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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

CHARACTERISTICS

AND

USE OF NE W-GENERATION

39.5”C (Harada el al., 1984). The counterion can be either Cl- or Br- and there are no indications that the anion affects the adjuvanticity. DDA is present in raw materials known as di(hydrogenated tallow) dimethylammonium salts (Arquad 2HT or Quaternium-18) with a typical alkylchain distribution of C12:C14:C16:C18 of 1:4:31:64. These quaternary amines are produced in large quantities and applied in cosmetic products and laundry softeners. Their affinity for negatively charged surfaces and for proteinaceous substrates are employed in antistatics and hair preparations (Reck, 1983). Dispersed in water, DDA can form complexes with multivalently negatively charged compounds, e.g. phosphate ions and polycarboxylates (Duphar, 1988). Adjuvant

activity for humoral

immune

responses

DDA stimulated humoral immune responses against antigens of distinct nature and complexicity (table I). Responses against whole cells, whole microbial organisms, viruses, proteins, chemically defined haptens conjugated to proteins, peptide-protein conjugates, synthetic antigens, etc. have been shown to be augmented by DDA. There are no indications that DDA is not effective with certain types of antigens. Most studies have been performed in laboratory animals and a few have been carried out in larger animals such as chickens (Rijke et al., 1988), pigs (Rijke et al., 1988; Gualandi et al., 1988; Dzata et al., 1991a, 1991b) and cattle (Thomas et al., 1986; Goubau et al., 1989; Roberts et al., 1990). Vaccines supplemented with DDA can be prepared by simply mixing an antigen solution with a freshly prepared solution of DDA. The effective dose range of DDA varies from tens to thousands of pg per animal which equalizes about 1 to 10 mg per kg body weight in small laboratory animals and from 0.01 to 1 mg per kg body weight in large animals. DDA stimulated preferentially IgG2a and IgG2b antibody titres (Katz et al., 1991). Despite the efficacy of DDA in many different models, comparative studies with different adjuvants demonstrated that other adjuvants are sometimes more effective in stimulating antibody responses. Examples are dextran sulphate for SRBC in mice (Hilgers et al., 1985a), saponin for antibody responses against sheep red blood cells (SRBC) in mice (Bomford, 1980) and against Trepanosoma cruzi cell surface glycoprotein in mice (Scott et al., 1984), AI( in an oil-in-water emulsion and Carbopol 934P for porcine parvovirus in pigs (Gualandi et al., 1988), Freunds’ incomplete adjuvant (FIA) and muramyl dipeptide for B. abortus antigen in cattle (Dzata et al., 1991b), Freund’s complete adjuvant (FCA) for humoral responses against gonadotropinreleasing hormone peptides in cattle (Goubau et al., 1989) and FIA, Ribi adjuvant system plus an oil-inwater emulsion and Quil-A plus an oil-in-water emul-

ADJUVANTS

495

sion for anti-luteinizing hormone antibody responses in cattle (Roberts et al., 1990), avridine for Newcastle disease virus in chickens (Rijke et al., 1988), and water-in-oil emulsion for pseudorabies virus in pigs (Rijke et al., 1988). A few studies with DDA have been performed in humans with tetanus toxoid (Veronesi et al., 1970; Stanfield et al., 1973) or autologous lymphoblasts as antigens (Chambers, 1980) and DDA further enhanced antibody response to tetanus toxoid adsorbed to aluminium hydroxide (Veronesi et al., 1970; Stanfield et al., 1973). Effect on cell-mediated

immune

responses

The number of adjuvants that can induce a cellmediated immune response is limited. DDA is very effective in inducing delayed type hypersensitivity (DTH) reactions in both laboratory animals (mice and guinea pigs) and larger animals (cattle) (table II). DDA enhanced the DTH response against various antigens including Brucella abortus antigens (Dzata et al., 1991a,b), keyhole limpet haemocyanin (Limpens and Scheper, 1989), bovine serum albumin (BSA; Gordon and Prager, 1980; Dailey and Hunter, 1977), Semliki Forest virus (Kraaijeveld et al., 1984), tetanus toxoid (Gall, 1966), arsonate-phosphatidylethanolamine conjugate (Hilgers et al., 1986c), Listeria monocytogenes (Willers et al., 1982), arsonateovalbumin (van Houte et al., 1981), SRBC (Chiba and Egashira, 1978) and dinitrophenylated BSA (Snippe et al., 1982b). In several separate studies, it was shown that DDA is at least as effective as FCA (Snippe et al., 1982a,b). Respecting the efficacy on the basis of the dose, DDA is about lOOO-fold more effective than FCA, since 100 pg of DDA were as effective as 100 mg of oil in FCA. Studies with several distinct adjuvants indicated that for DTH responses, no adjuvant was stronger than DDA of FCA. Effects of DDA on the activation of other parts of cellmediated immunity, e.g. T helper cells type 2 and cytotoxic T cells, are not known. Since a few other lipophilic adjuvants have been proven to be capable of stimulating cytotoxic T cell responses (Deres et al., 1989; Takahashi et al., 1990), DDA is worthwhile looking at. Interference

with other immunomodulating

agents

One of the most interesting aspects of well defined adjuvants is the activity of combination of different agents. In the literature, there are several examples of beneficial and disadvantagous collaboration of DDA with other adjuvants or immunomodulating compounds. DDA stimulated the antibody responses against tetanus toxoid adsorbed to aluminium hydroxide (Veronesi et al., 1970; Stanfield et al., 1973). Selenium administered in drinking water had

496

44th FORUM

Table I. Adjuvanticity

of DDA for antibody

Type of antigen

IN IiWMfNOLOGY

responses against various antigens in different animal species. Antibody

(Glyco)proteins (from) BSA Gonococcal protein I Brucella abortus Brucella abortus Tetanus toxoid Tetanus toxoid Tetanus toxoid Diphtheria toxoid Viruses Semliki Forest virus Encephalomyocarditis virus Porcine parvovirus Porcine parvovirus Porcine parvovirus Pseudorabies virus Newcastle disease virus Newcastle disease virus Newcastle disease virus Bovine viral diarrhoea

T T T T T T T T T/G1 /G2a/G2b T

Species

References

M M Ca Ca G Hu Hu G

Dailey, 1977 Jiskoot, 1986 Dzata, 1991a Dzata, 1991b Gall, 1966 Veronesi , 1970 Stanfield, 1973 Gall, 1966

M M G G Pi Pi Ch Ch M Ca

Kraaijeveld, 1980 Kraaijeveld, 1983 Molitor, 1984/1985 Gualandi, 1988 Gualandi, 1988 Rijke, 1988 Rijke, 1988 Katz, 1990 Katz, 1991 Thomas, 1986

Bacteria L. monocytogenes

T

M

Willers,

Mammalian SRBC SRBC SRBC SRBC Tumour

T T T T T

M M M M M

Bomford, 1980 Gordon, 1980 Hilgers, 1984 Hilgers, 1985 Prager and Gordon,

Hapten-carriers (including peptide-protein conjugates) Dinitrophenyl-tripeptide-BSA T Dinitrophenyl-BSA T Dinitrophenyl-BSA T Luteinizing hormone-OVA T Luteinizing hormone-KLH T Luteinizing-hormone-releasing hormone T Gonodatropin-releasing hormone-OVA T Gonodatropin-releasing hormone-ESA T

M G Ra Ca Ca M Ca Ca

Snippe, 1980 Snippe, 1982a Snippe, 1982b Roberts, 1990 Roberts, 1990 Silversides, 1988 Goubau, 1989 Goubau, 1989

Parasites Eimeria

Ch

Lillehoj,

1979

cells

cells

spp.

T

1980

1990

BSA = bovine serum albumin ; ESA = equine serum albumin ; T = total antibody titre ; SRBC = sheep red blood cell ; G = IgG ; G 1 = IgG 1; G2a=IgGZa; G2b=IgGZb; M=mice; G=guinea pigs; Ra=rabbits; Pi=pigs; Ca=cattle; Ch=chicken; Hu=humans.

CHARACTERISTICS

Table II. Adjuvanticity

AND

USE OF NE W-GENERATION

ADJUVANTS

497

of DDA for CM1 responses against various antigens after subcutaneous injection into different animal species.

Type of antigen

Response

Species

(Glyco)proteins (from) : BSA Lysozyme Keyhole limpet haemocyanin Listeria monocytogenes Brucella abortus Brucella abortus

DTH bTH DTH DTH DTH DTH

M M M M Ca Ca

Dailey, 1977 Kraaijeveld, 1986a Limpens, 1989 Antonissen, 1986a Dzata, 1991a Dzata, 1991b

Viruses Semliki Forest virus Encephalomyocarditis virus Infectious bronchitis virus Measles virus Pseudorabies virus

DTH DTH DTH DTH DTH

M M M M M

Kraaijeveld, 1980 Kraaijeveld, 1983 Rijke, .1983 Smith, 1986 Rijke, 1988

Bacteria L. monocytogenes L. monocytogenes L. monocytogenes

DTH DTH DTH

M M M

van der Meer, 1977 Willers, 1979 Willers, 1982

Mammalian cells SRBC SRBC SRBC SRBC SRBC Autologous lymphoblasts

DTH DTH DTH DTH DTH MLR

M M M M Rt Hu

Gordon, 1980 Chiba, 1978 Snippe, 1977 Snippe, 1981 Brocades Zaalberg, Chambers, 1980

Recombinant DNA antigens of Semliki Forest virus Infectious bronchitis virus

DTH DTH

M M

Snijders, 1989 Boots, 1992

Hapten-carriers Dinitrophenyl-BSA Dinitrophenyl-BSA Arsonate-BSA Sulphonate-BSA Dinitrophenyl-BSA Dinitrophenyl-BSA Dinitrophenyl-BSA Arsonate-PE Arsonate-PE Arsonate-PE liposomes Oligopeptide-beta-galactosidase

DTH DTH DTH DTH DTH DTH DTH DTH DTH DTH DTH

M M M $9 GP Ra M M M M

Dailey and Hunter, Snippe, 1978 Snippe, 1978 Snippe, 1978 Snippe, 1980 Snippe, 1982 Snippe, 1982 van Houte, 1981 Hilgers, 1984 van Houte, 1981 Snijders, 1992

CM1

Ch

Parasites Eimeria

spp.

(“)

References

Lillehoj,

1980

1974

1990

BSA = bovine serum albumin; ESA = equine serum albumin ; Pe = phosphatidylethanol amine; SRBC = sheep red blood cell ; DTH = delayed type hypersensitivity ; MLR = mixed lymphocyte reaction ; M = mice ; Gp = guinea pigs ; Ra = rabbits ; Rt = rats ; Ca = cattle ; Ch = chicken; Hu = humans. (*) Different mouse strains were tested. (**) Peptide of Semliki Forest virus conjugated to beta-galactosidase.

44th FORUM an additional effect on the protective activity of immunization with killed Plasmodium berghei antigen plus DDA (Desowitz and Barnwell, 1980). Dextran sulphate combined with DDA interacted synergistically in stimulating the antibody response against SRBC in mice (Hilgers et al., 1985a). The number of antibody-producing cells was increased more than 500-fold by the combination, while the constituting adjuvants evoked about 30- and 160-fold increases. In contrast, dextran sulphate decreased the adjuvanticity of DDA for DTH responses against a synthetic antigen (Hilgers et al., 1986~). Trehalose dimycolate plus DDA was more effective than DDA alone in inducing antibody titres against Brucella abortus antigen in cattle, but the effect of trehalose dimycolate alone was not measured (Dzata et al., 1991a). Treatment of mice with cyclophosphamide prior to immunization with antigen plus DDA augmented the DTH response (Smith and Ziola, 1986) and an additional rather than synergistic collaboration was observed. Local administration of cytostatic drugs, cyclophosphamide-derivative 27557 and the plant alkaloid VP-16 improved the adjuvanticity of DDA in a synergistic fashion (Limpens and Scheper, 1989). Thus, additional or synergistic effects can be observed with DDA and other adjuvants. The two interacting compounds can be injected in conjunction with one other (DDA plus DXS), or separately with respect to route (selenium and 27557 or VP-16) or time (cyclophosphamide).

Adjuvanticity

for local immune

responses

Only a few reports describe the effects of DDA on local responses. lntravaginal administration of horse ferritin induced a low IgA response in mice, but DDA did not enhance this response (Thapar et al., 1990a). Subcutaneous injection of the same antigen induced significantly higher IgA antibody titres in vaginal fluid and DDA was capable of stimulating the antibody titre (Thapar et al., 1990b). The DDA analogue N,N-dioctadecyl-N’,N’-bis(2-hydroxyethyl)-propanediamine (also known as CP-20,961 or avridine) displayed adjuvanticity for local responses against various antigens including SRBC (Anderson, MacDonald and Rubin, 1985), cholera toxin (Anderson, MacDonald and Rubin, 1985), procholeragenoid (Pierce and Sacci, 1984) and reovirus (Anderson, MacDonald and Rubin, 1985).

Adjuvanticity

for protective immunity

Induction of protective immunity is the ultimate goal of several vaccine development programs. DDA stimulated protection against various infection upon injection with the appropriate antigen (table Ill).

IN IA4MUNOLOGY Adjuvanticity

of analogues of DDA

Various other lipophilic amines displayed adjuvanticity, including primary, secondary and tertiary amines with alkyl chains of 10 or more C atoms, and quaternary di-alkyldimethyl-, aIkyltrimethyland alkylbenzyldimethylammonium-derivatives with 12 to 18 carbon atoms per alkyl chain (Gall, 1966, 1967). A lipophilic amine which has also attracted much attention as an adjuvant is avridine (review, Jensen, 1986). In chemical respects, it differs from DDA as it is a diamine with two tertiary amines and two alkyl chains 18 carbon atoms. Effect upon non-specific protection A unique property of DDA is the induction of protective immunity against infections with related and unrelated bacteria by combinations of DDA with ribosomal RNA of Listeria monocytogenes (Antonissen et al., 1985) or Pseudomonas aeruginosa (Gonggrijp et al., 1985). The mechanism underlying this phenomenon was thought to be T-cell-independent activation of macrophages (Van den Bosch et al., 1986). Modes of action Physicochemical interaction of DDA with different types of antigens including red blood cells (Baechtel and Prager, 1984), proteins (Dailey and Hunter, 1977) and synthetic antigens (Hilgers et al., 1986~) has been demonstrated. The relationship between degree of binding of DDA to the synthetic antigen arsonate-phosphatidylethanolamine conjugate and immunogenicity (Hilgers et a/., 1986b) and the absence of adjuvant activity if DDA and antigen were administered by different routes (Hilgers et al., 1985a) suggest that binding is crucial. In contrast, other observations indicate that physical association of DDA with certain antigens is not an absolute requirement . Antibody responses against proteins were enhanced by DDA administered via a separate route (Smith and Ziola, 1986) and responses against SRBC were augmented by giving DDA 6 h earlier via the same route (Hilgers et al., 1984). In the latter experimental models, it cannot be excluded, however, that antigen and adjuvant form complexes in the body after trapping into the same compartment, i.e. lymphoid organs and cells. Binding of DDA to antigens results in two effects, namely increased overall hydrophobicity of the antigen and neutralization of negatively charged moieties. Covalent conjugation of lipid groups to proteins augmented the immunogenicity for DTH-responses (Coon and Hunter, 1973 ; Daily and Hunter, 1974) supporting the suggestion that increasing the hydrophobicity of an antigen is

CHARACTERISTICS

Table III. Adjuvanticity

AND

USE OF NE W-GENERA

of DDA for protective immunity

Type of antigen

Nucleic acids; ribosomal

RNA

499

against various antigens in different animal species.

Protection

(Glyco)proteins (from) : Plasmodium berghei Trepanosoma cruzi

TION ADJUVANTS

Species

References

+

M M

Desowitz, 1980 Scott, 1984

+ +

M M

Antonissen, 1986a,b Gonggrijp, 1983

M M M M Ch

Kraaijeveld, 1983 Kraaijeveld, 1983 Rijke, 1988 Rijke, 1988 Rijke, 1988

M M M M M M

Willers, 1979 Willers, 1980 Gonggrijp, 1985 van der Meer, 1980 van der Meer, 1977 Woodward, 1980

of

Listeria monocytogenes Pseudomonas aerigunosa Viruses

Semliki Forest virus Encephalomyocarditis virus Rabies virus Pseudorabies virus Newcastle disease virus Bacteria L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes Brucella abortus Parasites Plasmodium yoelli Eimeria spp.

+

McColm, Lillehoj,

1982 1990

M = mice; Ch = chicken.

important to immunostimulation. On the other hand, it has been demonstrated that the immunogenicity of a protein can also be increased by neutralization of negatively charged groups by reaction of carboxylic groups with diamines (Muckerheide et al., 1987). DDA influences various components of the host immune system. Intraperitoneal injection of DDA induced an influx of cells into the peritoneal cavity, including polymorphonuclear cells, during the first 6 h and mononuclear phagocytes from 24 to 96 h (Gonggrijp et al., 1985; Hilgers et al., 1985b). The total activity of the phagocytic system was stimulated 24 h after administration (Willers et al., 1979; Bloksma et al., 1983) and returned to normal levels. The inflammatory reaction elicited by DDA is affirmed by the appearance of serum amyloid P protein in the blood 24 h after injection (Hilgers et al., 1988). Transfer experiments demonstrated no so-called transfer factor (considered to be interleukin-1) as demonstrated for lipopolysaccharides and suggested an alternative pathway for the induction of serum amyloid protein P (SAP). Comparison of results of

studies in different mouse strains suggested a relationship between levels of SAP induced by DDA (Hilgers et al., 1988) and adjuvanticity for DTH responses (Snippe et al., 1980a) but not for humoral immune responses (Hilgers et al., 1988). Injection of high doses of DDA resulted in the induction of low but significant levels of interferon in both serum and peritoneal cavity (Kraaijeveld et al., 1982). Stability at low pH suggested interferon of the alpha or beta type. In vitro, DDA caused a time- and dosedependent reduction of overall complement activity and of activation of the complement system via the alternative pathway (Klerx et al., 1988). Toxicity

and safety of DDA

Little is known about the toxicity of DDA. Parenteral administration induced a local inflammatory reaction with slight, transient swelling. Severe local or systemic side effects were not observed. Quaternium-18 comprising various lipophilic quater-

500

44th FORUM

IN Ih4MUNOLOGY

nary amines including DDA is considered to be a potential eye and skin irritant (Reck, 1983). Solution of 2 to 10 % quaternium-18 applied for 21 consecutive days induced mild irritation (Reck, 1983). Acute toxicity (LD,,) of quaternium-18 in rats is > 0.5 g per kg body weight. Chronic oral toxicity studies in dogs and rats revealed that dietary levels of 2,800 ppm for 90 days did not result in abnormalities in food consumption, body weight, reaction, mortality or urinalysis, nor in haematologic, blood chemistry, gross pathology, or histopathology (Armak Co, 1973). Administration of Arquad 2HT comprising DDA in humans did not induce apparent side effects (Veronesi et al., 1970; Stanfield et al., 1973; Chambers et al., 1980). The limited toxicity of the combinations of quaternary amines which contain considerable amounts of DDA suggests that DDA is relatively safe.

Summary and conclusions As compared to other adjuvants, DDA is a moderate or strong adjuvant for humoral responses and a strong adjuvant for CMI, especially DTH responses, against different types of antigens and in both laboratory animals and larger animals. DDA can collaborate with other immunomodulating compounds resulting in further enhanced responses. Mechanisms include interactions with both antigen and components of the host immune system and possibly, multiple beneficial effects contribute to the relatively strong adjuvanticity of DDA. Toxicity of DDA is not known but severe detrimental side effects were not seen. This adjuvant can be applied in experimental vaccines and in commercial vaccines for veterinary purposes, especially if cell-mediated immunity is considered to be important. In immunology, DDA can be of use to study T helper cells responsible for DTH responses (T helper cells type 1) and to characterize T helper cell epitopes on antigens (Snijder et al., 1992).

References Anderson, A.O., MacDonald, T.T. & Rubin, D.H. (1985),

Effect of orally administeredavridine on enteric antigen uptake and mucosal immunity. Inf. J. Immunother., 1, 107-I15. Antonissen,A.C. J.M., Lemmens,P.J.M.R., Gonggrijp, R., van denBosch,J.F. & van Boven, C.P.A. (1985), RNase-sensitive andRNase-insensitive protectivecomponentsisolatedfrom Listeria monocytogenes.Anhoniev. Leeuwenhoek,51, 227-240. Antonissen, A.C. J.M., Lemmens,P.J.M.R., van den Bosch,J.F. &van Boven, C.P.A. (1986a),Dissocia-

tion between enhanced resistance and delayed hypersensitivityinducedwith subcellularpreparationsfrom

Listeria monocytogenesand the adjuvant dimethyldioctadecyl-ammonium-bromide. Antonie v. Leeuwenhoek,52, 75-84. Antonissen, A.C.J.M., Lemmens, P.J.M.R., van den Bosch, J.F. &van Boven, C.P.A. (1986b),Transfer of enhancedresistance againstListeriamonocytogenes induced with ribosomalRNA and DDA. Immunol. Letters, 14, 21-28. Armak Co. (1973), Product data bulletin No. 73-6. Baechtel,F.S. & Prager, M.D. (1982),Interaction of antigenswith dimethyldioctadecylammonium bromide,a chemically defined biological responsemodifier. CancerRes., 42, 4959-4963. Bioksma,N., de Reuver, M. J. & Willers, J.M.N. (1983), Impaired functions as a possible basis of immunomodificationby microbial agents,tilorone and dimethyldioctadecylammonium bromide.Antonie, v. Leeuwenhoek,49, 13-22. Bomford, R. (1980),Saponinand other haemolysins(vitamin A, aliphatic amines,polyeneantibiotics) asadjuvants for SRBC in the mouse.Int. Arch. Allergy, 63, 170-177. Boots, A.M.H., Benaissa-Trouw,B.J., Hesselink,W., Rijke, E., Schrier, C. & Hensen,E.J. (1992), Induction of immunity to avian coronavirusIBV by immunization with recombinant-DNA encoded nucleocapsidprotein. Vaccine, 10, 119-124. Van den Bosch, J.F., Kanis, I.Y.R., Antonissen, A.C.J.M., Buurman, W.A. & van Boven, C.P. (1986), T-cell-dependentmacrophageactivation in mice induced with rRNA from Lisferia monocyfogenesand dimethyldioctadecylammonium bromide. Infecf. Immun., 53, 611-615. BrocadesZaalberg,0. & Gerbrandy, J.L.T. (1980),Evaluation of immunologicmethodsfor toxicity studiesin animals. Bull. Medical Biological Lab. TNO, Rijswijk, The Netherlands. Carmona-Ribeiro,A.M. & Chaimovich,H. (1983),Preparation and characterization of large dioctadecyldimethylammonium chloride liposomes and comparisonwith smallsonicatedvesicles.Biochim. biophys. Acta (Amst.), 733, 172-179. Carmona-Ribeiro,A.M., Yoshida, L.S., Sesso,A. & Chaimovich, H. (1984), Permeability and stabilities of large dihexadecylphosphate and dioctadecyldimethylammoniumchloride vesicles.J. CON.Znferface Sci., 100, 433-443. Chambers,J.D., Thomas,C.R. & Hobbs, J.R. (1980),Induction of specifictransplantation tolerancein man by autoblast immunization. Blut, 41, 229-236. Chiba, J. & Egashira,Y. (1978), Adjuvant effect of cationic surface-activelipid, dimethyl dioctadecyl ammonium bromide, on the induction of delayed-type hypersensitivityto sheepred blood cellsin mice. Jap. J. med. Sci. Biol., 37, 361-364. Coon, J. & Hunter, R.L. (1973). Selectiveinduction of delayedhypersensitivityby a lipid conjugatedprotein antigenswhich is localizedin thymusdependentlymphoid tissue.J. Immunol.. 110, 183-190. Dailey, M.O. & Hunter, R.L. (1974),The role of lipid in the induction of hapten-specificdelayedhypersensitivity and contact sensitivity. J. Immunol., 122, 1526-1534. Dailey, M.O. & Hunter, R.L. (1977). Induction of cellmediatedimmunity to chemicallymodified antigens

CHARACTERISTICS

AND

USE OF NEW-GENERATION

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Adjuvant properties of stable water-in-oil emulsions : evaluation of the experience with Specol W.J.A. Boersma cl), W.J.C. Bogaerts (I), A.T.J. Bianchi t2) and E. Claassen (l) (‘) Dept. of Immunology and Medical Microbiology, TN0 Medical Biological Laboratory, PO Box 45, 2280 AA Rijswijk (The Netherlands), and (2’ Central Veterinary Institute, Department of Immunology, Lelystad (The Netherlands)

Introduction We will discuss in this paper the various modalities of the use of oil-based adjuvants with emphasis on stable water-in-oil emulsions (WIO). The ability of oil-based adjuvants to support humoral (level, affinity, isotype) as well as cellular immune responses and memory induction leading to protective vaccination is evaluated. Emphasis is laid on the use of the oil-based adjuvant “Specol”, the formulation of which is based on constituents which all are approved for animal use by the USA Food and Drug Administration. The relationship between specific formulation of adjuvants and the effects on the well-being of experimental animals is discussed.

Composition, physical and chemical properties of oilbased adjuvant formulations A search among international patents revealed a number of physical and chemical parameters that are of importance for establishing a functional adjuvant formulation. WI0 formulations of various kinds have been registered. Most are rather broadly defined. Oil phases of 20-90 ‘70 and water or hydrophilic phases of lo-80 ‘70 are often described. The antigen to which an immune response has to be elicited is, in general, taken up in the hydrophilic phase. In practice, however, most adjuvants contain over 40 Vo (v/v) oil phase. The oil phase consists mainly of three types of