Adhesion-mediated enhancement of the adjuvant activity of alum

Adhesion-mediated enhancement of the adjuvant activity of alum Danna L. Skea and Brian H. Barber* Alum, the only adjuvant currently licensed for use in humans, fails to adsorb influenza virus haemagglutinin ( BHA ) and is a poor adjuvant for this antigen. A specific monoclonal anti-BHA antibody adsorbed to alum promoted adhesion of the antigen to the adjuvant. The 'alum-anti-BHA-BHA' complex was found to be 1500-fold more immunogenic in mice and 5-fold more immunogenic in rabbits than a mixture of alum and BHA lacking the anti-BHA antibody. The biotin-binding protein, avidin, also mediated adsorption of biotinylated BHA to alum, and enhanced its irnmunogenicity to a comparable extent. These results indicate that the adjuvant activity of alum can be markedly enhanced by promoting a physical association between the antigen and the adjuvant. Keywords:Alum; vaccine adjuvant; influenza haemagglutinin

Recent advances in the fields of protein purification, peptide synthesis and recombinant DNA technology have made possible the creation of a new generation of defined subunit vaccine candidates. Although such vaccines have the potential to be much safer than vaccines composed of killed or attenuated whole micro-organisms, vaccines that contain only soluble proteins or peptide as immunogens tend to be poorly immunogenic. Thus, for a subunit vaccine to induce an adequate immune response, the concomitant administration of a substance with adjuvant activity is required. The only adjuvant that is currently licensed for use in humans is alum. Although alum is considered a safe adjuvant, it is not always an effective one 1. For some antigens the adjuvanticity of alum is minimal or non-existent 2"3. For example, in clinical trials, alum failed to enhance the immunogenicity of purified influenza virus haemagglutinin4,5. The main mode of action of alum is thought to be the conversion of a soluble protein to a particulate form by adsorption of the protein to the surface of the precipitated aluminium salt 6. This adsorption creates a depot effect which delays resorption of the antigen from the site of injection, thus prolonging the period of antigenic stimulation 7. In addition, protein-alum particles may be taken up by antigen-presenting cells more readily than soluble antigens are 6, thus intensifying the interaction of the antigen with the immune system. These mechanisms require that the antigen be bound to the alum, and it is considered that 'effective adjuvanticity depends on complete adsorption of the antigen to the aluminium salt '1.

Department of Immunology, University of Toronto, Toronto, Ontario, Canada, M5S 1A8. *To whom correspondence should be addressed. (Received 2 September 1992; revised 20 October 1992; accepted 20 October 1992) 0264-410X/93/10/1018-09 © 1993 Butterworth-Heinemann Ltd

1018 Vaccine, Vol. 11, Issue 10, 1993

During immunization studies involving the bromelain fragment of influenza virus haemagglutinin (BHA), we observed that alum failed to adsorb BHA and was a poor adjuvant for this antigen. As a result, efforts were made to improve the adjuvanticity of alum for BHA by adsorbing the antigen to the adjuvant via a specific monoclonal anti-BHD antibody. This immuno-adhesion of BHA to alum enhanced the immunogenicity of BHA in mice by 1500-fold; a finding which could have significant practical importance for the improvement of the adjuvant properties of alum. MATERIALS AND METHODS

Animals Female (B6 x C3H)F1 mice were purchased from Charles River Canada (St Constant, Quebec, Canada). The mice were 5-7 weeks old at the start of experiments. At the end of experiments, mice were bled from the retro-orbital plexus and serum was collected by centrifugation of the clotted blood. Female New Zealand White rabbits were purchased from Riemans Fur Ranches (St Agatha, Ontario, Canada). The rabbits were approximately 12 weeks old at the beginning of experiments. The rabbits were bled from the marginal ear vein and serum was collected by centrifugation of the clotted blood.

Antigen The bromelain fragment of influenza virus haemagglutinin (BHA), prepared from X31 strain virus as previously described a, was generously provided by Dr J. Skehel of the National Institute for Medical Research, Mill Hill, London, UK. For biotinylation, an 18-fold molar excess of NHS-LC-biotin (sulfosuccinimidyl 6-(biotinamido) hexanoate; Pierce, Rockford, Illinois, USA) (1 mg ml-1

Enhancement of the adjuvant activity of alum: D.L. Skea and B.H. Barber

in dimethyl sulfoxide) was incubated with BHA (1.5 mg ml- t in 0.1 M NaHCO3, pH 8.3) for 2 h at room temperature. Unreacted reagent was removed by dialysis against the same buffer. A colorimetric HABA (4hydroxyazobenzene-2'carboxylic acid; Sigma Chemicals, St Louis, Missouri, USA) assay9 indicated 6 moles of biotin per mole of BHA.

300 x 7.8mm, fractionation range 1000-300000 Mw (BioRad Laboratories, Toronto, Ontario, Canada) was used in conjunction with an HPLC system from Water Associates (Mississauga, Ontario, Canada). The column was calibrated with a set of molecular weight standards obtained from BioRad Laboratories. The eluant was monitored by measuring A28o.

Antibodies and other proteins The hybridoma 33A1 (EX6 - mouse IgG2a reactive with the H3 subtype haemagglutinin of X31 strain virus) was made by Dr W. Tamminen in our laboratory 1°. The hybridoma, HB 66 (73/1 - mouse IgG2~ reactive with the H1 subtype haemagglutinin of Bangkok 1/79 strain virus) was obtained from the American Type Culture Collection (Rockville, Maryland, USA). The hybridomas were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, L-glutamine, penicillin and streptomycin (Gibco, Grand Island, New York State, USA). The monoclonal antibodies were .purified from culture supernatants by affinity chromatography on protein A-Sepharose (Pharmaeia Canada, Baie D'Urfe, Quebec, Canada). F(ab')2 and Fab fragments of 33A1 were prepared by digestion with pepsin and papain (Sigma Chemicals) as previously described TM. The fragments were purified by passing the digests over protein A-Sepharose affinity chromatography columns to remove Fc-derived fragments and undigested molecules. Avidin and streptavidin were purchased from Sigma Chemicals.

Adjuvants Aluminium phosphate (18.5mgml-1), abbreviated as alum, and aluminium hydroxide (31-5 mg ml- 1) were obtained from Connaught Laboratories Ltd (Toronto, Ontario, Canada). Freund's complete adjuvant was obtained from BDH, Inc (Toronto, Ontario, Canada). Equal volumes of 0.2 mgm1-1 BHA and Freund's complete adjuvant (FCA) were mixed to form a stable emulsion, immediately prior to immunization.

Enzyme-linked immunosorbent assay An indirect enzyme-linked immunosorbent assay (ELISA) 12 was used to measure serum antibody responses to the antigen, BHA. Antibody binding was determined using goat-anti-mouse IgG (Fc~-specific)alkaline phosphatase (ICN Biological, Lisle, Illinois, USA), goat-anti-rabbit IgG (FcT-specific)-alkaline phosphatase (Jackson Immunoresearch Laboratories Inc, West Grove, Pennsylvania, USA) or one of the following mouse isotype-specific, alkaline phosphatase conjugates: GaMIgG1-AP, GaMIgG2a-AP, GaMIgG2bAP (Cedarlane Laboratories Ltd, Hornby, Ontario, Canada), GaMIgG3-AP (Zymed Laboratories, San Francisco, California, USA), G a M I g M - A P (Jackson Immunoresearch Laboratories Inc) or GaMIgA-AP (Sigma Chemicals). A standard curve of antibody binding was constructed using a pool of serum from five mice immunized with alum-anti-BHA-BHA. The standard serum was assigned a value of 10 000 arbitrary antibody units per ml. Serial 2-fold dilutions of the standard serum were run in ELISA, simultaneously with dilutions of other unknown sera. A sigmoidal curve was fit to the standard curve of absorbance versus antibody concentration, using a computer program 13. The binding activity in a serum sample was computed from its absorbance value by interpolation of the fitted curve. For the rabbit experiment, a standard curve was constructed using serum from a rabbit that had been immunized with BHA emulsified in FCA. This serum was assigned a value of 10000 arbitrary antibody units per ml.

Statistics Preparation of alum-antibody-BHA complexes Equal volumes (0.75 ml) of 6.2 mg m1-1 aluminium phosphate in water and 1.3 mgml -~ 33A1 (anti-BHA) in 0.1 M NaHCO3, pH 8.3, were mixed on a rotator overnight at 4°C. The mixture was centrifuged at 500g for 5 min at 4°C and washed twice with 1.5 ml of 0.1 M NaHCO 3, pH 8.3. The absorbance at 280 nm (A2ao) of the supernatant indicated that approximately 80% of the antibody had adsorbed to the mineral gel. Following the last wash, 1.5 ml of 0.1 mg ml- 1 BHA in 0.1 MNaHCO3, pH 8.3, was added to the pelleted alum, the alum was resuspended and the suspension was mixed on a rotator overnight at 4°C. An aliquot of this mixture was centrifuged, as before, and the supernatant was analysed by gel filtration high performance liquid chromatography (HPLC). Mice were immunized, subcutaneously, with 0.2 ml of the mixture.

High performance liquid chromatography Gel filtration HPLC was used to analyse the binding of proteins to alum. A Bio-Sil SEC 250-5 column,

Prior to statistical analysis, a logarithmic transformation of the data was conducted. Student's t test was performed using StatView (Abacus Concepts Inc, Berkeley, California, USA) on a Macintosh computer. This test was used for experiments in which the means of two groups only were to be compared. For experiments that involved more than two groups, the one-factor analysis of variance test was performed using SuperANOVA (Abacus Concepts Inc). The post hoc test used to compare the means was the Dunnett t test ~4. In this test, one group is assigned the status of 'control group', and the means of all other groups are tested against the mean of the control group for a significant difference. In Figures 2 and 7, the control group consisted of mice primed with BHA alone (no adjuvant); in Figures 3 and 4, the control group consisted of mice primed with BHA and alum (no binding protein). The one-tail Dunnett t test was used to determine whether the mean of each other group in the experiment was significantly greater than the mean of the control group. The level of significance, denoted as the p-value, is given in the figure legends.

Vaccine, Vol. 11, Issue 10, 1993 1019

Enhancement of the adjuvant activity of alum: D.L. Skea and B.H. Barber

RESULTS Binding of BHA to alum pre-coated with anti-BHA antibody For alum to enhance the immunogenicity of an antigen by converting it to a particulate, it must be able to adsorb the antigen in question. The large, water-soluble fragment of influenza virus haemagglutinin (BHA) is not bound by aluminium phosphate. However, aluminium phosphate does bind mouse immunoglobulin G. Thus, we investigated whether a mouse monoclonal IgG anti-BHA antibody could be used as an intermediary to promote adhesion of this antigen to alum. Alum-anti-BHA-BHA complexes were prepared as described. As a control, the same amount of alum was mixed with buffer (0.1 u NaHCO3, pH 8.3 ) instead of antibody, but was otherwise treated in an identical manner. The supernatants were analysed by gel filtration HPLC; the separation of components by HPLC allowed us to distinguish A2ao due to unbound BHA from A28o due to free antibody or immune complexes that might have dissociated from the alum. The results are presented in Figure 1. Figure l(a) shows the elution profile for 0.10ml of 0.1 mg ml-1 BHA; the elution time for the single peak

was 8.7 rain and the integrated area under the peak was 263mV. s. Figure1(b) shows the elution profile for 0.10 ml of the control preparation supernatant (alum + BHA, no antibody); the elution time of the single peak was 8.7 rain and the integrated area under this peak was 273 mV. s. These results indicate that none of the BHA was adsorbed to the alum. Figure 1(c) shows the elution profile for the test supernatant (alum + antibody + BHA). There was no peak at 8.7 min, indicating that all of the BHA had bound to the alum pre-coated with anti-BHA antibody. The small peak at 9.8min corresponds, in terms of molecular weight, to IgG and thus indicates that a tiny fraction of the antibody had detached from the alum. The peak at the void volume of the column (6.5 min) probably represents a small amount of immune complex; this complex may have formed between the detached antibody and the added BHA, or may have formed on the alum and then detached. In either case, this peak represents only a very small fraction of the BHA added. These results indicate that BHA, which does not itself bind to alum, can be bound to alum via a mouse monoclonal IgG anti-BHA antibody.

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Having achieved a method whereby BHA could be bound to alum, we next compared the immunogenicity of alum-anti-BHA-BHA and alum + BHA (i.e. BHA simply mixed with, and not adsorbed to, alum). Preparations were made, as described above, and used as immunogens immediately after the second overnight incubation. Groups of five mice were immunized subcutaneously with 0.2 ml of alum-anti-BHA-BHA, or alum + BHA, or with BHA alone in phosphate-buffered saline (PBS). The priming dose was 20/~g of BHA. Four weeks later, each mouse was boosted intraperitoneally with 10/~g of BHA in PBS. One week later, the mice were bled from the retro-orbital plexus, and the sera were analysed for IgG anti-BHA antibody by ELISA. The results are presented in Fi#ure 2. Mice that were primed with alum-anti-BHA-BHA showed a geometric mean IgG anti-BHA antibody response that was 1500-fold greater than that shown by mice that were primed with alum + BHA. The response by the latter group of mice was not significantly different from that by mice that were primed with BHA alone. These results indicate that alum-anti-BHA-BHA is Immunization

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elution time (minutes) Rgure 1 Gel filtration HPLC profiles of (a) BHA, (b) alum + BHA and (c) alum-anti-BHA-BHA, prepared as described in the text. Equal volumes were loaded for each run. Thus, the range of the y axis is constant between the three chromatograms

1020

Vaccine, Vol. 11, Issue 10, 1993

Rgure 2 IgG anti-BHA antibody response of mice immunized as described in the text. The data represent the geometric mean antibody response of five mice, plotted on a logarithmic scale. The error bars represent the upper limit of the range of the standard error of the mean. *p < 0.01, Dunnett's one-tail t test, mean > control, control = BHA

Enhancement of the adjuvant activity of alum: D.L. Skea and B.H. Barber

much more immunogenic than alum + BHA, which is not significantly different from BHA without any adjuvant.

Determination of the amount of anti-BHA antibody required for optimal immunogenicity The next question was how much anti-BHA antibody was required to optimally enhance the immunogenicity of BHA with alum. Preparations were made exactly as before except that the concentration of the antibody solution was varied. The amount of anti-BHA antibody that was used ranged from a 20-fold molar excess of antibody-combining sites over BHA to 0.5 times the amount of antibody-combining sites relative to BHA. A control preparation in which no antibody was used was also included. The amounts of alum and BHA were held constant. Mice were immunized and boosted as described above. The results are presented in Figure 3. Preparations of alum-anti-BHA-BHA that contained a twofold molar excess of antibody combining sites over BHA, or greater, were optimally immunogenic. The preparation containing equal amounts of antibodycombining sites and BHA was 10-fold less immunogenic than the maximum but was still significantly more immunogenic than the control preparation lacking antibody. The preparation that contained fewer combining sites than antigen was no more immunogenic than the control preparation. The immunogenicity results were related to HPLC measurements of the proportion of BHA bound. In the 20-fold and 10-fold preparations, 100% of the added BHA was adsorbed; the HPLC profiles were very similar to the one shown in Figure l(c). In the twofold preparation, 31% of the added BHA was adsorbed, and in the onefold preparation, 28% was adsorbed. In the

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0.5-fold preparation, less than 20% of the added BHA was adsorbed. Thus, there appears to be a threshold at approximately 30% binding (corresponding to 6 of 20 ktg per mouse), above which optimal immunogenicity is attained.

Effectiveness of binding proteins other than intact anti-BHA In order to investigate whether the enhanced immunogenicity of the alum-anti-BHA-BHA was due simply to adsorption of the BHA to alum or to some other property of the monoclonal antibody, we tested the ability of other proteins to mediate this adsorption and enhance immunogenicity. The principal antibody used in this study, 33A1, is specific for the H3 subtype of influenza virus haemagglutinin, the same subtype as the X31 BHA that was used. Another anti-haemagglutinin antibody, HB 66, is specific for the H1 subtype and does not cross-react with H 3 - B H A (data not shown). HB 66 bound to alum somewhat less well than 33A 1. Nonetheless, after washing there was still a greater than twofold molar excess of antibody-combining sites over antigen. As expected, alum-33A1-BHA adsorbed all of the BHA added, while alum-HB 66 adsorbed none (data not shown). As shown in Figure 4, the alum-33A1-BHA complex was significantly more immunogenic in mice than was the al um -H B 66-BHA mixture. The latter was no more immunogenic than a control preparation of alum and BHA lacking antibody. These results indicate that the mere presence of an antibody in the preparation is insufficient to enhance the immunogenicity of BHA with alum. To accomplish this, the antibody must be able to bind the BHA. To determine whether the Fc portion or the bivalency

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[anti-BHA IgG] arbitrary units/ml Fl~lure 3 IgG anti-BHA antibody response of mice immunized as described in the text. The data represent the geometric mean antibody response of five mice, plotted on a logarithmic scale. The error bars represent the upper limit of the range of the standard error of the mean. "dose of aluminium phosphate per mouse, ~dose of BHA per mouse, °molar excess of anti-BHA antibody over BHA. * p < 0.05, * * p < 0.001, Dunnett's one-tail t test, mean > control, control = 0 x

Vaccine, Vol. 11, Issue 10, 1993 1021

Enhancement of the adjuvant activity of alum: D.L. Skea and B.H. Barber

Binding Protein 33A1 HB 66 33A1-intact 33A1-F(ab~2 33A1-Fab 33A1 Avidin Streptavidin none

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[anti-BHA IgG] arbitrary units/ml Figure 4 IgG anti-BHA antibody response of mice immunized as described in the text. The data represent the geometric mean antibody response of five mice, plotted on a logarithmic scale. The error bars represent the upper limit of the range of the standard error of the mean. 33A1, anti-BHA (H3); HB 66, anti-BHA (H1). *p < 0.05, **p < 0.01, Dunnetrs one-tail t test, mean > control, control = none

of the anti-BHA antibody were important, we used pepsin-derived F(ab')2 fragments and papain-derived Fab fragments of 33A1 in place of the intact antibody. The fragments bound less well to alum than the intact antibody (24% for both F(ab')2 and Fab versus 82% for the intact antibody). However, for both kinds of fragments, after washing the alum, there was still a greater than twofold molar excess of antibody-combining sites over BHA bound. Nevertheless, the alum-anti-BHA-F(ab')2 and alum-anti-BHA-Fab adsorbed BHA poorly. Neither preparation bound more than 15% of the added BHA, while 100% was bound by alum-anti-BHA-intact IgG. As shown in Figure 4, alum-anti-BHA-F(ab')2BHA and alum-anti-BHA-Fab-BHA were significantly less immunogenic in mice than alum-anti-BHA-intact IgG-BHA. The alum-anti-BHA-F(ab')2-BHA was somewhat more immunogenic than the alum-antiB H A - F a b - B H A ; the former was significantly more immunogenic than the control preparation, alum + BHA (no binding protein ), while the latter was not significantly different from the control. These results indicate that F(ab')2 and Fab fragments of the anti-BHA antibody are less efficient than the intact antibody in terms of enhancing the imunogenicity of BHA with alum. This diminished effectiveness correlated with the reduced ability of the antibody fragments to mediate adsorption of BHA to alum. To test the efficacy of binding proteins other than immunoglobulins, BHA was biotinylated (bBHA) and avidin and streptavidin were used in place of the anti-BHA antibody. Avidin bound well to alum ( > 90% ), but streptavidin did not (approximately 20% adsorbed). Moreover, HPLC analysis revealed that, when mixed with bBHA, the alum-avidin preparation adsorbed the

1022 Vaccine, Vol. 11, Issue 10, 1993

antigen completely. However, the supernatant from the alum-streptavidin-bBHA preparation revealed a large peak of unbound bBHA and a large, broad peak at a shorter elution time (6.5-7.5 min) which may represent soluble complexes of streptavidin and bBHA dissociated from the alum. These results suggest that avidin, but not streptavidin, acts as an efficient intermediary in the adhesion of bBHA to alum. As shown in Figure 4, alum-avidin-bBHA was as immunogenic in mice as alum-anti-BHA-BHA, while the immunogenicity of alum-streptavidin-bBHA was not significantly different from the control preparation, alum + BHA with no binding protein. Collectively, these results indicate that adsorption of BHA to alum, via an antibody or a non-immunoglobulin binding protein, significantly enhances its immunogenicity.

Comparison of aluminium phosphate and aluminium hydroxide In the experiments described so far, the alum that was used was aluminium phosphate. Another aluminium salt that is currently used in vaccine preparations is aluminium hydroxide. The two aluminium salts have different protein-binding properties 15, therefore it was important to examine whether the concepts described above were also applicable to aluminium hydroxide. For this experiment, concentrations of the aluminJum salts were used such that each mouse received the same amount of aluminium (0.136mg A13+) as in previous experiments. BHA and the anti-BHA antibody, 33A1, were used as described for Fioures 1 and 2. Unlike aluminium phosphate, aluminium hydroxide adsorbed some of the BHA added (approximately 40% ).

Enhancement of the adjuvant activity of alum: D.L. Skea and B.H. Barber Table 1 Immunogenicity of BHA with aluminium phosphate and aluminium hydroxide pre-coated with anti-BHA antibody

Immunization

Anti-BHA IgG response (arbitrary units per ml)

Aluminium phosphateanti-BHA-BHA Aluminium phosphate + BHA

4635" (3500-6100)"

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Alum - anti-BHA - BHA Significance BHA + FCA

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However, like aluminium phosphate, aluminium hydroxide pre-coated with anti-BHA antibody adsorbed 100% of the BHA added. Mice were immunized as previously described, with aluminium phosphate-anti-BHA-BHA or aluminium phosphate + BHA, or with aluminium hydroxide-antiBHA-BHA or aluminium hydroxide + BHA. The IgG anti-BHA antibody responses of these mice are reported in Table l. As expected, the immunogenicity of aluminium phosphate-anti-BHA-BHA was three orders of magnitude greater than that of aluminium phosphate + BHA. Similarly, the immunogenicity of aluminium hydroxide-anti-BHA-BHA was statistically significantly enhanced relative to that ofaluminium hydroxide + BHA. The latter preparation however, unlike aluminium phosphate + BHA, was itself significantly immunogenic. Hence, the enhancement of immunogenicity resulting from the use of anti-BHA antibody with aluminium hydroxide and BHA was approximately one order of magnitude. These results indicate that increasing the adhesion of BHA to aluminium hydroxide via an anti-BHA antibody increases its immunogenicity. However, of the four preparations tested, the aluminium phosphate-antiBHA-BHA was the most immunogenic; the mouse antibody response to aluminium phosphate-antiBHA-BHA was more than twofold higher than the response to aluminium hydroxide-anti-BHA-BHA.

Quantity and isotype distribution of the antibody response to alum-anti-BHA-BHA For most antigens, Freund's complete adjuvant is the most powerful immunological adjuvant available. Thus, it was of interest to compare the immunogenicity of the alum-anti-BHA-BHA complex with that observed for BHA delivered in FCA. Mice were primed subcutaneously with 20/~g of BHA in the form of alum-anti-BHA-BHA prepared as described for Figures 1 and 2, or in a water-in-oil emulsion prepared by mixing equal volumes of 0.1 mg ml-1 BHA and FCA. Four weeks later the mice were boosted intraperitoneally with 10 #g of BHA alone, in PBS. One week later the mice were bled and the serum IgG anti-BHA antibody levels were measured by ELISA. The results are presented in Figure5. Although the geometric mean response of mice primed with BHA in FCA was somewhat higher than that of

Figure 5 IgG anti-BHA antibody responses of mice immunized as described in the text. The data represent the geometric mean antibody response of five mice, plotted on a logarithmic scale, The error bars represent the upper limit of the range of the standard error of the mean. p = 0.375, Student's t test

mice primed with alum-anti-BHA-BHA, this difference was not statistically significant. The isotype distribution of the antibody response to alum-anti-BHA-BHA was determined by ELISA, using subclass-specific conjugates. These results are presented in Figure 6. Of four mice that had been immunized with alum-anti-BHA-BHA, all four made IgG x and IgM anti-BHA antibody, two made IgG2b anti-BHA antibody and none made anti-BHA antibody of the IgG2a, IgG 3 or IgA isotypes. Four mice that were immunized with alum + BHA made IgM anti-BHA antibody but no IgG or IgA anti-BHA. These results are consistent with published data regarding the isotype distribution of the antibody response to antigens delivered with alum 16 and with data presented in this paper showing that alum-anti-BHA-BHA is far more immunogenic in mice than alum + BHA.

Immunogenicity of alum-anti-BHA-BHA in rabbits To test the immunogenicity of alum-anti-BHA-BHA in a species other than mice, we chose rabbits. Groups of three rabbits each were immunized subcutaneously with alum-anti-BHA-BHA, alum + BHA or BHA alone. The priming dose was 20/~g of BHA. Four weeks later each rabbit was boosted subcutaneously with 10/~g of BHA alone, in PBS. A serum sample was obtained from each rabbit one week prior to priming, one week prior to boosting and one week after boosting. The anti-BHA IgG levels in these sera are shown in Fioure 7. Unlike mice, rabbits primed with BHA alone, or with BHA + a l u m , showed a moderate anti-BHA IgG response. The response was similar regardless of the presence of alum. However, the immunogenicity of BHA in rabbits was enhanced when the BHA was adsorbed to alum pre-coated with anti-BHA antibody. In rabbits, this enhancement was less marked (approximately fivefold) than in mice (approximately 1500-fold), probably because the BHA alone was immunogenic in rabbits, but not in mice. DISCUSSION The data in this paper clearly indicate that the immunogenicity of influenza virus haemagglutinin is markedly enhanced in mice and rabbits when it is adsorbed to alum by means of a previously bound anti-haemagglutinin monoclonal antibody. The antiBHA antibody response in mice to alum-antiBHA-BHA was 1500 times more potent than the response to a simple mixture of alum and BHA. This

Vaccine, Vol. 11, Issue 10, 1993

1023

Enhancement of the adjuvant activity of alum: D.L. Skea and B.H. Barber

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enhanced immunogenicity seemed to be due to the binding of BHA to alum via the antibody, and not to any other intrinsic property of the immunoglobulin itself, since avidin could mediate the binding of biotinylated BHA to alum and the anti-BHA antibody response to alum-avidin-bBHA was similar to the response to alum-anti-BHA-BHA. Other proteins with the potential to bind BHA to alum, namely Fab and F(ab')z fragments of the anti-BHA antibody, and streptavidin in the case of biotinylated BHA, were significantly less effective in their ability to enhance the adjuvanticity of alum for BHA. These results probably reflect the unstable adsorption of these linkage proteins to alum. For maximum immunogenicity, a twofold molar excess (or greater) of intact anti-BHA antibody-combining sites over BHA was required. The

1024 Vaccine, Vol. 11, Issue 10, 1993

excess may be necessary because a proportion of the molecules may bind to alum in such an orientation that prevents their subsequent association with antigen. The Fab and F(ab')2 antibody fragments and the streptavidin bound poorly to alum; in each case, less than 25% of the added protein was bound. This low level of binding notwithstanding, the amount of protein that was bound still provided a greater than twofold molar excess of binding sites over BHA. Nevertheless, these proteins failed to mediate an immunologically productive adsorption of BHA to alum. A possible explanation for these observations is that the molecules that did adsorb to the alum did so preferentially in the orientation that blocked their binding sites for BHA (or bBHA). In addition, it was noted that after the incubation of alum-streptavidin with bBHA, more streptavidin

Enhancement of the adjuvant activity of alum: D.L. Skea and B.H. Barber

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-

,~ ~

~

.

,

BHA

BHA + alum

alum anti-BHA BHA

Rgure 7 IgG anti-BHA antibody response of rabbits immunized as described in the text. The data represent the geometric mean for three rabbits, plotted on a linear scale. The white bars represent pre-immune sera, the shaded bars represent pre-boost sera and the hatched bars represent post-boost sera. * p < 0 . 0 5 , Dunnett's one-tail t test, mean > control, control = BHA, post-boost sera only

became dissociated from the alum. This desorption may also have contributed to the failure of streptavidin to mediate binding of bBHA to alum. Randall and colleagues recently introduced an alternative immunization protocol utilizing what they term 'solid matrix antigen-antibody' (SMAA) complexes1 ~-21. The solid matrix in this system is composed of fixed and killed S t a p h y l o c o c c u s a u r e u s Ccwan strain A bacteria ~7. These bacteria bear a surface molecule, called protein A, which binds with high affinity to the Fc portion of certain subclasses of mouse immunoglobulins 22. Randall and Young la found that this matrix, pre-coated with specific monoclonal antibodies and mixed with the appropriate antigen, was highly immunogenic. This system has been shown to be effective for a number of different viral antigens 19. In one study involving the paramyxovirus, simian virus 5 (SVS), they substituted alum for the S. a u r e u s bacteria, and found that SV5 antigen recovered from infected cell lysates by alum-anti-SV5 complexes induced high titres of virus-neutralizing antibody in mice 21. Our results clearly support the strong immunogenicity which can be observed when viral antigens are immuno-adsorbed to alum particles in this way. In addition, our findings extend the system to the use of purified viral antigens, clarify the role of the antibody in the immunoenhancement process, and also indicate that equally effective augmentation of immunogenicity can be achieved when adhesion of antigen to alum is mediated by other means, specifically the avidin-biotin linkage. In some cases, alum has been found to be a reasonably good adjuvant for antigens that are intrinsically able to bind to it, such as polio virus 23 and diphtheria and tetanus toxoids 2. In contrast, Sacco e t al. 3 found that alum was a very poor adjuvant for porcine zona pelludica (a potential contraceptive vaccine). These authors concluded that the poor adjuvanticity of alum for this antigen was due to unstable adsorption of the antigen to the adjuvant. Callahan e t al. ~ made a study of the binding of recombinant malarial peptides to alum. They found that peptides that bore a net negative charge bound very well to aluminium hydroxide which has a positive surface charge, and poorly to aluminium phosphate which has a negative surface charge. Conversely, a peptide that bore a net positive charge bound well

to aluminium phosphate and poorly to aluminium hydroxide. The binding of this peptide to aluminium hydroxide was improved in the presence of phosphate ions; the authors concluded that the negatively charged phosphate ions adsorbed to the positively charged surface of the aluminium hydroxide, reversing the surface charge and subsequently promoting the binding of the positively charged peptide. Unfortunately, this study did not include experiments to examine the imunogenicity of the various peptide-alum preparations. Our prediction would be that the negatively charged pcptides would be more immunogenic when combined with aluminium hydroxide than when combined with aluminium phosphate, and that the positively charged peptide would be more immunogenic when combined with aluminium phosphate or with aluminium hydroxide in the presence of phosphate ions, than when combined with aluminium hydroxide (in the absence of phosphate ions). BHA, whose isoelectric point is not known, is not adsorbed by aluminium phosphate at all, and is adsorbed poorly by aluminium hydroxide. In both cases, complete adsorption was attained by pre-coating the aluminium salts with the anti-BHA antibody, and in both cases complete adsorption resulted in a significant enhancement of immunogenicity. Mice that were immunized with BHA plus aluminium hydroxide showed a small, but significant, anti-BHA antibody response, consistent with partial adsorption of BHA to aluminium hydroxide. Collectively, our data support the hypothesis that complete adsorption of an antigen to alum is required for maximum adjuvanticity. There have been two reports in the literature that suggest that an adjuvant effect can be attained even when the antigen and the alum are injected separately. However, in one of these reports 24, the antigen ( D N P - K L H ) and the alum were injected separately, but simultaneously, into the peritoneal cavity of mice. Therefore it is possible that the antigen adsorbed to the alum in vivo. The authors did note that the immune response was significantly higher when the antigen was adsorbed to the alum prior to the injection. In the other report 25, 5 mg of antigen (bovine gamma globulin) were injected intraperitoneally into mice, one day prior to an intraperitoneal injection of 5 mg of alum. Since the doses were so large it is possible that sufficient antigen was retained in the peritoneal cavity to associate with the alum injected on the following day. Once again adsorption of the antigen to the alum in v i v o may have accounted for the observed adjuvant effect. No adjuvant effect was observed if the antigen was injected intraperitoneally and the alum subcutaneously. Additional aspects of the alum-antibody-antigen system that were investigated in the present study include the quantity of antibody induced, its isotype distribution, and whether the system worked in a species other than mice. In terms of quantity, the antibody response in mice immunized with alum-anti-BHA-BHA was not significantly different from that in mice immunized with the same dose of BHA emulsified in FCA. Enhancing the efficacy of alum to the level of response achieved with FCA is a significant achievement for most antigens, and one which could have important practical applications. As expected, most of the antibody produced in response to alum-anti-BHA-BHA was IgG1; there was a small amount of IgG2b antibody in some of the mice, but no IgG2a or IgG 3 antibody was detected. Mice that were immunized with alum + BHA (no anti-BHA

Vaccine, Vol. 11, Issue 10, 1993 1025

Enhancement of the adjuvant activity of alum: D.L. Skea and B.H. Barber

antibody to mediate adhesion) did not show any IgG anti-BHA antibody response. However, these mice, and those immunized with alum-anti-BHA-BHA, made anti-BHA antibody of the IgM isotype. Since the immunizations were systemic, neither group made any IgA antibody response. The isotype distribution of the antibody response in mice to alum-anti-BHA-BHA is consistent with published data concerning the isotype distribution of the antibody response to alum-adsorbed vaccines 16,2 6. Finally, it was demonstrated that the adhesion of BHA to alum via a specific anti-BHA antibody enhanced the immunogenicity of BHA in rabbits, as well as in mice. Thus, the enhancement phenomenon is not speciesspecific, indicating that the potential exists to utilize this manipulation of alum to develop both new and significantly improved subunit vaccines for use in humans and other animals. One important possibility would be the use of monoclonal anti-gp 120/gp 160 antibodies to augment the immunogenicity of the recombinant human immunodeficiency virus envelope antigens currently being assessed as alum-adjuvanted vaccine candidates 2~.

8 9 10

11 12 13 14

15 16

17

ACKNOWLEDGEMENTS D.L.S. is the recipient of a Medical Research Council of Canada Post-doctoral Fellowship. This work was supported by Connaught Laboratories Limited and the Province of Ontario Technology Fund.

19

REFERENCES

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1 Edelman, R. Vaccine adjuvants. Rev. Infect. Dis. 1980, 2, 370-383 2 Aprile, M.A. and Wardlaw, A.C. Aluminum compounds as adjuvants for vaccines and toxoids in man: a review. Can. J. Public Health 1966, 57, 343-354 3 Sacco, A.G., Yurewicz, E.C. and Subraminian, M.G. Effect of varying dosages and adjuvants on antibody response in squirrel monkeys (Saimiri sciureus) immunized with the porcine zona pellucida M, = 55000 glycoprotein (ZP'3). Am. J. Reprod. Immuno/. 1989, 21, 1-8 4 Davenport, F.M., Hennessy, A.V. and Askin, F.B. Lack of adjuvant effect of AIPO4 on purified influenza virus hemaggiutinins in man. J. Immunol. 1968, 100, 1139-1140 5 Nicholson, K.G., Tyrrell, D.A.J., Harrison, P., Potter, C.W., Jennings, R., Clark, A. et al. Clinical studies of monovalent inactivated whole virus and subunit A/USSR/77 (HIN1) vaccine: serological responses and clinical reactions. J. Biol. Stand. 1979, 7, 123-136 6 Edsall, G. Application of immunological principles to immunization practices. Med. C/in. North Am. 1966, 49, 1729-1743 7 Leeling, J.L., Muni, I.A., Helms, R.J. and Johnson, N. Rates of release of subcutaneously injected antigens in the rat. Allergy 1979, ~4, 339-344

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Brand, C.M. and Skehel, J.J. Crystalline antigen from the influenza virus envelope. Nature New Biol. 1972, ~ , 145-147 Green, N.M. Spectrophotometric determination of avidin and biotin. Methods Enzymol. 1970, 18, 418-427 Tamminen, W.L., Wraith, D. and Barber, B.H. Searching for MHC-restricted anti-viral antibodies: antibodies recognizing t h e nucleoprotein of influenza virus dominate the serological response of C57BL/6 mice to syngeneic influenza-infected cells. Eur. J. Immunol. 1987, 17, 999-1006 Harlow, E. and Lane, D. Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988 Goding, J.W. Monoclonal Antibodies: Principles and Practices, Academic Press Inc, London, 1983 Davis, S.E., Jaffe, M.L., Munson, P.J. and Rodbard, D. RIA data processing with a small programmable calculator. J. Immunoassay 1980, 1, 15-20 Dunnett, C. and Goldsmith, C. When and how to do multiple comparisons. In: Statistics in the pharmaceutical industry (Eds Buncher, C.R. and Tsay, J.Y.) Marcek Dekker Inc, New York, 1981, pp. 397-433 Callahan, P.M., Shorter, A.L. and Hem, S.L. The importance of surface charge in the optimization of antigen-adjuvant interactions. Pharm. Res. 1991, 8, 851-858 Kenney, J.S., Hughes, B.W., Masada, MP. and Allison, A.P. Influence of adjuvants on the quantity, affinity, isotype and epitope specificity of murine antibodies. J. Immuno/. Methods 1989, 121, 157-166 Randall, R.E., Young, D.F. and Southern, J.A. Immunization with solid matrix-antibody-antigen complexes containing surface or internal virus structural proteins protects mice from infection with the paramyxovirus, Simian virus 5. J. Gen. Viro/. 1988, 00, 2517-2526 Randall, R.E. and Young, D.F. Humoral and cytotoxic T cell immune responses to internal and external structural proteins of Simian virus 5 induced by immunization with solid matrix-antibody-antigen complexes. J. Gen. Virol. 1988, 00, 2505-2517 Randall, R.E. and Young, D.F. Immunization against multiple viruses by using solid matrix-antibody-antigen complexes. J. Virol. 1989, 63, 1808-1810 Randall, R.E. Solid matrix-antibody-antigen complexes for constructing multivalent subunit vaccines. Immunol Today 1989, 10, 336-339 Randall, R.E. and Young, D.F. Solid matrix-antibody-antigen complexes induce antigen-specific CDS-t- cells that clear a persistent paramyxovirus infection. J. Virol 1991, 6~, 719-726 Kronvall, G., Seal, U.S., Finstead, J. and Williams R.C. Phylogenetic insight into evolution of mammalian Fc fragment of 7G globulin using Staphylococcal protein A. J. Immunol. 1970, I(N, 140-147 Henneberg, G., Drescher, J., Godgluck, G. and Grutzner, L. Immunogenic activity of aqueous and aluminum oxide adsorbed poliovirus vaccine in Macaca mulatta. Am. J. Hygiene 1982, 75, 44-55 Flebbe, L.M. and Braley-Mullen, H. Immunopotentiating effects of the adjuvants SGP and Quil A. 1. Antibody responses to T-dependent and T-independent antigens. Ce#. Immunol. 1986, 00, 119-127 Dresser, D.W. An assay for adjuvanticity. C/in. Exp. Immunol. 1968, 3, 877-888 Beck, L. and Spiegelberg,.H.L. The polyclonal and antigen-specific IgE and IgG subclass response of mice injected with ovalbumin in alum or complete Freund's adjuvant. Cell Immunol. 1989, 123, 108 Spalding, B.J. In hot pursuit of an HIV vaccine. Bio/Technology 1992, 10, 25-29