W. C. Hanly, Ph.D., Department of Microbiology and Immunology,
B. Taylor Bennett, D.V.M., Ph.D., and James E. Artwohl, D.V.M.,
Biologic Resources Laboratory,
College of Medicine, University of Illinois
Chicago, Illinois

The following was adapted from a series of short articles published in the August and September 1994 issues of the BRL Bulletin. These articles were intended to explain briefly the function of adjuvants; guidelines for use of adjuvants, particularly Freund's adjuvants; and to introduce alternative adjuvants. For a more in depth discussion of adjuvants and antibody production patrons are referred to the ILAR Journal, volume 37, number 3, 1995.

Need for adjuvants

The production of antibodies [Ab] in laboratory animals is a tool used in many fields of biomedical research. Antibodies are routinely made to proteins, carbohydrates, complex lipids, and nucleic acids isolated from natural sources. In addition, modern biochemical, biosynthetic, and recombinant DNA techniques have created increasingly pure antigens [Ag]. Many of these newer antigens are small or generally weak immunogens. The small polypeptides (<10 kDa) and nonprotein antigens usually need to be conjugated to a large immunogenic carrier protein to become good immunogens. These as well as most other protein antigens (especially when administered in small quantity) need to be administered with an adjuvant to assure a high quality/high quantity, memory-enhanced antibody response by the laboratory animal. In the past few years a number of new adjuvants have become available for use in laboratory animals, although Freund's adjuvants continue to be the most commonly used despite their potential hazards.

Functions of adjuvants

In general, g to mg quantities of a protein Ag are needed to elicit an antibody response in a laboratory animal. This range, which may differ from Ag to Ag and from species to species, is called the "window of immunogenicity". Too much or too little Ag may induce tolerance rather than an active response for the given Ag. Compared to injection of Ag alone, injection of antigen plus an adjuvant generally permits use of a much smaller quantity of the Ag and greatly enhances the Ab titer (Kaeberle, 1986). Within the window of immunogenicity, with or without adjuvant, larger Ag doses generally result in greater Ab responses up to a point at which suppressive activities become exaggerated. However, production of the highest titer is not always the best goal, for a moderate titer of high affinity Ab may be preferable to a high titer of low affinity. High affinity Ab generally results from immunization with smaller quantities of Ag than needed for production of the highest titer.

For an animal to sustain an Ab response, a continual or intermittent supply of Ag is needed. One way an adjuvant may aid the immune response is by forming a depot of Ag at the injection site resulting in the sustained release of small quantities of Ag over a long period of time. This approach gives sustained stimulation while minimizing suppressive effects. Even with an adjuvant that forms a depot of Ag, at some point in time the quantity of Ag is diminished and the Ab titer declines. At this time a second injection of Ag (a booster dose) may be given. When an animal that has responded maximally is given a booster dose of Ag too soon, suppression rather than enhancement of the immune response may ensue. Ideally, one follows the serum Ab titer in a hyperimmunized animal and gives a booster injection of Ag only after the Ab titer has begun to decline. However, when an animal has responded less than maximally (which is the more usual situation for small doses of Ag), a booster dose of Ag given at 3 to 6 weeks after the first Ag dose will usually increase the serum Ab titer (Cooper et al., 1991; Siskind et al., 1968; Herbert, 1978; Hu and Kitagawa, 1990). Booster doses of Ag are typically equal to or less than (~1/2) the priming dose. The advantage of using a smaller Ag dose is that only the higher affinity clones of B cells are stimulated, thus improving the quality of the Ab produced.

A second way an adjuvant can work is to serve as a vehicle to help deliver the Ag to the spleen and/or lymph nodes where Ag is trapped by the follicular dendritic cells and where most of the necessary cell to cell interactions take place to generate plasma cells ( the Ab-secreting cells). For example, microdroplets of oil containing Ag, such as those formed in an oil-in-water adjuvant emulsion, are readily ingested by macrophage and taken to draining lymph nodes or spleen. Additionally, emulsions aid tissue dendritic cells in their capture of Ag. Ag-loaded tissue dendritic cells rapidly emigrate via lymphatics to draining lymph nodes. Substances that activate complement enhance trapping of Ag by the follicular dendritic cells through their surface complement receptors. It is, in fact, the retention of Ag in the follicles of the spleen and/or lymph nodes that is essential for Ab production and for the maintenance of memory. The terminally differentiated plasma cells survive only a few days to a few weeks, and thus must be replaced by reactivated memory cells. Memory cell activation takes place only in the Ag-loaded follicles.

A third way an adjuvant can work is to activate the various cells involved in the immune response, either directly or indirectly. Surfactants, components of all emulsion adjuvants, may serve this function as well as helping to stabilize oil-water emulsions. Also, many bacteria contain substances that activate cells of the immune system, particularly the macrophage. The activated macrophage in turn helps activate T and B cells. Thus some adjuvants contain bacteria, bacterial products, or derivatives of bacterial products. Although the activation of macrophages indeed aids in the antibody response, excessive activation of macrophages also causes excessive inflammation, so that bacterial components cannot be used in excess. In recent years, a number of bacterial products have been modified in ways that maximize their desirable activation potential and minimize their inflammatory potential with the goal of finding ideal adjuvant components. For example, some of the new generation adjuvants incorporate a chemical variant of endotoxin called monophosphoryl lipid A [MPL] or a modified muramyl dipeptide [thr-MDP] or other "detoxified" cell wall constituents of bacteria (Rudbach et al., 1988).

Advantages and disadvantages of Freund's adjuvants

Freund's Complete Adjuvant (FCA), a mixture of a non-metabolizable oil (mineral oil), a surfactant (Arlacel A), and mycobacteria (M. tuberculosis or M. butyricum) has been used for many years to enhance immunologic responses to antigens, and even today is considered to be one of the most effective adjuvants. It is prepared as a water-in-oil emulsion by combining one volume FCA with one volume aqueous antigen solution. In the emulsion, Ag is distributed over a large surface area thereby increasing the potential for interaction with relevant cells. Antibody production is enhanced by FCA primarily because of: a) the depot effect and b) nonspecific immunopotentiation of macrophages by surfactant and the mycobacteria.

The preparation of an appropriate and stable emulsion is critical for the proper functioning of the adjuvant, and methods to prepare the emulsion and check its stability have been described (Herbert, 1978; Moncada et al., 1993). Some important points are emphasized here. A source bottle of FCA needs to be mixed very thoroughly before an aliquot is withdrawn in order to assure even distribution of the mycobacteria. Then the aqueous phase, in stepped portions, is introduced into the oil, rather than vice-versa, assuring that the oil becomes the continuous phase. Mixing follows addition of each portion of aqueous phase and can be accomplished with two glass syringes connected with a double hubbed needle or a 3-way stopcock ( the latter being advantageous for stepped addition of the aqueous phase by a third syringe). The goal is to achieve water droplets <1 m diameter dispersed in the continuous oil phase without introduction of air. A drop of a stable emulsion will remain cohesive when floated on cold water.

Although FCA is a very effective adjuvant for production of antibodies, there are problems and hazards associated with its use (Broderson, 1989; Kleinman et al., 1993; Claassen et al., 1992; Steiner JW et al, 1960; Stills and Bailey, 1989; Stills 1994). At the site of injection, FCA causes a chronic inflammatory response that may be severe and painful for the animal depending on the site as well as the quantity and quality of adjuvant injected. The inflammatory response may result in formation of chronic granulomas, sterile abscesses, and/or ulcerating tissue necrosis. Adjuvant-induced lesions may appear to be metastatic when excessive amounts of the emulsion are injected in a single site. Emulsion injected subcutaneously on the dorsal region of some species (rabbit, in particular) may migrate by fistulous tracts to the ventral region of the animal. Emulsion injected intramuscularly may spread along fascial planes to distant muscular sites or may travel to lung, liver or other organs, apparently by a hematogenous route. Monitoring the site of injection as an index of health of the animal may be inadequate when excessive quantities of emulsion are injected.

Measures to limit the severity of the inflammatory reaction include: a) choosing or making preparations of FCA with a lower mycobacterial concentration, i.e., 0.05 to 0.1 mg/ml, rather than 1 mg/ml; b) adding a concentrated antigen solution to the adjuvant to obtain an antigen-rich emulsion, thereby reducing the quantity of emulsion injected; c) using multiple injection sites with limitation of volume injected at any one site; d) separation of injection sites to avoid fusion of inflammatory lesions; and e) maintaining sterility of the Ag solution.

FCA is also a potential hazard for laboratory personnel. Accidental self-inoculation can result in tuberculin sensitization followed by chronic local inflammation which responds poorly to antibiotic treatment (Chapel and August, 1976). Accidental splashing of FCA in the eye, a risk for unguarded eyes during preparation of the emulsion or during injection of the animal, can result in severe ocular irritation and even blindness. For individuals already sensitized to mycobacterial antigens (i.e., those having a positive tuberculin skin test), an accidental inoculation can cause a severe necrotizing lesion that persists for months or that may require surgical excision of the injection site for ultimate resolution. Systemic effects persisting for weeks or months such as fever and neurological and arthritic symptoms have also been described.

Freund's Incomplete Adjuvant (FIA) has the same oil/surfactant mixture as FCA but does not contain any mycobacteria. It is frequently used to boost animals that received a primary antigen injection in FCA, but it can be used as the adjuvant for the primary injection as well. It has adjuvant properties that favor humoral immunity without cell-mediated immunity, but is generally considered to be less potent than FCA (although exceptions exist). FIA is capable of causing abscesses and granuloma formation, but such reactions are generally less severe than those that accompany the use of FCA.

Prior to using Freund's adjuvants, particularly FCA, investigators should consider the use of an alternative adjuvant system. Several new generation adjuvants are commercially available, and some others may be constructed in an investigator's laboratory.

Alternative adjuvants

Montanide ISA Adjuvants [Seppic, Paris, France] are a group of oil/surfactant based adjuvants in which different surfactants are combined with either a non-metabolizable mineral oil, a metabolizable oil, or a mixture of the two. They are prepared for use as an emulsion with aqueous Ag solution. The surfactant for Montanide ISA 50 [ISA = Incomplete Seppic Adjuvant] is mannide oleate, a major component of the surfactant in Freund's adjuvants. The surfactants of the Montanide group undergo strict quality control to guard against contamination by any substances that could cause excessive inflammation, as has been found for some lots of Arlacel A used in Freund's adjuvant. The various Montanide ISA group of adjuvants are used as water-in-oil emulsions, oil-in-water emulsions, or water-in-oil-in-water emulsions. The different adjuvants accommodate different aqueous phase/oil phase ratios, because of the variety of surfactant and oil combinations. The performance of these adjuvants is said to be similar to Incomplete Freunds Adjuvant [IFA] for antibody production; however the inflammatory response is usually less.

Ribi's Adjuvants (Ribi ImmunoChem Research, Inc., Hamilton, MT) are supplied by the manufacturer as mixtures of oil, detergent, and immunostimulator(s); the investigator need only add aqueous Ag and mix by vortexing to form a stable oil-in-water emulsion. To minimize the inflammatory response, the adjuvant utilizes a metabolizable oil (squalene) and contains modified bacterial products designed to provide immunopotentiation without excessive inflammation (Rudbach et al., 1988). One mycobacterial component, trehalose dimycolate [TDM], serves as a surfactant and an immunostimulator as well as an adherence factor in binding protein Ag to the oil droplets. Also, because significantly less oil is needed for a stable oil-in-water emulsion than for a stable water-in-oil emulsion, a Ribi's adjuvant presumably causes less tissue damage than water-in-oil adjuvants, since less oil is administered. However, the depot effect is not as great as with water-in-oil emulsions and thus booster injections are needed more frequently. Ribi's adjuvants provide a choice of different detoxified bacterial products as immunostimulators for different species, and thus the manufacturer refers to the products as Ribi's Adjuvant System [RAS]. A Ribi's adjuvant may not be applicable to all antigens or all species, but may in some cases be superior to FCA. Variable results have been reported, but experience with this adjuvant is not as extensive as with FCA. In general, a Ribi's adjuvant emulsion (or other oil-in-water emulsion) is better for protein antigens that have some hydrophobic aspects or are amphipathic than for very hydrophilic proteins. This is because the adjuvant's effectiveness is dependent on adsorption of the protein antigen to the oil droplets of the oil-in-water emulsion. This mode of antigen presentation to B cells should bias the antibody response to epitopes of the native protein rather than to epitopes of the denatured protein. You should consider whether antibodies to native or denatured protein antigen are desired when choosing an adjuvant.

Hunter's TiterMax (CytRx Corp., Norcross, GA) is an oil/surfactant-based adjuvant prepared as a water-in-oil emulsion in a manner similar to that used for Freund's adjuvants. However, it uses a metabolizable oil (squalene) and a nonionic surfactant that has good protein antigen-binding capacity as well as adjuvant activity. The adjuvant activity may relate, in part, to the surfactant's ability to activate complement and bind complement components, as this helps target the Ag to follicular dendritic cells in the spleen and lymph nodes. The surfactant used in the commercially available adjuvant is one of a number of synthetic nonionic block copolymers of polyoxyethylene and polyoxypropylene developed by Hunter (Hunter et al., 1991). It was found to be superior for most, but not all proteins. Although experience with TiterMax is limited, some reports show it to be superior or equal to Freund's adjuvants with some protein antigens, particularly in rabbits and mice (Bennett et al, 1992). It may not be as successful in rats. When compared to FCA, TiterMax can be used in smaller quantities for initial injections, which minimizes the inflammatory reaction at the injection site. The inflammatory reaction that does develop is said to be due primarily to an Arthus type reaction. The utilization of copolymer-coated microparticles to stabilize the emulsion permits formation of stable emulsions with less than 20% oil, a big factor in minimizing total adjuvant injected. Also, booster injections may be needed less frequently than with Freund's adjuvant, making TiterMax after the initial investment, a cost effective adjuvant for antibody production.

Aluminum Salt Adjuvants are used with protein antigens in two ways: a) as alum-precipitated vaccines and b) as alum-adsorbed vaccines (Harlow and Lane, 1988; Nicklas, 1992). Investigator generated or commercially available Al(OH)3 [Alhydrogel - Superfos of Denmark/Accurate Chemical and Scientific Co., Westbury, NY] can be used to adsorb proteins in a ratio of 50 - 200 g protein/mg aluminum hydroxide. Adsorption of protein is dependent on the pI (Isoelectric pH) of the protein and the pH of the medium. A protein with a lower pI adsorbs to the positively charged aluminum ion more strongly than a protein with a higher pI. Aluminum salts are generally weaker adjuvants than emulsion adjuvants; however, because of their generally mild inflammatory reactions, safety, and efficacy for generating memory, they are the primary adjuvants utilized in humans. When used in larger quantity in laboratory animals, the inflammatory reactions that may occur at the site of injection will generally resolve within a few weeks although chronic granulomas may occasionally form. The mineral adjuvants work by establishing a depot of Ag which is released slowly over a period of 2-3 weeks, nonspecific activation of macrophages and complement activation. The effectiveness of aluminum salt adjuvants has been increased in experimental studies by the addition of gamma-inulin (Cooper et al., 1991), detergents, or Bordetella pertussis, but the inflammatory reaction is generally increased as well. Due to the short-term depot effect, the alum booster injections may be needed more frequently than with water-in-oil emulsions.

Nitrocellulose-Adsorbed Protein can be used for immunization without desorption of the protein in vitro, as desorption of protein from nitrocellulose [NC] paper will occur in vivo giving a desirable slow release of Ag over a period of 2 weeks to 2 months (Nilsson and Larsson, 1992). The nitrocellulose itself is essentially inert, causing little if any inflammatory response and no anti-NC antibody response. Either intrasplenic or subcutaneous deposition of the nitrocellulose paper with ng to g quantities of Ag has been successfully used for antibody production. On the order of 100 g of protein can bind to 1 cm2 of NC, so that only small quantities of NC need be introduced into the animal. The antibody response is not as vigorous as with FCA/antigen emulsions and may require 1 or more booster immunizations to be detected, but this method is particularly advantageous for situations in which only small quantities of pure Ag can be obtained as in a band from an electroblot. Antibodies have even been raised to NC-adsorbed protein administered after the protein had been stained with Coomassie Blue. However, you should be aware that antibodies raised to reduced or otherwise denatured protein may not react well with the native protein. On the other hand, such antibodies may be desirable for use on electroblots (Western blots).

Encapsulated Antigens have been prepared in several ways permitting sustained slow release of Ag and, in some cases, release of immunostimulators as well. Examples include liposome-entrapped Ag, nondegradable ethylene-vinyl acetate copolymer [EVAc]-entrapped Ag (Niemi et al., 1985), and degradable polymer-entrapped Ag. Among the biodegradable, biocompatible polymers used for encapsulation, poly(DL-lactide-co-glycolide) demonstrates very favorable characteristics for use in bulk-prepared vaccines, in that construction and administration of different size microspheres in a single dose may accomplish timed release of Ag in a way that mimics primary and booster injections (Eldridge et al., 1991). However, the preparation is rather complex for use with the occasional antigens prepared for injection by individual investigators, particularly when the antigen is available in very limited quantity. The complexity of preparation is a drawback for essentially all of the encapsulated Ag preparations, but in special situations the potential of an encapsulated Ag to generate a significant immune response may make its preparation worthwhile.

Gerbu Adjuvant [Gerbu Biotechnik GmbH, Gaiberg, Germany/C-C Biotech, Poway, CA] is a new aqueous phase adjuvant that does not have a depot effect. It utilizes immunostimulators in combination with zinc proline. Although it requires frequent boosting to achieve a high-titered response, the inflammatory effect at the site of injection is minimal. Preliminary experiments reported by a member of the company indicate that the adjuvant can be safely used for footpad injection of rabbits.

Preparation of Ag solutions for injection

When the aqueous Ag is prepared for vaccine use, it is important that it be prepared in a manner to prevent or eliminate contamination, particularly extraneous bacteria or bacterial products that may cause sepsis or extensive inflammatory reaction. Most protein antigens can be filtered through a microporous filter (0.22 m pore size) of a type that has minimal adsorption of protein to achieve sterility of the preparation just before its addition to adjuvant. It is also important for you to take measures at all stages of Ag preparation to minimize contamination of the preparation by bacterial endotoxin which is inflammatory and pyrogenic and may not be removed by filtration.


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