CHARACTERISTICS

AND

USE OF NE W-GENERA

Aluminum

TION ADJUVANTS

salts

W. Nicklas Central Animal

Laboratories,

German Cancer Research Centre, D-6900 Heidelberg

Introduction Among various mineral salts which have been used as adjuvants, aluminum salts are the most widely used adjuvants in humans. In addition, they are part of a number of veterinary vaccines. In many countries, a large proportion of the population has been successfully and safely vaccinated with different aluminum-adjuvanted vaccines against numerous microorganisms. Various clinical studies demonstrate that good serological responses can be obtained in humans with acceptable clinical reactions (Nicholson et al., 1979; Scolnick et al., 1984). However, in many cases, the adjuvant effect is dependent on the antigen used, and in certain vaccines it has not always been satisfactory (Aprile and Wardlaw, 1966). Nevertheless, the immense experience which results from the worldwide use of aluminum salts has led to a great deal of knowledge about its efficiency and compatibility, and makes it the ideal adjuvant for experimental studies in humans. Aluminum compounds have even been successfully used in the first recombinant vaccines and were shown to induce antibodies and immunity against hepatitis B virus (Murray et al., 1984 ; Scolnick et al., 1984). In addition, they were included in the first recombinant (Ballou et al., 1987) and synthetic (Herrington et al., 1987) malaria vaccines which were tested in man. Compared to other existing adjuvants, aluminum salts such as, for example, aluminum hydroxide, are rarely used for experimental immunization of laboratory animals. However, for the reasons mentioned above, aluminum compounds are frequently used for comparative purposes in studies intended to evaluate other adjuvants destined for use in man. Preparation

of aluminum

adjuvants

Different methods can be employed to prepare vaccines containing aluminum adjuvants. One involves the addition of a solution of aluminum salts to the antigen to form a precipitate of protein aluminate in the presence of the antigen. These products have been called “alum-precipitated vac-

(Germany)

cines”. Such precipitates may be of variable composition depending on both the anion present during precipitation and the extent of interaction between the aluminum cation and antigen. Therefore, the nature of aluminum-precipitated vaccines is not necessarily defined qualitatively or quantitatively. Using another method, the solution of antigen is added to preformed precipitates of aluminum compounds. Such preparations have been termed “aluminum-adsorbed vaccines”. Most commonly, commercial preparations of AI( or AlPO, are used. Details about the preparation have been described by Dresser (1986) and by Hu and Kitagawa (1990) for alum-precipitated vaccines and by Herbert (1978) and Harlow and Lane (1988) for aluminum-adsorbed vaccines. In general, 1 mg Al(OH), will bind about 50-200 pg of protein. Proteins are readily absorbed on alumen. However, the pH conditions may be important. Hu and Kitagawa (1990) reported that aluminum phosphate (AP) absorbs bovine serum albumin (BSA) completely when pH is acidic, whereas the optimal pH value of aluminum hydroxide (AH) was neutral (table I). Aluminum-containing vaccines may have different properties depending on the composition and mode of preparation. Levine et al. (1955) found that the immunization of mice and guinea pigs was more effective with precipitated tetanus toxoid than with adsorbed toxoid, and AI( was more potent than AIPO,. However, no differences were seen for diphtheria toxin. Other studies showed that AH is a more effective adjuvant than AP (Berman et al., 1985). Beh and Lascelles (1985) immunized sheep against ovalbumin and found that AH-precipitated antigen produced a lower antibody response than AP-precipitated antigen. Adsorbed preparations yielded even lower antibody responses than the respective precipitated preparations. Combination

with other adjuvants

Aluminum salts can be combined with other adjuvants to increase adjuvanticity. Teerlink et al.

scd scd scd ip

-

Mycopl.

SRBC Leishmania Trypanosoma

Bacteria

Cell Protozoans

(*) Antigen/adjuvant. G = guinea pig ; Go = goat ; M = mouse. NA = not available.

ip NA

BSA -

Viomycin HBV

Peptide Virus F 38

scd scd scd, ip

-

-

-

BSA BSA HBsAg

(foreign)

Protein

Carrier route

Name

Antigen

mg

lOs/O.4 mg lo’/25 mg 10 pg/O.5 mg

5 mg/lO

10 pgjO.2 mg 25 Kg/O.2 pg

100 pg/? 50 pgiO.4 mg 1.2 ug/?

Dose (‘)

Partial protect. NA Protect. Protect.

NA IgGl,2 Wl, little IgG2 NA NA

salts.

NA NA NA

NA Cell. resp. NA

NA NA Low resp.

T cells

of aluminum

Isotypes

Table I. Characteristics

M M M

Go

M G

M M M

Species

NA NA NA

et al. (1988) Bomford (1980) Jarecki-Black et al. (1988) Gonzalez et al. (1991)

Mulira

Hu and Kitagawa (1990) Sanchez et al. (1980)

NA NA NA

Woodard (1989) Bomford (1980) Byars et al. (1991)

Reference

NA NA NA

Side effects

CHARACTERISTICS AND USE OF NE W-GENERATION ADJUVANTS (1987) found that the adjuvant effect of several detergents can be increased by combination with AP. The combination of detergent and AIPO, induced higher antibody titres to bacterial proteins in mice than Freund’s complete adjuvant (FCA). Richards et al. (1988) immunized rabbits with cloned malaria sporozoite antigen. They found the strongest antibody response when the antigen was given with aluminum adsorbed liposomes containing lipid A. Therefore, they assumed that these adjuvants may have an additive or synergistic effect. Mannhalter et al. (1991) immunized chimpanzees with a recombinant glycoprotein of human immunodeficiency virus (HIV) and found that an AH-adjuvanted vaccine was not very efficient in inducing primed T cells or neutralizing antibodies. However, the adjuvanticity could be improved by addition of deoxycholate or by subsequent immunizations with antigen given in a lipid-based adjuvant . Most commonly, aluminum salts are combined with killed bacteria, usually heat-killed Bordetella pertussis,to induce non-specific stimulation. This can easily be achieved by adding around 2-4 x lo9 organisms per 100 ~1 of material to be injected (Dresser, 1986; Harlow and Lane, 1988). However, the addition of B. pertussis may reintroduce some of the potential side effects seen with complete Freund’s adjuvant.

Mode of action Like many potent adjuvants, aluminum salts exhibit a depot effect. Compared to water-in-oil adjuvant mixtures, the repository effect is much lower. Beh and Lascelles (1985) even found that the resorption from the site of injection of an aluminumprecipitated ovalbumin was similar to antigen injected in saline. According to Osebold (1985), the antigen persists in alumina-gel-induced granulomas for 2 to 3 weeks. Cellular responses are induced for a period of 2 months, whereas the antigen persists for months at the site of injection when given with Freund’s adjuvant. Sacco et al. (1989) explain the poor antibody responses to a porcine glycoprotein in squirrel monkeys with the rapid dissociation of the immunogen from the Al(OH), gel. The poor depot effect might be one reason for the usually short-lived antibody response to antigens given with aluminum adjuvants. Antibody titres reach a maximum after 2 to 3 weeks and already decrease at 3 to 4 weeks after injection (Osebold, 1982). Therefore, repeated injections are necessary to obtain a more prolonged effect (WHO, 1976). However, the booster effect can depend on properties of the antigen like the antigen dose. Hu and Kitagawa (1990) found that with optimal or higher antigen doses, the more boosters given, the weaker the response in mice. With low an-

491

tigen dosage, more boosters increased the amount of IgG produced. Some additional mechanisms are responsible for the adjuvanticity of aluminum salts. Ramanathan et al. (1979) found out that aluminum hydroxide can stimulate the immune system by activation of the complement system in guinea pigs. In addition, there is a direct stimulatory effect on cells involved in the immune response, as an augmentation of the antibody response can occur even when the adjuvant is given separately from a soluble antigen (Flebbe and Braley-Mullen, 1986). However, in contrast to many other adjuvants, alumen is deficient in boosting cellmediated immunity (Allison and Byars, 1986). In studies reported by Flebbe and Braley-Mullen (1986), Al(OH), augmented the responses to T-celldependent antigens but had little or no effect on responses to T-cell-independent antigens. Properties

of vaccines containing

aluminum

salts

The addition of adjuvants to vaccines potentiates the immune response so that a greater amount of antibody is produced or a smaller quantity of antigen is required to obtain a sufficient immune response. Kuwert et al. (1978) demonstrated that AH can compensate for a 90 070reduction in antigen content and, hence, contribute to a reduction in production costs. However, Byars et al. (1991) report in a recent paper that a ten-fold reduction in the dose of antigen required is possible when adjuvants are used which are more potent than alum. Aluminum compounds are themselves not immunogenic nor do they act as haptens ; thus they are unlikely to cause harmful immune complex reactions or clinically important hypersensitivity reactions in immunized individuals. However, aluminum adjuvants are very efficient in enhancing the synthesis of IgE antibodies in rabbits and rodents (Allison and Byars, 1986; WHO, 1976). Following immunization of mice with bovine serum albumin (BSA), Bomford (1980) found only antibodies of the IgGl isotype during the primary reaction, whereas during the secondary response, an IgGl and IgG2a response was stimulated by AH. Byars et al. (1991) confirm that, in contrast to several other adjuvants, predominantly the IgGl isotype and only a little IgG2a and IgG2b are produced. The authors suggest that this is of special importance when a protective immunity is required, as antibodies of the IgG2a subclass act synergistically with complement and antibody-dependent effector cells. After systemic immunization with an alumadjuvanted vaccine, antibodies of the IgG and the IgA type are detectable not only in the blood, but also in other body fluids like urine (Kruze et al., 1989)

492

44th FORUM

IN IMMUNOLOGY

and in vaginal fluid (Thapar et al., 1990a). However, local immunization with Al(OH), results in the production of higher titres of local IgA and IgG (Thapar et al., 1990a,b). Use of aluminum-containing

vaccines

The formation of granuloma is very common with alum-adjuvanted vaccines. In humans, they should therefore be injected intramuscularly. In animals, immunizations are usually given subcutaneously or intramuscularly. Alum can, in addition, be injected intravenously (mainly in rabbits); in mice, the intraperitoneal route is common. When B. pertussis is added, the vaccine should not be injected intravenously (Harlow and Lane, 1988). Other sites of injection are possible. Thapar et al. (1990a) report that aluminum adjuvants can be administered intravaginally and thereby increase local immune responses. Using this route, Al(OH), was the most effective of 5 adjuvants tested. Side effects In general, local reactions following the use of aluminum salts do not cause severe clinical problems. In humans, they only occasionally produce granulomas and nodules at the injection sites, particularly following subcutaneous injection (Aprile and Wardlaw, 1966; Butler et al., 1969; Etlinger et al., 1988). In guinea pigs, intradermal injection of AH produces persistent granuloma in the skin (Turk and Parker, 1977). In mice, Goto and Akama (1980) observed that intramuscular injection of AH with tetanus toxoid caused injury to muscle fibres and infiltration of neutrophils around the residues of the material injected. In some cases, microabscesses and granulomas were formed. Alum alone caused similar, but less marked lesions. In guinea pigs, the authors found macroscopically visible nodule formation in the pancreas after intraperitoneal injection of an aluminum adjuvanted toxoid. After intraperitoneal application of Al(OH), in mice, Thapar et al. (1990b) detected adhesions linking several organs in the peritoneal cavity. Recent studies on the efficiency of aluminum in laboratory animals

salts

Comparative studies which seek to evaluate adjuvants have been predominantly performed in mice. Aluminum hydroxide can be used to increase the antibody response to particulate antigens. Bomford (1980) found that Al(OH), provoked a higher antibody response in mice immunized with sheep red

blood cells than FCA. However, saponin was even more potent. With BSA as an antigen, the addition of AH elicited a strong antibody response equivalent or superior to FCA. Woodard (1989) confirms that the adjuvant activity of Al(OH), with 100 pg of BSA is comparable to FCA. Hu and Kitagawa (1990) found that 10 pg of a small peptide coupled to BSA was the optimal dose for the immunization of mice. Amounts of less than 5 pg did not result in antibody production. This might explain why Hong et al. (1989) were not able to induce significant immune responses with 1 pg of human serum albumin and alum although the antigen was administered into lymph nodes and intrasplenically. However, the use of FCA resulted in a strong response with the same amount of antigen when given in both routes. Smithers et al. (1989) tested 8 adjuvants for their potency to induce antibody production and a protective immunity against Schistosoma mansoni in mice. Alum was not among those adjuvants which stimulated significant protective immunity although antibodies against a few of the antigens used were induced. A protective immunity to Trypanosoma infections was induced by the use of a purified antigen by Gonzalez et al. (1991). Of different adjuvants tested, alumen was the most effective. Mice immunized with antigen plus alum were protected against an acute infection, and sera of such mice lysed the flagellates in contrast to sera of mice immunized with antigen only or with antigen together with FCA. JareckiBlack et al. (1988), too, produced significant resistance in mice by immunization with kiIled protozoans. With small amounts of recombinant hepatitis B virus surface antigen, Byars et al. (1991) found only low antigen titres. Larger amounts of antigen were necessary to induce a significant immune response with alum. Other adjuvants were already effective already with lower antigen dosage, or mice responded more quickly or with higher titres. Similar results were obtained in guinea pigs. Animals vaccinated with a cloned herpes simplex virus (HSV) glycoprotein in FCA were completely protected against a challenge infection, whereas vaccination with alum preparations resulted in a partial protection only (Berman et al., 1985). However, antibody production was induced. The authors conclude that incorporation of the antigen in alum may result in the loss of antigenic determinants. Sanchez et al. (1980) demonstrated that the adjuvant activity of alum is sufficient to induce a moderate antibody response when guinea pigs are injected with whole virus particle of HBV, whereas only a weak response was seen with a polypeptide surface antigen. Again, the highest immune response was obtained with FCA. Additional papers confirm that FCA and several

CHARACTERISTICS

AND

USE OF NEW-GENERATION

more recently developed adjuvants are superior to alum in various animal species, such as, for example rabbits (Richards et al., 1988), primates (Sacco et al., 1989; Mannhalter et al., 1991), goats (Mulira et al., 1988) and sheep (Beh and Lascelles, 1985). Conclusion

Unlike commercial vaccines, frequently only partially defined antigens are used for research purposes. Only rarely is sufficient information available on the suitability of an adjuvant for a specific immunogen. For that reason, only those adjuvants able to induce a strong immune reaction to different antigens in a high percentage of individuals can be recommended for experimental immunization of laboratory animals. In addition, an adjuvant for universal use should be effective in different animal species. Minimal side effects are observed when aluminum salts are used. There exist many examples of a good immunostimulatory effect of aluminum alone or in combination with other adjuvants. Aluminum compounds may be suited for antigens which are highly immunogenic and available in large amounts. But they cannot be recommended when only weak immunogens are used or when the amount of antigen available is limited. At present, FCA is most commonly used for experimental immunization because its action is reliable and reproducible. Unfortunately, it is toxic and should therefore be avoided whenever possible. However, the efficiency of aluminum salts is insufficient for many antigens. Therefore, alumen is unlikely to replace FCA as an adjuvant. References Allison, A.C. & Byars, N.E. (1986),An adjuvant formulation that selectivelyelicitsthe formation of antibodies of protective isotypes and of cell-mediated immunity. J. Immunol. Methods, 95, 157-168. Aprile, M.A. & Wardlaw, A.C. (1966),Aluminium compoundsasadjuvantsfor vaccinesand toxoids in man. Canad. J. Publ. Hlth, 57, 343-354. Ballou, W.R., et al. (1987),Safety andefficacy of a recombinant DNA Plasmodiumfalciparum sporozoite vaccine. Lancet, I, 1277-1288. Beh, K.J. & Lascelles, A.K. (1985),The effect of adjuvants and prior immunizationon the rate and modeof uptake of antigen into afferent popliteal lymph from sheep.Immunology, 54, 487-495. Berman,P.W., et al. (1985),Protectionfrom genitalherpes simplex virus type 2 infection by vaccination with clonedtype 1glycoproteinD. Science,227, 1490-1492. Bomford, R. (1980),The comparativeselectivity of adjuvants for humoral and cell-mediatedimmunity. I. Effect on the antibody responseto bovine serum albuminandsheepred blood cellsof Freund’sincom-

ADJUVANTS

pleteandcompleteadjuvants,alhydrogel,Corynebacteriumparvum, Bordetellapertussis,muramyl dipeptide and saponin.Clin. exp. Immunol., 39,426-434. Butler, N.R., et al. (1969), Advantages of aluminium hydroxide adsorbedcombined diphtheria, tetanus, and pertussisvaccinesfor the immunization of infants. Brit. Med. J., 1, 663-666. Byars, N.E., et al. (1991),Improvementof hepatitisB vaccineby the useof a newadjuvant. Vaccine,9, 309-318. Dresser, D.W. (1986), Immunization of experimental animals,in “Handbook of experimentalimmunology in four volumes, 4. Ed., Volume 1: Immunochemistry,S. 8.1-8.21” (Weir, D.M.). Blackwell Scientific PubI., Oxford. Etlinger, H.M., et al. (1988),Assessment in humansof a syntheticpeptide-based vaccineagainstthe sporozoite stageof the humanmalariaparasite,Plasmodiumfalciparum. J. Immunol., 140, 626-633. Flebbe, L.M. & Braley-Mullen, H. (!986), Immunopotentiating effects of the adjuvants SGP and Quil A. I. Antibody responsesto T-dependent and Tindependentantigens.Cell. Immunol., 99, 119-127. Gonzalez, J., et al. (1991), Resistanceto acute Trypanosomacruzi infection resultingfrom immunization of mice with a 90-kilodalton antigen from metacyclic trypomastigotes.Infect. Immun., 59, 863-867. Goto, N. & Akama, K. (1982),Histopathologicalstudies of reactionsin mice injected with aluminium - adsorbed tetanus toxoid. Microbial. Immunol., 26, 1121-1132. Harlow, E. & Lane, D. (1988),Antibodies. A laboratory manual.Cold SpringHarbor Laboratory, New York. Herbert, W.J. (1978), Mineral-oil adjuvants and the immunization of laboratory animals,in “Handbook of experimentalimmunology in three volumes,3. Ed., Volume 3 : Application of immunologicalmethods, S. A3.1-A3.15” (Weir, D.M.). Blackwell Scientific Publ., Oxford. Herrington, D.A., et al. (1987),Safetyand immunogenicity in man of a synthetic peptide malaria vaccine againstPlasmodiumfalciparum sporozoites.Nature (Lond.), 328, 257-259. Hong, T.H., et al. (1989), The production of polyclonal and monoclonalantibodiesin mice usingnovel immunization methods. J. Immunol. Methods, 120, 151-157. Hu, J.-G. & Kitagawa, T. (1990), Studieson the optimal immunization scheduleof experimentalanimals.VI. Antigen dose-response of aluminum hydroxideaidedimmunizationand boostereffect underlow antigen dose. Chem. Pharm. Bull., 38, 2775-2779. Jarecki-Black,J.C., et al. (1988),Resistance againstLeishmaniadonovani inducedwith an aluminumhydroxide vaccine. Ann. Clin. Lab. Sci., 18, 72-77. Kruze, D., et al. (1989),Urinary antibody responseafter immunizationwith a vaccineagainsturinary tract infection. Ural. Res., 17, 361-366. Kuwert, E.K., et al. (1978),Antigenicity of low concentrated HDCS vaccine with and without adjuvant as comparedto the standardfluid formulation. Develop. Biol. Stand., 40, 29-34. Levine, L., et al. (1955), Factors affecting the efficiency of the aluminumadjuvant in diphtheria and tetanus toxoids. J. Immunol., 75, 301-307. Mannhalter, J.W., et al. (1991), Immunization of chim-

494

44th FORUM

IN IA4A4lJNOLOG

panzees with the HIV-l glycoprotein gpl60 induces long-lasting T-cell memory. AIDS Res. Hum. Retrovir., 7, 485-493. Mulira, G.L., et al. (1988), Efficacy of different adjuvants to potentiate the immune response to mycoplasma strain F-38. Trap. Anim. Hith Prod., 20, 30-34. Murray, K., et al. (1984), Hepatitis B virus antigens made in microbial cells immunise against viral infection. EMBO J., 3, 645-650. Nicholson, K.G., et al. (1979), Clinical studies of monovalent inactivated whole virus and subunit A/USSR/77 (H 1N 1) vaccine : serological responses and clinical reactions. J. biol. Stand., 7, 123-136. Osebold, J.W. (1982), Mechanisms of action by immunologic adjuvants. J. Amer. vet. med. Ass., 181, 983-987. Ramanathan, V.D., et al. (1979), Complement activation by aluminium and zirconium compounds. Immunology, 37, 881-888. Richards, R.L., et al. (1988), Liposomes, lipid A, and aluminum hydroxide enhance the immune response to a synthetic malaria sporozoite antigen. Infect. Immun., 56, 682-686. Sacco, A.G., et al. (1989), Effect of varying dosages and adjuvants on antibody response in squirrel monkeys (Saimiri sciureus) immunized with the porcine zona pellucida M, = 55,000 glycoprotein (ZP3). Amer. J. Reprod. Immunol., 21, 1-8. Sanchez, Y ., et al. (1980), Humoral and cellular immuni-

ty to hepatitis B virus-derived antigens : comparative activity of Freund complete adjuvant, alum, and Iiposomes. Infect. Immun., 30, 728-733. Scolnick, E.M., et al. (1984), Clinical evaluation in healthy adults of a hepatitis B vaccine made by recombinant DNA. J. Amer. med. Ass., 21, 2812-2815. Smithers, S.R., et al. (1989), Protective immunization of mice against Schistosoma mansoni with purified adult worm surface membranes. Parasite Immunol., 11, 301-318. Teerlink, T., et a/. (1987), Synergistic effect of detergents and aluminium phosphate on the humoral immune response to bacterial and viral membrane proteins. Vaccine, 5, 307-314. Thapar, M.A., et a/. (1990a), The effect of adjuvants on antibody titers in mouse vaginal fluid after intravaginal immunization. J. Reprod. Immunol., 17, 207-216. Thapar, M.A., et a/. (1990b), Secretory immune responses in mouse vaginal fluid after pelvic, parenteral or vaginal immunization. Immunology, 70, 121-125. Turk, J.L. &Parker, D. (1977), Granuloma formation in normal guinea pigs injected intradermally with aluminum and zirconium compounds. J. invest. Dermatol., 68, 336-340. Woodard, L.F. (1989), Adjuvant activity of water-insoluble surfactants. Lab. Anim. Sci.. 39. 222-225. Report of a WHO scientific group (1976), Immunological adjuvants. Technical report Series No. 595, World Health Organisation, Geneva 1976.

DDA as an immunological L.A.T.

Hilgers

Y

(‘1 and H. Snippe

adjuvant t2)

“’ Solvay S A., Central Laboratory, Applied Immunology, Rue de Ransbeek 310, B-1120 Brussels, and (2J Utrecht ‘University, Eijkman- Winkler Laboratorium for Medical Microbiology, Heidelberglaan 100, 3584 CX, Utrecht (The Netherlands)

Ger,eral

introduction

More than 25 years ago, Gall demonstrated that di(hydrogenated tallow) dimethylammonium chloride comprising various quaternary amines was an effective adjuvant for both humoral and cell-mediated immune responses (Gall, 1966). Most quaternary amines of this mixture demonstrated significant adjuvanticity, and dimethyldioctadecylammonium bromide (DDA) appeared to be the most active one. Since then, many investigators confirmed and extended these findings in different experimental models,

resulting in numerous publications of which most are cited in this review. DDA belongs to the group of lipophilic quaternary amines. It is a positively charged compound with a monovalent counterion and has a molecular weight of 63 1 D. Due to the presence of two long alkylchains it has a hydrophobic character and is poorly soluble in cold water. It is, however, dispersible in warm (> 40°C) water in which it forms liposomal structures (Carmona-Ribiero et al., 1983; CarmonaRibiero et al., 1984; Harada et al., 1984). Gel-liquid phase transition temperature was determined to be