The Production of Polyclonal Antibodies in Laboratory Animals

The Report and Recommendations of ECVAM Workshop 351,2

Reprinted with minor amendments from ATLA 27, 79-102.

Appendix 2

Overview of Adjuvants

An overview of adjuvant categories used for routine polyclonal antibody (pAb) production is given in Table I.

Table I: Overview of Categories of Adjuvants that May Be Used for Routine Polyclonal Antibody Production

Category Examples (references)
Immunostimulatory oil emulsions (for example, water-in-oil, oil-in-water, water-in-oil-in-water Freund's incomplete adjuvant, Montainde®, Specol (18)
Mineral salts Al(OH)3, AlPO4
Microbial (like) products LPS, MDP, MPL, TDM (10)
Saponins Quil A (19)
Synthetic products DDA (13), ISCOMs, NBP (12)
Adjuvant formulations Oil emulsion + NBP (TiterMaxTM), Oil emulsion + bacterial products (Freund's complete adjuvant; RIBITM, Gerbu (11)

LPS = lipopolysaccharide; MDP = muramyl dipeptide; MPL = monophosphoryl lipid A ; TDM = trehalose dimycolate; DDA = dimethyldioctadecylammonium bromide; NBP = non-ionic block polymer; ISCOMs = immune stimulating complexes.

Immunostimulatory Oil Emulsions

Water-in-oil emulsions, which include Freund-type adjuvants, are the adjuvants most commonly used to produce pAbs in laboratory animals. Although most investigators and commercial vendors still refer to the Freund-type adjuvants in use today as Freund's complete adjuvant (FCA, i.e. it contains mycobacteria) or Freund's incomplete adjuvant (FIA, i.e. it does not contain mycobacteria), it would be desirable to replace these terms to reflect the differences between the components in the original formulation and those in the modern formulation, as well as the differences in reactogenicity of the different formulations. Few laboratories would be in a position to make the original FCA, because it was formulated with heat-killed Mycobacterium tuberculosis of high virulence, a mineral oil of low quality manufactured before 1969, and a surfactant, predominantly mannide mono-oleate, of variable purity and quality. Due to a change in the oil refining procedure in the early 1970s, the mineral oil component of the original FCA is no longer available (1). It has been replaced by a higher quality oil with less-irritant properties. The mannide mono-oleate currently in use is also of higher quality. Today, only a few adjuvant immunologists retain the use of the old FCA, which is manufactured by the StatensSerum Institute (Copenhagen, Denmark) as a "gold standard" for comparison against a new adjuvant. The workshop participants agreed that this formulation is unsuitable as an adjuvant for use in routine pAb production, due to severe side-effects. The product that is quoted as FCA in the more recent literature can be obtained from, for example, Difco Laboratories, Sigma, ICN Biomedicals, and Pierce, and consists of a refined oil and a high quality mannide mono-oleate preparation, with heat-killed M. butyricum or M. tuberculosis H37Ra, an avirulent human strain. This FCA has less-irritant and less-inflammatory properties than the original FCA, but still induces considerable side-effects in animals (2, 3).

Currently, there are immunostimulatory oil emulsions that are acceptable or even superior to the original FIA with regard to enhancing antibody responses. Moreover, the purlfied components in these formulations produce fewer and less-severe adverse reactions after injection, for example, the Montanide® ISA (Incomplete Seppic Adjuvant) series (Seppic, Paris, France) and NUFCA Guildhay oil (Guildhay, Guildford, Surrey, UK).

Montanide ISA 740 adjuvant is composed of highly purlfied mannitol octadecenoic esters (Montanide ISA 80) as surfactant, in a mixture of a metabolisable oil and a refined non-metabolisable light mineral oil classified pharmacologically as an excipient. This mixture can form a stable emulsion (especially under nitrogen storage), in the weight ratio 70:30 Montanide ISA 740:aqueous phase antigen. When properly formulated, the emulsion will remain in a single phase for at least 2 years. This adjuvant emulsion is easy to inject and is well-tolerated by the recipient animal. When injected subcutaneously into mice or guinea-pigs in accordance with the European Pharmacopoeia, there are no serious adverse effects. The Montanide ISA series has been accepted for use in all food-producing species (4), as a pharmacologically active substance generally regarded as safe.

A non-ulcerative oil (NUFCA Guildhay oil) that can be administered by the intramuscular, subcutaneous or intradermal routes at multiple sites has been introduced by Guildhay. The intramuscular site creates a focus of stimulus with fewer adverse reactions than the classical FIA on the market today. However, as NUFCA Guildhay oil has only recently been introduced, limited information on its use is currently available.

Mineral Salts

Aluminum adjuvants in the form of aluminum hydroxide or aluminum phosphate hydrated gels can be injected subcutaneously or intramuscularly for priming an immune response in the recipient. These adjuvants are generally regarded as safe and they have been used for human vaccination for more than 50 years (5). Priming immunizations with aluminum adjuvants can be followed by boosters with or without adjuvant (6, 7). The biological function of these adjuvants is related to their ability to adsorb protein antigens, thereby ensuring that soluble proteins will be taken up as particulate antigens by antigen-presenting cells (8). Due to this adsorption/function relationship, it is strongly recommended that investigators ascertain that adsorption of the antigen to the gel has been successfully accomplished prior to its injection (9).

Microbial (like) Products

Micro-organisms such as M. butyricum and microbial products can exhibit strong adjuvant activity. The innate vertebrate immune system has evolved mechanisms for the recognition of, and response to, certain microbial products. Although the innate immune system itself is not highly efficient, some of its response components, once stimulated, help energise the specific antibody response. The microbial products involved (primarily cell wall components) usually induce considerable undesirable inflammatory side-effects, as well as an adjuvant effect. Investigators have identified active fractions or subunits of bacterial products, for example, trehalose dimycolate, and have in some cases mod)fied the bacterial products, for example, threonyl-muramyl dipeptide, or monophosphoryl lipid A, to achieve a balance of immunostimulatory properties and diminished inflammatory properties (10, 11).

Saponins

Saponins are triperpene glycosides which are derived from the bark of the Quillaja saponaria tree and which have detergent and adjuvant properties. Saponin preparations intended for use as immunological adjuvants (for example, Quil A or QS-21) are purlfied to reduce the presence of components which cause adverse local reactions. Food-grade saponin preparations should not be used for immunization schemes. In general, saponins should not be injected intraperitoneally or intravenously, but only subcutaneously or intramuscularly, due to their haemolytic activity.

Synthetic Products

Synthetic adjuvants are a rather heterogeneous group of products, because their classification has no single chemical, physical, or functional basis. This group includes nonionic block polymers (NBP), dimethyldioctadecylammonium bromide (DDA), immune stimulating complexes (ISCOMs), and liposomes. NBPs can contain different hydrophobic and hydrophilic regions, which influence their surfactant and immunopotentiating properties (12). The adjuvant effect of a given NBP also depends on the antigen used in combination with it, and, as such, different NBPs may be needed for different antigens for optimal effects. DDA is not an optimal adjuvant for antibody responses (13), but is rather better for Tcellmediated cytotoxic responses. DDA has a lipophilic character, which might be responsible for its capacity to enhance T-cell responses. It is a representative of the quaternary amines (also classified as a cationic detergent). ISCOMs are small (40 nm diameter) cage-like structures prepared from Quil A, cholesterol, and phospholipids. The antigen to be inserted into ISCOMs must be amphipathic (14). ISCOMs can be recommended as an excellent first choice for viral vaccines, based on past successes. In part, this is because ISCOMs can deliver the antigen to the cytosolic compartment of antigenprocessing/antigen-presenting cells, and thus direct the immune response to a cytotoxic T-cell response, which is effective against many viruses. However, ISCOMs may also facilitate antibody responses. There is generally some resistance to the use of ISCOMs, because of the perceived, but misconceived, difficulty in their preparation; as an alternative, there are commercially available "honeycomb structures", to which the antigen can be added (15). Liposomes are unilamellar or multilamellar vesicles artificially constructed from natural products. The bilayer membranes mimic those of cells. The adjuvanticity of liposomes is influenced by charge, composition, and method of preparation. The antigen can be encapsulated in the water phase or the lipid phase, or it can be coupled to the surface (see reviews by Buiting et al [16] and Alving [17]).

Adjuvant Formulations

Combining different immunostimulatory agents can increase the potency of an adjuvant. Oil emulsions are frequently combined with other agents (for example NBP in TiterMaxTM, or bacterial products in FCA and RIBITM adjuvants). Immunostimulatory agents such as muramyl dipeptide can be incorporated along with antigen into liposomes. In fact, many adjuvants have more than one immunostimulatory substance and more than one mechanism of action.

References

  1. Stewart-Tull, D.E.S. (1995). The Theory and Practical Application of Adjuvants, 380pp. Chichester, UK: John Wiley & Sons.
  2. Smith, D.E., O'Brien, M.E., Palmer, V.J. & Sadowski, J.A. (1992). The selection of an adjuvant emulsion for polyclonal antibody production using a low molecular weight antigen in rabbits. Laboratory Animal Science 42: 599-601.
  3. Leenaars, P.P.A.M., Koedam, M.A., Wester, P.W., Baumans, V., Claassen, E. & Hendriksen, C.F.M. (1998). Assessment of side-effects induced by injection of different adjuvant/antigen combinations in rabbits and mice. Laboratory Animals 32: 387-406.
  4. EC (1995). Commission Regulation (EC) No. 2796/95 of 4 December 1995 amending Annex II of Council Regulation (EEC) No. 2377/90 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin (Text with EEA relevance). Official Journal of the European Communities L290: 1-4.
  5. Gupta, R.K., Relyveld, E.H., Lindblad, E.B. Bizzini, B., Ben-Efraim, S. & Gupta, C.K. (1993) AdJuvants: a balance between toxicity and adjuvanticity. Vaccine 11: 293-306.
  6. Collier, L.H., Polakoff, S. & Mortimer, J. (1979). Reactions and antibody responses to reinforcing doses of adsorbed and plain tetanus vaccines. Lancet 8131: 1364-1367.
  7. Bomford, R. (1980). The comparative selectivity of adjuvants for humoral and cell-mediated immunity. 1. Effect on the antibody response to bovine serum albumin and sheep red blood cells of Freund's incomplete and complete adjuvants, alhydrogel, Corynebacterium parvum, Bordetella pertussis, muramyl dipeptide and saponin. Clinical and Experimental Immunology 39: 426-434.
  8. Mannhalter, J.W., Neychev, H.O., Zlabinger, G.J., Ahmad, R. & Eibl, M.N. (1985). Modulation of the human immune response by the nontoxic and non-pyrogenic adjuvant aluminium hydroxide: effect on antigen uptake and antigen presentation. Clinical and Experimental Immunology 61: 143-151.
  9. Lindblad, E.B. (1995). Aluminium adjuvants. In The Theory and Practical Application of Adjuvants (ed. D.E.S. Stewart-Tull), pp. 21-35. Chichester, UK: John Wiley & Sons.
  10. Warren, H.S., Vogel, F.R. & Chedid, L.A. (1986). Current status of immunological adjuvants. Annual Review of Immunology 4: 369-418.
  11. Grubhofer, N. (1995). An adjuvant formulation based on N-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutamine with dimethyldioctadecylammonium chloride and zinc-Lproline complex as synergists. Immunology Letters 44: 19-24.
  12. Verheul, A.F.M. & Snippe, H. (1992). Non-ionic block polymer surfactants as immunological adjuvant. Research in Immunology 143: 512-519.
  13. Hilgers, L.A.T. & Snippe, H. (1992). DDA as an immunological adjuvant. Research in Immunology 143: 494-503.
  14. Dalsgaard, K., Lovgren, K. & Stewart-Tull, D.E.S. (1995). Immune stimulating complexes with Quil A. In The Theory and Practical Application of Adjuvants (ed. D.E.S. Stewart-Tull), pp. 129-144. Chichester, UK: John Wiley & Sons.
  15. Morein, B., Sundquist, B., Hoglund, S., Dalsgaard, K. & Osterhaus, A.D M.E. (1984). ISCOM, a novel structure for antigenic presentation of membrane proteins from enveloped viruses. Nature, London 308: 457-459.
  16. Buiting, A.M.J., Van Rooijen, N. & Claassen, E. (1992). Liposomes as antigen carriers and adjuvants in vivo. Research in Immunology 143: 541-548.
  17. Alnng, C.R. (1997). Liposomes as adjuvants for vaccines. In New Generation Vaccines, 2nd edn (ed. M.M. Levine, G.C. Woodrow, J.B Kaper & G.S. Cobon), pp. 207-213. New York, USA: Marcel Dekker.
  18. Boersma, W.J.A., Bogaerts, W.J.C., Bianchi, A.T.J. & Claassen, E. (1992). Adjuvant properties of stable water-in-oil emulsions: evaluation of the experience with Specol. Research in Immunology 143: 503-512.
  19. Campbell, J.B. & Peerbaye, Y.A. (1992). Saponin. Research in Immunology 143: 526-530.

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