Oil-based adjuvants are formulated as emulsions, which are dispersions of an antigen-containing aqueous phase with an oil phase. Water-in-oil (W/O) emulsions contain higher concentrations of oil, typically 30-80%, consist of water droplets immersed in an oil milieu, and are quite viscous. In contrast, oil-in-water (O/W) emulsions are composed of oil droplets immersed in an aqueous phase, and are predominantly aqueous in nature, with oil concentrations of only 2-10%. W/O emulsions are more apt to induce cellular immune responses, whereas O/W emulsions predominantly induce Th2 responses unless they are formulated with an additional immunomodulator. Both types of emulsions contain surfactants that, through their amphipathic nature, aid in the stabilization of the emulsion by interacting at the interface between the oil and water.
The adjuvant basis of emulsions is strongly related to their interaction with the antigen. They make the antigen particulate in nature through aggregate formation, and form depots allowing for slow release of the antigen. The degree of depot formation is dependent on the type and concentration of the oil used. Thicker, more viscous W/O emulsions cause significant depot formation and substantially prolong release of antigen. Additionally, emulsions induce inflammation at the injection site attracting macrophages to the area, which engulf the antigen and carry it to the draining lymph nodes (18).
Jules Freund was the first to develop oil-based adjuvants, and these formulations have been the principle adjuvants used in research laboratories for generations (19). Complete Freund's adjuvant (CFA) is a mineral oil W/O emulsion, which includes killed M. tuberculosis as an added immunostimulant. CFA is still considered one of the most potent adjuvants available, but the associated toxicity precludes its use in the clinic. Incomplete Freund's adjuvant (IFA) is a similar emulsion to CFA but lacking the mycobacterium component. IFA is demonstrably less toxic than CFA and has been utilized in modern vaccines, notably as an adjuvant for peptide-based strategies. These trials have reported the induction of immune responses in immunized cancer patients (20-23), but these responses have often been of low frequency (23) or have not been associated with a clinical improvement (20,22,24), suggesting the need for additional immunostimulants to improve the potency of the induced response. Indeed it has been shown that the coadministration of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), or CpG with these formulations has improved immune responses and/or clinical outcome over those seen with IFA alone (21,25-27).
Mineral oils such as Drakeol, Bayol, or paraffin were traditionally used to formulate CFA and similar W/O emulsions (19). These complex mixtures of lipids with varying-length fatty acids are nonmetabolizable, leading to longer retention at the injection site and some local reactogenicity. Several groups continue to develop novel oil-based emulsions with the aim of retaining their potent adjuvant activity while reducing toxicity. Seppic's Montanide ISA 51 is a W/O emulsion analogous to IFA, but it is made with a more refined mineral oil. ISA 51 has been tested extensively in the clinics where it has demonstrated an acceptable level of toxicity (28). There are many ongoing peptide-based clinical trials in cancer patients using the ISA 51 adjuvant, alone or in combination with other immunostimulants (29). Published studies to date suggest the success of these types of formulations is modest and sporadic, similar to what has been seen with IFA (28,30).
Another W/O emulsion being tested in the clinics is Montanide ISA 720, also from Seppic, which is prepared with a metabolizable oil to reduce injection site reactivity associated with mineral oil (31-35). This adjuvant has also shown an acceptable toxicity profile for disease indications such as cancer.
O/W emulsions like those first developed by Ribi (36) provide another mechanism by which to reduce the toxicities associated with the more viscous and reactogenic W/O emulsions. Chiron Corporation's MF-59 adjuvant has an oil concentration of only 4.3% (w/v) requiring a microfluidization process to make the emulsion stable, yet fluid in nature. In addition to the low oil concentration, MF-59 is made with the metabolizable oil, squalene. MF-59 has been studied extensively in clinical trials with infectious disease vaccines (37,38) and is approved for use in Italy as part of an influenza vaccine (39).
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