Online Issues

<< All Back-issues

<< This Issue's Table of Contents

ILAR Journal V37(3) 1995
Adjuvants and Antibody Production

Review of Selected Adjuvants used in Antibody Production
Veronica M. Jennings
Veronica M. Jennings, D.V.M., has completed her residency in laboratory animal science and is now a graduate student in immunology at Emory University, Atlanta, Georgia.

INTRODUCTION

Immunologic adjuvants are agents that nonspecifically increase immune responses to specific antigens that are weakly immunogenic. The purpose of this article is to inform investigators and research staff about three commonly used and commercially available adjuvants for antibody production in laboratory animals and to briefly cover other adjuvants that may be used for this purpose. The three adjuvants that will be emphasized in this article are Freund's-type mineral oil adjuvant emulsions, Ribi Adjuvant System, and TiterMax®. Other reports have studied these adjuvants, or combinations thereof, when administered with specific antigens (Lipman et al., 1992; Smith et al., 1992; [Johnston et al., 1991).

Whether used for research or production of antisera or for developing vaccines, the objective of using adjuvants differs greatly. Adjuvants intended for production of antisera need to induce high titers of high avidity antibody within a short time. Adjuvants intended for vaccine use need only induce protective titer, although the duration of the response and induction of immunologic memory are critical for maintaining protection. Induction of cell-mediated immunity is a requirement for protection against many etiologic agents, but is also partly responsible for side effects of adjuvants.

Adjuvants or immunopotentiators were initially thought of as agents capable of promoting an augmented and more sustained antibody response. However, new evidence has shown that adjuvants influence the titer, duration, isotype, and avidity of antibody, as well as affecting properties of cell-mediated immunity (CMI) (Table 1) (Hunter et al., l995). For example, adjuvants have been shown to induce class I-restricted CD8-positive cytotoxic T lymphocytes and modulate the specificity of antibody among available epitopes on protein antigens (Takahashi et al., 1990; Kenney et al., 1989).

Adjuvants can be been categorized according to their origins (whether they are derived from mineral; bacterial; plant; synthetic; or host product, such as Interleukin 1 and 2), and according to their proposed mechanism of action. Certain adjuvants such as aluminum compounds, oil emulsions, liposomes, and synthetic polymers act through the effect of antigen localization ("depot" effect), which leads to slow delivery of the antigen. Most adjuvants also induce complex cell interactions between macrophages and lymphocytes.

Investigators and research staff should not overlook the pain and distress that adjuvant-related protocols can cause in laboratory animals. Most of these protocols are accompanied by undesirable side effects that range in severity and duration and are usually caused by the adjuvant. To date, there is no agreement as to the level of discomfort adjuvants induce in laboratory animals with respect to the U.S. Department of Agriculture classification scheme on pain and distress. Aside from general signs of discomfort such as anorexia, weight loss, and lethargy, underlying conditions such as irritation at the site of injection and abdominal distention due to ascites production in mice require veterinary care and monitoring. If neither of these measures can be provided, affected animals should be euthanized immediately according to an approved method.

FREUND'S COMPLETE AND INCOMPLETE ADJUVANTS

In 1937, Jules Freund reported the discovery of elevated titers in tuberculous guinea pigs injected with sheep erythrocytes (Freund et al., 1937). This finding led him to examine the effect of emulsifying an aqueous solution of the antigen ovalbumin in paraffin oil containing killed tubercle bacilli with the aid of a surfactant. After further development, this resulted in Freund's complete adjuvant (FCA), a water-in-oil emulsion of mineral oil, mannide monooleate (a surfactant), and heat-killed Mycobacterium tuberculosis organisms or components of the organism (Freund, 1951). Some commercial preparations contain M. butyricum instead of M. tuberculosis (Stewart-Tull, 1995). FCA is a potent adjuvant that stimulates both humoral and cell-mediated immunity, and preferentially induces antibody against epitopes on denatured proteins. However, its toxicity has limited its use to laboratory animals only. The toxicity occurs because the mineral oil cannot be metabolized and the mycobacterial elements can elicit severe granulomatous reactions.

Freund's incomplete adjuvant (FIA) is the same as FCA, except that killed mycobacterial cells or cellular components are absent. Both FCA and FIA contain the antigen in the aqueous phase of the emulsion. FIA is less effective for inducing high antibody titers and enhancing cell-mediated immunity than FCA, but it is still an effective adjuvant and is much less toxic than FCA. In fact, its use in human vaccines is being revived.

Water-in-oil emulsions sequester the antigen and release it over long periods, up to 6 months or more after injection, which induces high titers as long as the antigen is stable. This is an important factor to consider when planning booster schedules. Also, because these emulsions contain antigen in the aqueous phase, it is important to know the chemical nature of the antigen being used. The depot effect is the hallmark of these types of emulsions, but other complex effects on cells of the immune system, such as nonspecific stimulation and alteration on cellular interactions, also occur. Furthermore, FCA not only localizes the antigen to its initial depot, but also disseminates it to lymph nodes, which may be more important for its adjuvant activity (Waksman, 1979). FCA is normally administered with equal volumes of antigen, that is, one part FCA or less with one part antigen. The concentration of mycobacteria varies greatly among commercial brands; if kept at 0.5 mg/ml or lower, less severe inflammatory reactions result. A general rule is that FCA should be used only for weakly immunogenic antigens and only for initial immunizations. FIA should be used for subsequent immunizations. Numerous reports of animal studies in which FCA was administered using multiple subcutaneous injections have shown not only that severe systemic reactions and local ulcerations are likely, but also that immune response to protein antigens can be decreased rather than increased (Stewart-Tull, 1995).

The biological activities of FCA and FIA have to do in part with the stable emulsions they form when properly prepared. This provides the slow release of antigen and also protects it from rapid degradation. In addition, FCA and FIA may contain substances that directly stimulate the immune system (Osebold, 1982). The inclusion of heat-killed mycobacteria in FCA induces the aggregation of macrophages at the site of injection and culminates in a delayed-type hypersensitivity (DTH) granulomatous reaction which, on occasion, may metastasize. Therefore, the combination of the nonmetabolizable mineral oil and the mycobacterial elements make FCA too toxic for use other than in a preclinical setting, although there has been increasing pressure to discontinue its use altogether.

RIBI ADJUVANT SYSTEM

It has been recognized since the nineteenth century that certain microbes and microbial products, especially mycobacteria and lipopolysaccharides (LPS), can be nonspecifically immunostimulatory (Nauts et al., 1946; Mastrangelo and Berd, 1982). Advances in cellular immunology, microbial physiology, and vaccine development have since led to a better understanding of how these substances act as adjuvants and to the development of Ribi Adjuvant System (RAS). Unlike Freund's adjuvants, Ribi adjuvants are oil-in-water emulsions in which the antigen is blended with a minimal volume of oil and these droplets are then emulsified in a saline solution containing the surfactant Tween 80 (Ribi et al., 1975). Although oil-in-water emulsions are less viscous than water-in-oil emulsions and, therefore, easier to inject, they are rather poor adjuvants when given alone. For this reason, immunostimulators are added to enhance the immunogenicity of the emulsion (Altman and Dixon, 1989). Some of these agents include two refined products from mycobacteria, trehalose 6,6'-dimycolate (TDM) and cell wall skeleton (CWS), and a purified gram-negative bacterial product, monophosphoryl lipid A (MPL®).

Among the first microbial products investigated for immunostimulant activity were components of the tubercle bacillus and other related mycobacteria (Bekierkunst et al., 1969). A series of biochemical studies ultimately led to the identification and isolation of TDM, also known as cord factor, which has adjuvant properties (Noll et al., 1956). Ribi adjuvants contain this high-molecular weight glycolipid, which is extracted from saprophytic, noncording M. phlei cells (Rudbach et al., 1995). CWS is a mycobacterial cell wall extract in which the loosely bound lipids, proteins, and carbohydrates have been removed. The remaining compound is a potent immunostimulator that works independently of free TDM (Azuma et al., 1974). The immunostimulatory activities of CWS can be attributed in part to the polymerized form of muramyl dipeptide (MDP) that it contains (Vosika, 1983). MDP is largely responsible for the adjuvant activity of whole mycobacterial cells. It has a wide variety of effects on immune responses, including enhancement or suppression of antibody production, depending on time of antigen administration; enhanced cell-mediated immunity; increased nonspecific immunity to bacteria, viruses, fungi, and parasites; stimulation of natural resistance to tumors; induction of autoimmunity; and increased cytokine release (Johnson, 1994).

LPS is a component of endotoxin of gram-negative bacteria, such as Escherichia coli, which among other effects induces shock. Use of LPS as an adjuvant stems from observations that Haemophilis pertussis enhances immune responses to diphtheria vaccines and tetanus toxoid (Landy et al., 1955; Webster et al., 1955). LPS, a complex amphipathic molecule, is a component of the outer membrane of gram-negative organisms and is a particularly potent adjuvant for protein antigens (Johnson et al., 1956). Both humoral and cell-mediated immunity can be enhanced by LPS, but the severe toxicities of endotoxins render them unacceptable for human use. One component of LPS, a covalently bound lipid, "lipid A," is responsible for most of the biological activities of LPS (Takada and Kotani, 1989). Lipid A, and certain synthetic derivatives with diminished toxicity resulting from the substitution of fatty acids and removal of phosphate groups, increase humoral and cell-mediated immune responses (Chiller et al., 1973; Kotani et al., 1986). MPL® (monophosphoryl lipid A) is a key component of most Ribi adjuvants (Table 2).

Three formulations of RAS are commercially available for either polyclonal or monoclonal antibody production: (1) TDM emulsion recommended for use with strong immunogens in mice, guinea pigs, and rats; (2) MPL® + TDM emulsion recommended for use in mice, guinea pigs, and rats; and (3) MPL® + TDM + CWS emulsion recommended for use in rabbits, goats, and large animals. The components of each are contained in a metabolizable oil, squalene.

The biological activities of Ribi adjuvants can be attributed to several factors, some of which are specific and others non-specific. MPL® and LPS-like materials are amphipathic and interact with membranes of cells of the immune system (Morrison and Rudbach, 1981). LPS and TDM bind proteins weakly; however, LPS interacts with specific receptors, and activates multiple cytokines and other mediators of toxic reactions (Morrison, 1990). LPS is also a B-cell mitogen, which induces production of tumor necrosis factor (TNF). The interaction of LPS and MPL® with cells results in production of interleukin 1 (IL-1), IL-2, IL-6, IL-8, TNF, granulocyte-macrophage colony-stimulating factor (GMCSF), and gamma-interferon (gamma-IFN) (Henricson et al., 1990). These cytokines enhance antigen uptake, processing, and presentation, which facilitates cellular interactions and immunostimulation (Unanue and Allen, 1987). Furthermore, MPL® negatively regulates antigen-activated suppressor T cells (Baker, 1988). Trehalose diester, such as TDM, when emulsified with oil, binds antigen to oil droplets, enhancing their uptake by macrophages (Adam et al., 1973). Finally, CWS affects macrophages in two ways: lysozyme-mediated digestion of CWS peptidoglycan results in release of soluble MDP particles, which are immunostimulants, and CWS contains high concentrations of mycolic acids, which are also immunostimulatory (Ribi et al., 1978). RAS is usually less potent than FCA, but is less toxic and has satisfactory adjuvant activity for many purposes.

TITERMAX®

Studies on the role of lipids in the induction of delayed-type hypersensitivity (DTH) reactions led to the discovery of nonionic block copolymers in the early 1980s (Hunter et al., 1981). This work deviated from the traditional and dominant paradigm of modern biology, which views biological functions as being controlled to a large extent by specific receptor-ligand interactions. The novel conclusion in this case was that surface free energy, a non-specific property, can influence biological reactions in diverse ways. These copolymers are a special type of surfactant composed of linear blocks or chains of hydrophobic polyoxypropylene (POP) and hydrophilic polyoxyethylene (POE) in different proportions (Hunter and Bennett, 1986). A broad range of surface-active properties can be obtained by varying the size of POP blocks and the ratio of the two components. Because the structure of nonionic block copolymers is flexible, is weak in hydrophobic activity, has limited capacity for intermolecular interactions, and lacks charged groups, these copolymers are less toxic than other surface-active agents (Hunter et al., 1995). High molecular weight copolymers with hydrophilic moieties flanking hydrophobic moieties form a particular type of adhesive surface and tend to have potent adjuvant properties by favoring chemotaxis, complement activation, and antibody production (Hunter and Bennett, 1986; 1984). The copolymer CRL-8941 is the immunomodulator component of TiterMax® (Table 2).

TiterMax® adjuvant forms a microparticulate water-in-oil emulsion with CRL-8941 and squalene, a metabolizable oil. CRL-8941 is coated with silica particles, which stabilizes the emulsion. This stability is a key property of TiterMax® in that it enables the emulsion to contain a wide variety of antigens without the use of large amounts of toxic emulsifying agents. No sugars, fatty acids, or any other biologically derived materials are incorporated. Some of the antigens that have been tested with TiterMax® include proteins, peptides, polysaccharides conjugates, whole killed viruses, and recombinant proteins (Kalish et al., 1991; van-de-Wijgert et al., 1991; Byars and Allison, 1987).

The most basic biological activity of TiterMax®, of which there are several, is that copolymers are able to concentrate more antigen on their surface than can be achieved by comparable concentrations of free antigen in solution. This occurs because the particular surface active characteristics of copolymers allow them to form special adhesive surfaces that are necessary for adjuvant activity. Copolymers with adjuvant properties are insoluble and able to form hydrophilic surfaces by the action of the hydrophilic moieties displacing nearly all of the hydrophobic moieties from the aqueous surface. These surfaces have been shown to activate complement, which influences the localization and retention of antigen in lymphoid tissue and the activation of immunoreactive cells (Hunter and Bennett, 1984). These copolymers also induce increased expression of class II (Ia) major histocompatibility molecules (MHC) on macrophages, resulting in an increased ability to present antigen to T cells (Howerton et al., 1990). Therefore, the antigen bound by adjuvant copolymers on the surface of oil droplets is presented to cells of the immune system in a highly concentrated form. Overall, TiterMax® usually induces antibody titers as high or higher than FCA and with less toxicity.

OTHER ADJUVANTS

Aluminum compounds and liposomes are two nonbacterial types of adjuvants that have been used primarily for vaccine development. Alum is the only adjuvant approved for use in human vaccines and is also used in some veterinary vaccines. It is occasionally used in laboratory animal protocols as an adjuvant for antibody production. These compounds appear to induce their effect by the slow release of antigen, as well as by other immunostimulatory properties such as attraction of immunocompetent cells to the injection site (Warren et al., 1986). Alum has not been widely used for production of antiserum because it is a relatively weak adjuvant for many antigens and it is deceptively difficult to prepare. Antigens, normally proteins, are physically precipitated with hydrated insoluble salts of aluminum hydroxide or aluminum phosphate. This process can be critically influenced by diverse factors including the concentration of materials, presence of salts, and temperature.

Liposome-type adjuvants are spheres consisting of phospholipid bilayers separated by an aqueous compartment. The charge, composition, method of preparation, and number of layers appear to influence the adjuvant activity, depending on the antigen. Incorporation of protein antigens within such spheres not only protects the antigen from rapid degradation, but also creates a depot effect (Altman and Dixon, 1989). Furthermore, liposomes mobilize the antigen from the area of injection to the draining lymph nodes, where they interact with antigen-presenting macrophages (Allison and Gregoriadis, 1976). Incorporating antigens within liposomes can enhance both humoral and cell-mediated immunity, and adding other immunostimulatory agents, such as LPS and MDP, can further increase their immunogenicity (Sanchex et al., 1980; Desiderio and Campbell, 1985). As with alum compounds, liposomes are undesirable candidates for antibody production because they are difficult to prepare and are not very effective with most antigens.

Adjuvants that are surface-active agents rely on surface free energy of cells to bind to hydrophobic surfaces and bring about their immunostimulatory properties (Allison, 1979). Lipoteichoic acid of gram-positive organisms, lipid A, and TDM are some of the most potent natural surface-active adjuvants known. Reduction in their adjuvant activity has been associated with alterations in structure that leads to disturbances in surface-active properties. On the other hand, Quil A and QS-21 (saponin-type adjuvants), monophosphoryl lipid A, and lipophilic MDP derivatives have hydrophilic and hydrophobic domains from which their surface-active properties arise (Hunter et al., 1995). Compounds normally found in the body such as vitamin A and E, and lysolecithin are also considered to be surface-active agents. Other classes of proposed adjuvants include glycan analog, coenzyme Q, amphotericin B, dimethyldioctadecylammonium bromide (DDA), levamisole, and benzimidazole compounds (Bomford, 1980). None of these are commonly used for production of antisera due to either lack of efficacy or lack of experience.

PAIN AND DISTRESS

FCA has been used in a wide variety of laboratory animals for over 50 years, and there is no question that it can induce chronic pain in both small and large animals (Amyx, 1987). Researchers have been reluctant to discontinue use of FCA because it is used as a gold standard due to its well known effectiveness with a wide variety of antigens. Therefore, it is important to be familiar with its side effects. Severe clinical pain has been reported in cases of accidental FCA injections (Chapel and August, 1976), and in cancer patients injected with autologous tumor extract in FCA (Hughes et al., 1970). In persons previously infected with tuberculosis, accidental injections of even very small amounts of FCA can produce painful lesions that persist for months. The painful reactions were attributed to tuberculin sensitivity. Therefore, in accordance with the U. S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training (REF), "unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings may cause pain or distress in other animals" (PHS, 1986). Other official guidelines for use of FCA can be obtained from the National Institutes of Health (Grumpstrup-Scott and Greenhouse, 1988) and the Canadian Council on Animal Care (CCAC, 1990).

GRANULOMA FORMATION AT INJECTION SITES

In 1954, Suter and White recognized that peptidoglycolipid from M. tuberculosis in a water-in-oil emulsion injected into the footpad of guinea pigs caused accumulation of epithelioid macrophages (Suter and White, 1954). Since then, similar inflammatory reactions have been described for intradermal, subcutaneous and intramuscular injections; granulomatous inflammation is consistently present at the sites of inoculation of either FCA or IFA (Broderson, 1989). In rabbits, intradermal FCA injections commonly cause ulcerations of the skin, and subcutaneous injections produce granulomas at both the injection site and adjacent or distant sites to which the inoculum has migrated. Draining abscesses can also occur at such sites. Intramuscular FCA injections can cause granuloma formation and necrosis of adjacent areas, and "metastatic" granulomas may be found in lymph nodes, lung, kidney, and spleen, depending on the distribution of the emulsion via the lymphatics and circulatory system (Broderson, 1989). These side effects may culminate in wasting and even death.

ADJUVANT ARTHRITIS

FCA-induced arthritis has been reported in small and large animals. The severity of the lesion varies, and can be very debilitating, especially in larger animals. Lewis rats injected with heat-killed mycobacterial cells suspended in mineral oil develop polyarthritis resulting in the term "adjuvant arthritis" (Pearson, 1956; Pearson and Wood, 1959; [Waksman et al., 1960). F344 and Buffalo rats are less susceptible. One case report describes posterior paresis in guinea pigs caused by inadvertent injections of FCA-antigen mixture into the paraspinal musculature, causing granulomatous infiltration into the spinal canal, which impinged on the spinal cord (Kleinman et al., 1993). TiterMax®-induced inflammatory reactions have been occasionally observed beginning at 2 or 3 weeks postinjection, intensifying for about another week, and then subsiding. High doses of adjuvant-antigen mixtures per site and toxic antigens may be associated with increased severity of the lesion. By itself, the toxicity of TiterMax® is comparable to that of FIA, which is much less than that of FCA and can be used in people. These lesions appear to be due to local antigen-antibody complement (Arthus) reaction rather than hypersensitivity granulomas typical of FCA. In subcutaneous injections, the reaction may resemble an abscess. Therefore, the toxicity of certain antigens may be exacerbated by TiterMax® (Pearson, 1956)). Arthus reactions have also been observed at sites of injection with RAS, although, being an oil-in-water emulsion, such reactions tend to be less severe due to brief retention in the area.

CONCLUSION

No one adjuvant works best with all antigens, animal species, or experimental conditions. Each adjuvant has advantages and disadvantages, and the antigen's properties must also be taken into account to select the adjuvant most likely to give the best results. Although FCA has been considered necessary because of the lack of effective alternatives, this is no longer true. Alternative adjuvants are now available that produce high antibody titers, in some cases exceeding those obtained with FCA and with less severe side effects.

ACKNOWLEDGMENT

The author would like to thank Dr. Robert L. Hunter for his assistance and guidance in the writing of this manuscript.

REFERENCES

Adam, A., R. Ciorbaru, J. F. Petit, et al. 1973. Preparation and biological properties of water-soluble adjuvant fractions from dilapidated cells of Mycobacteria smegmatis and Nocardia opaca. Infect. Immun. 7:855-861.

Allison, A. 1979. Mode of action of immunological adjuvants. J. Reticuloendothel. Soc. 26:619-630.

Allison, A. C., and G. Gregoriadis. 1976. Liposomes as immunological adjuvants. Recent Results Cancer Res. 56:58-64.

Altman, A., and F. J. Dixon. 1989. Immunomodifiers in vaccines. Adv. Vet. Sci. Comp. Med. 33:301-343.

Amyx, H.L. 1987. Control of animal pain and distress in antibody production and infectious disease studies. J. Am. Vet. Med. Assoc. 191:1287-1289.

Azuma, I., E. Ribi, T. J. Meyer, and B. Zbar. 1974. Biologically active components from mycobacterial cell walls: I. Isolation and composition of cell wall skeleton and component P3. J. Natl. Cancer Inst. 52:95-101.

Baker, P. J., J. R. Hiernaux, M. B. Fauntleroy, et al. 1988. Inactivation of suppressor T-cell activity by nontoxic monophosphoryl lipid A. Infect. Immun. 56:1076-1083.

Bekierkunst, A., I. S. Levij, E. Yarkoni, et al. 1969. Granuloma formation induced in mice by chemically defined mycobacterial fractions. J. Bacteriol. 100:95-102.

Bomford, R. 1980. The comparative selectivity of adjuvants for humoral and cell-mediated immunity. Clin. Exp. Immunol. 39:426-434.

Broderson, J. R. 1989. A retrospective review of lesions associatedwith the use of Freund's adjuvant. Lab. Anim. Sci. 39:400-405.

Byars, N. E., and A.C. Allison. 1987. Adjuvant formulation for use in vaccines to elicit both cell-mediated and humoral immunity. Vaccine 5:223-228.

Canadian Council on Animal Care (CCAC). 1990. CCAC guidelines on acceptable immunological procedures. Ottawa, Ontario: CCAC. (Available from CCAC 1000-151 Slater, Ottawa, Ontario, Canada K1P 5H3).

Chapel, H. M., and P. J. August. 1976. Report of nine cases of accidental injury to man with Freund's complete adjuvant. Clin. Exp. Immunol. 24:538-541.

Check, I. J., B. Bennett, and R. L. Hunter. 1991. Hunter's TiterMax®: Research adjuvant. Technical background from CytRx Corporation, Norcross, Georgia.

Chiller, J. M., B. J. Skidmore, D. C. Morrison, and W. O. Weigle. 1973. Relationship of the structure of bacterial lipopolysaccharides to its function in mitogenesis and adjuvanticity. Proc. Natl. Acad. Sci. 70:2129-2132.

Desiderio, J. V., and S. G. Campbell. 1985. Immunization against experimental murine salmonellosis with liposome-associated O-antigen. Infect. Immun. 48:658-663.

Freund, J. 1951. The effect of paraffin oil and mycobacteria on antibody formation and sensitization: A review. Am. J. Clin. Pathol. 21:645-656.

Freund, J., J. Casals, and E. P. Hosmer. 1937. Sensitization and antibody formation after injection of tubercle bacilli and paraffin oil. Proc. Soc. Exp. Biol. 37:509-513.

Grumpstrup-Scott, J., and D. D. Greenhouse, eds. 1988. NIH intramural recommendations for the research use of complete Freund's adjuvant. ILAR News 30(2):9.

Henricson, B. E., W. R. Benjamin, and S. N. Vogel. 1990. Differential cytokine induction by doses of lipopolysaccharide and monophosphoryl lipid A that result in equivalent early endotoxin tolerance. Infect. Immun. 58:2429-2437.

Howerton, D. A., R. L. Hunter, H. K. Ziegler, and I. J. Check. 1990. Induction of macrophage Ia expression in vivo by a synthetic block copolymer, L81. J. Immunol. 144:1578-1584.

Hughes, L. E., R. Kearney, and M. Tully. 1970. A study in clinical cancer immunotherapy. Cancer 26:269-278.

Hunter, R. L., and B. Bennett. 1984. The adjuvant activity of nonionic block polymer surfactants: II. Antibody formation and inflammation related to the structure of triblock and octablock copolymers. J. Immunol. 133:3167-3175.

Hunter, R. L., and B. Bennett. 1986. The adjuvant activity of nonionic block polymer surfactants: III. Characterization of selected biologically active surfaces. Scand. J. Immunol. 23:287-300.

Hunter, R. L., F. Strickland, and F. Kezdy. 1981. Studies on the adjuvant activity of nonionic block polymer surfactants. I. The role of hydrophile-lipophile balance. J. Immunol. 127:1244-1250.

Hunter, R. L., M. R. Olsen, and B. Bennett. 1995. Copolymer adjuvants and TiterMax®. Pp. 51-94 in The Theory and Practical Application of Adjuvants, D. E. S. Stewart-Tull, ed. New York: Wiley.

Johnson, A. G. 1994. Molecular adjuvants and immunomodulators: New approaches to immunization. Clin. Microbiol. Rev. 7:277-289.

Johnson, A. G., S. Gaines, and M. Landy. 1956. Studies on the O-antigen of Salmonella typhosa. V. Enhancement of the antibody response to protein antigens by the purified lipopolysaccharide. J. Exp. Med. 103:225-246.

Johnston, B. A., H. Eisen, and D. Fry. 1991. An evaluation of several adjuvant emulsion regimens for the production of polyclonal antisera in rabbits. Lab. Anim. Sci. 41:15-21.

Kalish, M. L., I. J. Check, and R. L. Hunter. 1991. Murine IgG isotype responses to the Plasmodium cynomolgi circumsporozoite peptide (NAGG)5. I. Effects of carrier, copolymer adjuvants and lipopolysaccharide on isotype selection. J. Immunol. 146:3583-3590.

Kenney, J. S., B. W. Hughes, M. P. Masada, and A. C. Allison. 1989. Influence of adjuvants on the quantity, affinity, isotype and epitope specificity of murine antibodies. J. Immunol. Methods 121:157-166.

Kleinman, N. R., A. B. Kier, E. Diaconu, and J. H. Lass. 1993. Posterior paresis induced by Freund's adjuvant in guinea pigs. Lab. Anim. Sci. 43:364-366.

Kotani, S., H. Takada, I. Takahashi, T. Ogawa, et al. 1986. Immunobiological activities of synthetic lipid A analogs with low endotoxicity. Infect. Immun. 54:673-682.

Landy, M., A. G. Johnson, M. E. Webster, and J. F. Sagin. 1955. Studies on the O antigen of Salmonella typhosa. II. Immunological properties of the purified antigen. J. Immunol. 74:466-478.

Lipman, N. S., L. J. Trudel, J. C. Murphy, and Y. Sahali. 1992. Comparison of immune response potentiation and in vivo inflammatory effects of Freund's and Ribi adjuvants in mice. Lab. Anim. Sci. 42:193-197.

Mastrangelo, M. J., and D. Berd. 1982. Immunotherapy with microbial products. Pp. 75-105 in Immunological Approaches to Cancer Therapeutics, Vol. 11., E. Mihich, ed. New York: Wiley.

Morrison, D. C. 1990. Diversity of mammalian macromolecules which bind to bacterial lipopolysaccharides. Pp. 183-189 in Cellular and Molecular Aspects of Endotoxin Reactions, A. Nowotny, J. J. Spitzer, and E. J. Ziegler, eds. Amsterdam: Elsevier.

Morrison, D. C., and J. A. Rudbach. 1981. Endotoxin-cell-membrane interactions leading to transmembrane signaling. Pp. 181-218 in Contemporary Topics in Molecular Immunology, P. Inman and W. J. Mandy, eds. New York: Plenum.

Nauts, H. C., W. E. Swift, and B. L. Coley. 1946. Treatment of malignant tumors by bacterial toxins as developed by the late William B. Coley, M. D., reviewed in light of modern research. Cancer Res. 6:205-216.

Noll, H., H. Bloch, J. Asselineau, and E. Lederer. 1956. The chemical structure of the cord factor of Mycobacterium tuberculosis. Biochem. Biophys. Acta 20:299-309.

Osebold, J. W. 1982. Mechanism of action by immunologic adjuvants. J. Am. Vet. Med. Assoc. 181:983-987.

Pearson, C. M. 1956. Development of arthritis, periarthritis and periostitis in rats given adjuvant. Proc. Soc. Exp. Biol. Med. 91:95-101.

Pearson, C. M., and F. D. Wood. 1959. Studies of polyarthritis and other lesions induced in rats by injection of mycobacterial adjuvant I. General clinical and pathologic characteristics and some modifying factors. Arthritis Rheum. 2:440-459.

Public Health Service (PHS). 1986. Public Health Service Policy on Humane Care and Use of Laboratory Animals. Washington, D.C.: U.S. Department of Health and Human Services. (Available from Office for Protection from Research Risks National Institutes of Health, 6100 Executive Boulevard, MSC 7507, Rockville, MD 20892-7507. Tel: 301-496-7163).

Ribi, E. T., J. Meyer, I. Azuma, R. Parker, and W. Brehmer. 1975. Biologically active components from mycobacterial cell walls. IV. Protection of mice against aerosol infection with virulent Mycobacterium tuberculosis. Cell. Immunol. 16:1-10.

Ribi, E., C. A. McLaughlin, J. L. Cantrell, et al. 1978. Immunotherapy for tumors with microbial constituents or their synthetic analogues: A review. Pp. 131-154 in Immunotherapy of Human Cancer. New York: Raven Press.

Rudbach, J. A., D. A. Johnson, and J. T. Ulrich. 1995. Ribi adjuvants: Chemistry, biology and utility in vaccines for human and veterinary medicine. Pp. 287-313 in The Theory and Practical Application of Adjuvants, D. E. S. Stewart-Tull, ed. New York: Wiley.

Sanchex, Y., I. Ionescu-Matiu, G. R. Dreesman, et al., 1980. Humoral and cellular immunity to hepatitis B virus-derived antigens: Comparative activity of Freund's Complete Adjuvant, alum and liposomes. Infect. Immun. 30:728-733.

Smith, D. E., M. E. O'Brien, V. J. Palmer, and J. A. Sadowski. 1992. The selection of an adjuvant emulsion for polyclonal antibody production using a low-molecular-weight antigen in rabbits. Lab. Anim. Sci. 42:599-601.

Stewart-Tull, D. E. S. 1995. Freund-type mineral oil adjuvant emulsions. Pp. 1-19 in the Theory and Practical Application of Adjuvants, D. E. S. Stewart-Tull, ed. New York: Wiley.

Suter, E., and R. G. White. 1954. Response of reticulo-endothelial system to injection of 'purified wax' and lipopolysaccharide of tubercle bacilli: Histologic and immunologic study. Am. Rev. Tuber. 70:793-805.

Takada, H. and S. Kotani. 1989. Structural requirements of lipid A for endotoxicity and other biological activities. Crit. Rev. Microbiol. 16:477-523.

Takahashi, H., T. Takeshita, B. Morein, S. Putney, R. N. German, and J. A. Berzofsky. 1990. Induction of CD8+ cytotoxic T cells by immunization with purified HIV-1 envelope protein in ISCOMS. Nature 344:873-875.

Unanue, E. R., and P. M. Allen. 1987. The basis for the immunoregulatory role of macrophages and other accessory cells. Science 236:551-557.

van-de-Wijgert, J. H., A. F. Verheul, H. Snippe, I. J. Check, and R. L. Hunter. 1991. Immunogenicity of Streptococcus pneumoniae type 14 capsular polysaccharide: Influence of carriers and adjuvants on isotype distribution. Infect. Immun. 59:2750-2757.

Vosika, G. J. 1983. Clinical immunotherapy trials of bacterial components derived from Mycobacteria and Nocardia. J. Biol. Resp. Modif. 2:321-342.

Waksman, B. H. 1979. Adjuvants and immune regulation by lymphoid cells. Springer Semin. Immunopathol. 2:5-9.

Waksman, B. H., C. M. Pearson, and J. T. Sharp. 1960. Studies of arthritis and others lesions induced in rats by injection of mycobacterial adjuvants. II. Evidence that the disease is a disseminated immunologic response to exogenous antigen. J. Immunol. 85:403-417.

Warren, H. S., F. R. Vogel., and L. A. Chedid. 1986. Current status of immunological adjuvants. Ann. Rev. Immunol. 4:369-388.

Webster, M. E., J. F. Sagin, M. Landy, and A. G. Johnson. 1955. Studies on the O antigen of Salmonella typhosa. I. Isolation and purification of the antigen. J. Immunol. 74:455-465.


TABLE 1 Factors modulated by adjuvants

Properties of antibody


Properties of cell-mediated immunity
Other characteristics


TABLE 2 Properties of three commercially available adjuvants

AdjuvantEmulsion typeMode of antigen incorporationImmunomodulatorOil component
Freund's Complete Adjuvantwater-in-oilencapsulation in oil with long retentionkilled whole MycobacteriaMineral oil
Ribi Adjuvant System®oil-in-wateradsorption to oil with brief retentionmycobacterial product (TDM) and/or endotoxin product (MPL®)Squalene
TiterMax®water-in-oilencapsulation in oil with long retentioncopolymer CRL-8941Squalene



Figure 1 The composition of FCA, RAS®, and TiterMax®





Copyright © 2008. National Academy of Sciences.
All rights reserved.
500 Fifth St. N.W., Washington, D.C. 20001.
Terms of Use and Privacy Statement