Review of Selected Adjuvants Used in Antibody Production

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. 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 ). Factors modulated by adjuvants Factors modulated by adjuvants 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. 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 ( Figure 1 ) ( 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. The composition of FCA, RAS®, and TiterMax® 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. 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 ). Properties of three commercially available adjuvants Properties of three commercially available adjuvants 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. 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. 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 with insoluble of aluminum or aluminum This can be by diverse including the concentration of of and adjuvants are of by an aqueous The of and of appear to influence the adjuvant activity, depending on the antigen. of protein antigens within such not only protects the antigen from rapid but also a depot effect ( Altman and Dixon, 1989 ). Furthermore, liposomes the antigen from the of injection to the lymph nodes, they interact with macrophages ( and ). antigens within liposomes can enhance both humoral and cell-mediated immunity, and other immunostimulatory agents, such as LPS and can further increase their immunogenicity ( et al., ; and ). with compounds, liposomes are undesirable for antibody production because they are difficult to and are not effective with most Adjuvants that are surface-active agents on surface free of cells to bind to hydrophobic surfaces and about their immunostimulatory properties ( Allison, 1979 ). of lipid A, and TDM are some of the most potent natural surface-active adjuvants in their adjuvant activity has been with in structure that leads to in surface-active the other A and monophosphoryl lipid A, and MDP derivatives have hydrophilic and hydrophobic from which their surface-active properties ( Hunter et al., 1995 ). normally in the such as A and and are also to be surface-active agents. Other of proposed adjuvants include and compounds ( ). of these are commonly used for production of antisera due to either of or of FCA has been used in a wide variety of laboratory animals for over and there is no that it can induce pain in both and large animals ( 1987 ). have been to discontinue use of FCA because it is used as a due to its well known with a wide variety of Therefore, it is important to be with its side pain has been reported in of FCA injections ( and and in injected with tumor extract in FCA ( et al., ). In with injections of amounts of FCA can that for The reactions were attributed to Therefore, in with the for the and of in and the is investigators should consider that that cause pain or distress in human may cause pain or distress in other ( 1986 ). Other for use of FCA can be obtained from the of ( and 1988 ) and the on ( 1990 ). In and recognized that from M. tuberculosis in a water-in-oil emulsion injected the of guinea pigs caused of macrophages ( and ). inflammatory reactions have been for subcutaneous and granulomatous is present at the of of either FCA or ( 1989 ). In rabbits, FCA injections commonly cause ulcerations of the and subcutaneous injections at both the injection site and or to which the has can also at such FCA injections can cause and necrosis of and may be in lymph nodes, and depending on the of the emulsion the and system ( 1989 ). These side effects may in and has been reported in and large animals. The severity of the and can be especially in animals. injected with heat-killed mycobacterial cells in mineral oil resulting in the ( 1956 ; and ; [ et al., ). and are less One case in guinea pigs caused by injections of the granulomatous the which on the cord ( et al., ). inflammatory reactions have been occasionally at 2 or for about and then High of site and toxic antigens may be with increased severity of the the toxicity of TiterMax® is comparable to that of which is from that of FCA. reactions have also been at of injection with RAS®, being an oil-in-water such reactions tend to be less severe due to retention in the No one adjuvant works with all animal or adjuvant has and and the properties also be to the adjuvant most to the Although FCA has been necessary because of the of effective this is no adjuvants are now available that high antibody in some obtained with FCA and with less severe side The to Hunter for and in the of this