-IN, gamma inulin; GTS, gelatin Tween saiine; Ig, immunoglobulin; i.p., intraperitoneal; PC-KLH, phosphorylcholine coupled to keyhole limpet haemocyanin; s.e, subcutaneous; SRBC, sheep red blood cells. (7). g-IN is a polymorph of inulin. a newly described crystalline form of higher molecular weight inulin fractions defined as being very slowly soluble at ?>TC (8). When it was identified as the active principle of inulin preparations (8) it was developed as a specific reagent for in vivo activation ofthe APC. Since APC activators had an antitumour action on the B16 melanoma in mice (9,10), a similar antitumour activity for g-IN in this system was predicted and found (11). In addition, there was a strong correlation among some 20 substances between ability to activate the APC and antitumour and adjuvant activities (12). It was thus expected that g-IN would have a vaccine adjuvant action, and the present results show that it is indeed a powerful adjuvant. The material has been formulated as 'gamma inulin for injection' (8), a purified endotoxin-free suspension of approximately 1 jam ovoids in saline. 346 P. D. COOPER ANDE. J. STEELE largely insoluble at 37T, plus phenylmercuric nitrate as preservative. MATERIALS AND METHODS Reagents The source of inuiin and the preparation of g-IN for injection are described by Cooper and Carter (8). ;>-Nitrophenyi-phosphorylcholine (Sigma Chem. Co.) was converted to the active p-diazonium intermediate DPPC (13) and reacted with either keyhoie limpet haemocyanin (KLH) (Calbiochem) or bovine serum albumin (BSA) foiiowing standard methods (14,15) to give ca. 2 moie phosphoryichoiine (PC)/moie BSA and i moic PC/iOO 000 moi. wt subunit KLH. IgG and IgM standards were affinity purified from ascitic iluid of BALB/c males hyperimmunized with PC-KLH (16) by binding to and eiution (3 moi/i NaSCN) from PCBSA-Sepharose 4B or KLH-Sepharose 4B coiumns, ibllowed by separation on Sephadex G-200. IgG was quantitated assuming an extinction coefficient of 13 5. The IgM standard, which contained non-Ig contaminants, was itself accurately standardized by reference to a pure IgM (The Binding Site Ltd, Birmingham, UK) in an ELISA using anti-mouse Ig-coated microtitre plates. The IgM and IgG standards were contaminated with ^ 7 % IgG and ^ 1 % IgM, respectively, and were stored at 100-500 ^g/ml in I%(w/v) BSA + O-J'% (w/v) sodium azide in PBS at 4°C. Live and gammairradiated (^0(^0, 1 26X 10^ rad) influenza virus preparations were a gift from Mr R. Tha HIa. ELISA To determine the ieveis of IgG- and IgM-specific antibodies, each serum was titrated in weiis of flat bottom ELISA plates (Titertek 'activated'. Flow Lab.) coated either with PC-BSA (50 ^i, 2 ^ig/mi m PBS) or KLH (50 \x\, 10 |ig/mi in PBS). Three-fold faiiing diiutions of sera in 1% BSA in PBS, 50 (ii/weii, were adsorbed for i6 h at 25°C and unbound antibodies washed away with GTS (0 5% w/v geiatin, 0 5% v/v Tween-20 m 0-85% w/v NaCi, pH 6 5-7). The weiis were given 50 |ai of a i :500 diiution (90 min, 25''C) of aikaline phosphatase-conjugated goat IgG (specific either for mouse IgM ^ chains or for mouse IgG; Sigma Chem. Co.) and washed again with GTS. Phosphatase substrate (100 |il/weii of i mg/mi p-nitrophenylphosphate (Sigma) in 1 moi/1 diethanolamine, pH 9 2) was reacted for 15-30 min at 25°C, and the ELISA endpoints were determined as the serum dilution giving an opticai density (OD) (4iO nm) = O i, using a Microeiisa Autoreader MR600, Dynateeh Labs. Inc., Aiexandria, VI. End-points were standardized with IgM and IgG standards titrated at the same time. Values show the arithmetic means and standard errors of IgG and IgM concentrations from seven individual sera; statistical significances of differences (Student's /-test) are between mean values from antigen in saline and from antigen with g-IN. Antibody titres of IgG subclasses and IgA specific for KLH and present in the 21 and 42 day sera were measured as reciprocal dilutions of sera giving an endpoint of OD (410 nm) - 0-1 in an ELISA assay like that just described, expressed as geometric means of seven individual sera and determined using the Mouse Ig Class (Subclass) Detection Kit from Chemicon International Inc. (El Scgundo, CA). Fresh sera (thawed once) were titrated on KLH coated plates. Alter adsorption (1 h, 25°C) and washing (GTS) the weiis were given 50 ^li ofthe recommended amount (diluted i: 3) of rabbit anti-mouse igG subciass-specific reagent (90 min, 25T), washed with GTS and coiour deveioped as before after adding 50 |ii of a i: 500 dilution of alkaiine phosphatase-conjugated goat anti-rabbit igG (Sigma Chem. Co.). PC-KLH inoculation Two portions of a batch of specific pathogen-free BALB/c mice (6-8 week old femaies) were injected intraperitoneaily (i.p.) with 0-i mi of saiine containing, respcctiveiy, either iO ^ig PC-KLH or 10 |ig PCKLH pius 100 i^g g-IN on days 0 and 14. Immediately before immunization and at days 7, 21, 28 and 42, groups of seven mice were bled out, the sera stored in dupiicate and diluted i: iO in PBS at - 2 0 T for iater ELISA assays. Measurement of DTH responses Batches of specific pathogen-free CBA mice, of matched age and sex, were inoculated subcutaneousiy (s.e.) (nape of neck) in groups of five per dose with 0 2 mi saline or varying doses of g-IN in saline, without or in admixture with SRBC. SRBC were washed three times in PBS before use and their concentration measured (an OD 415 nm of 1 = 1 -07 X i 0^ per ml) after diluting in water. Either 4 or 5 days after the primary SRBC the animals were injected s.e. into the piantar surface of the foot with 20 (ai of PBS containing iO^ chaiienge SRBC (right hind foot) and 20 ^i of PBS aione (ieft hind foot). Twenty hours later the specific footpad swelling was measured as the difference in thicicness (in 0- i mm units) between the ieft and right hind feet, using a diai-gauge caiiper (Schnelitaster, H.C, Kropiin GmbH, Hessen, FRG). Virus inoculation Specific pathogen-free BALC/c mice (6-8 week oid maies) were inocuiated s.e. (nape of neck) in groups of seven with 0 2 mi of PBS containing iive or gammairradiated influenza virus, strain A/JAP, with or without admixture with 50 |ig g-IN, and chaiienged intranasaiiy 29 days iater with iO^ or 5X iO^ EIDsoof iive influenza virus strain A/WSN (respectiveiy, 5 or 25 times the minimum dose required to kiii aii the mice in the group). Mice were monitored for 20 days when aii survivors had fuliy recovered. RESULTS Humoral immunity The antigen PC-KLH in BALB/c mice is a 347 INULIN AS ADJUVANT - CO 200 r O 20 • O O 30 1 0 2 0 3 0 3 0 40 4 0 1 0 20 30 40 4 0 TIME AFTER PRIMARY ANTIGEN (DAYS) Fig. 1. Kinetics ofthe serum antibody response in mice inocuiated i.p. with 10|ig PC-KLH in saiine (o) or mixed with iOO ^g g-IN (•) on days 0 and 14 (arrows), (a) IgG anti-KLH; (b) IgG anti-PC; (c) IgM anti-KLH; (d) IgM anti-PC. Bars represent standard errors. well-Studied antibody response system (1720). In our hands also, i.p. injection of PCKLH gives reproducible primary and secondary responses in both IgG and IgM classes of immunoglobulin. Admixture of PC-KLH with the lowest i.p. dose of g-IN found to give detectable systemic activation ofthe APC and an antitumour action against the B16 melanoma (100 |ig/mouse; 11) increased secondary IgG responses to KLH up to 28-fold (P<000\) and to PC up to nine-fold (P<0005). The IgM responses to both epitopes were increased up to seven-fold {P<0-001). Figure 1 shows one of two replicate tests. IgA was increased 6-4-fold (/*=0-001, measured in one test only). The IgG response to KLH + g-IN remained high up to day 42. The enhanced responses were statistically highly significant and followed kinetics closely similar to those of the responses from antigen delivered in saline. Analysis ofthe IgG subclasses present in the peak (21 day) responses to KLH (Fig. 2) showed in both tests that antigen alone elicited almost entirely IgG 1, while admixture with g-IN enhanced each of IgG 2a, 2b and 3 several hundred fold. In contrast, IgG 1 was enhanced up to 9-6-fold {P<0002). By day 42, IgG 2a, 2b and 3 in the g-IN sera had declined more than IgG 1 but were still greatly in excess of values from antigen alone. Cell-mediated immunity We have assessed the effect of g-IN on cellmediated responses by two types of test. First, certain strains of mice primed with SRBC produce a DTH reaction when challenged a few days later with SRBC. The 348 P. D. COOPER ANDE. J. STEELE o o LU CC SALINE g-IN SALINE 21 DAY SERA g-IN 42 DAY SERA Fig. 2. IgG subclasses present in the 21 and 42 day sera described in Fig. L enhancement of this reaction indicates increased T cell-mediated immunity (21,22). When g-IN was mixed with primary s.e. injections of SRBC the DTH response was significantly enhanced (Fig. 3), the optimum dose being 50-100 |ig per mouse. A similar enhancement was obtained when g-IN was given 5 days before the primary SRBC (Fig. 3), a stimulant effect that had waned if the interval was 7r 0.001 0.001 DOSE g-IN Fig 3 Effect of dose of g-IN on DTH response. The g-IN inocuia were either mixed with iO^ SRBC per mouse and* the mice were chaiienged 4 days later (A), or the g-IN inocula were injected 5 days before 10^ SRBC were injected into the same site and the mice were chaiienged 5 days iater (A). Points represent the arithmetic means ± standard errors of each group of five mice; P vaiues (Student's Mest) correspond to the differences from the corresponding saline values. 349 INULIN AS ADJUVANT (27,28) specific for the nucleoprotein antigen common to these influenza subtypes (29). We find that BALB/c mice primed s.e. with 30-300 HA units of live or gammairradiated (26) influenza virus (strain A/JAP, H2N2) do not survive an intranasal challenge with five or 25 lethal doses of influenza virus strain A/WSN (HlNl) given 2-4 weeks later (Fig. 5a). However, if the primary inoculum is mixed with 50 jig of g-IN, 50% of the animals survive (the aggregate of survivors from four tests with this protocol was 1/26 mice from antigen in saline and 13/26 mice from antigen with gIN;/^valueofthedifference<0 increased to 10 days (Table I). The enhancing effect of g-IN on DTH responses to SRBC was equivalent to increasing the antigen dose about 10-fold (Fig. 4). Second, primary inoculation of mice with influenza virus of a particular haemagglutinin and neuraminidase subtype, for instance, H2N2, produces an anti-H2N2 response that protects against re-infection with H2N2 virus but not against virus of another subtype, such as HlNl (23,24). If the primary inoculum is above a threshold level then cross-protection occurs (25,26), but such heterotypic immunity is mediated not by antibody but by cytotoxic T cells Table 1. Effect on DTH responses of increasing the interval between prior g-iN inoculation and subsequent injection of primary SRBC* Time of SRBC injection after g-IN (days) Footpad sweiiing (XOi mm) Mean s.e. P vaiue"*^ i 3 5 7 iO No g-IN 5-5 0-47 < 0 0i 6-7 04i <000i 4-6 0 i9 <0-0i 4-6 0-48 007 36 0-43 NS 3-4 029 *CBA mice were inocuiated s.e. (napeof neck) with 50 ^gg-iN in saiine at days — i to — iO, then on day Owere re-inocuiated in the same site witii iO^ SRBC and chaiienged 5 days later. tDifference from SRBC alone ('no g-IN'). 0.001 9 r LU ^ < LU CC CJ ^ fi ^ 5 0.001 O O 5.0 6.0 7.0 8.0 SRBC DOSE (LOGIO) Fig. 4. Effect of g-iN on the DTH response to varying primary doses of SRBC with (•) or without (o) admixture with 50 |ig g-IN; the mice were chaiienged 4 days iater. Points represent arithmetic means ± standard errors of pooied data from six replicate experiments involving a total of i28 mice, with i5-2O mice per point. The P values (Student's Mest) correspond to differences between g-IN and saiine values. 350 P. D. COOPER ANDE. J. STEELE 100 7.0 8.0 9.0 1 0.0 TIME AFTER CHALLENGE (DAYS) Fig. 5. Effect of g-IN on the protection afforded by pre-inoculation with (a) 30 or (b) 3 HA units of live influenza virus strain A/JAP from mortality caused by influenza virus strain A/WSN. Groups of seven mice were inoculated s.e. with A/JAP virus in saiine (o) or mixed with 50 \ig of g-iN (•) and chaiienged intranasaiiy 29 days iater with 5X iO5 EIDso of A/WSN virus. The P value is by x^ test. g-IN is mixed with a very low primary inoculum of A/JAP virus (3 HA units. Fig. 5b) then protection is lost, showing that the effect is dependent on dose of antigen and that g-IN is not of itself protective in these circumstances. The DTH tests thus show that g-IN markedly boosts T cell mediated immunity, while the influenza tests support this idea with the reservation that the relevant effectors are not yet identified. DISCUSSION Vaccine technology is currently advancing rapidly, drawing on modern recombinant DNA, peptide synthesis, monoclonal antibody and protein separation techniques applied to a wide range of antigens. The practical application of such preparations in vaccines, however, usually depends upon (or is much improved by) combining with a vaccine adjuvant. Adjuvant preparations boost the immune response in various ways, and are particularly necessary for isolated proteins and simpler peptides. Unfortunately only one type of adjuvant, aluminium hydrates, is currently licensed for human or veterinary use because of concern for possible side effects and other problems. Thus there is an important need for a vaccine adjuvant that is both effective and non-toxic. These experiments show that g-IN is an unusual adjuvant in that it potently stimulates both humoral and cell-mediated immunity, increasing IgG, IgM and IgA responses and activating T cells. KLH more closely resembles the type of antigen used in vaccines than does the hapten PC, and since the KLH-specific responses induced by g-IN remained high by day 42, its increase of memory to naturally occurring antigens may also be good. The IgG subclasses particularly desirable for protective immunity, namely IgG 2a and 2b, appear to be enhanced much more than IgG 1. The effect of g-IN on IgA suggests a useful application against mucosal infections. It is significant that g-IN is able to confer protection against a lethal infection in mice (intranasal influenza virus), in this case expected but not yet proven to be mediated by T cells. g-IN is reported to be non-antigenic (30). It is non-pyrogenic and non-toxic at adjuvant-active doses, at which granuloma formation is minimal (P. D. Cooper, unpubl. data); in its dissolved state it is already licensed for human injection. Its breakdown products are simple sugars (fructose and glucose), and it is inexpensive, abundant and easily processed. Because it is active if given several days before antigen, g-IN is a stimulant to immune cells rather than a vehicle or depot for antigen. Since g-IN is a specific reagent INULIN AS ADJUVANT for activating the APC, we expect the initial molecular effectors to involve APC activation products, especially C3 cleavage derivatives for which many leucocyte types carry surface receptors (4). g-IN may therefore intervene in a number of leucocyte functions. The present results show that g-IN stimulates immune responses to two T-dependent soluble antigens or epitopes (KLH and PC), a particulate antigen (SRBC) and cell-surface antigens (influenza virus gene products). Indirect (cytokinemediated) activation of, say, helper T cells by g-IN may be a cause of the enhanced humoral responses, as equally may be direct activation of B cells in the germinal centres of follicular lymphoid tissue (6). The proportions of the KLH-sensitive IgG subclasses found in the g-IN sera were similar to those of non-immune BALB/c sera (31), 351 suggesting that g-IN may stimulate all IgG subclasses in their natural ratio. 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