Comparison of alternatives to Freund’s Complete Adjuvant

Abstract

Studies on alternatives to Freund’s complete adjuvant (FCA) are important in order to scientifically support guidelines on immunization procedures. We evaluated several types of adjuvants in rabbits and mice. Weakly immunogenic antigens were used to discriminate between the efficacy of the adjuvants. The side effects were evaluated based on clinical findings, behavioural changes, physiological state and (histo)pathological lesions. Rabbits were immunized subcutaneously or intramuscularly with: FCA, Specol (water-in-oil emulsion), commercially available adjuvants (RIBI and TiterMax) or Iscom-matrix. Mice were immunized intraperitoneally, subcutaneously or in the dorsal side of the hind foot with: FCA, Specol, ISCOMS (preformed immune-stimulating complexes), Quil A orLactobacillus. In both studies the adjuvants were combined with three weak immunogenic antigens; a synthetic peptide of 3,2 kDa, a glycolipid and a particulate antigen (Mycoplasma pneumonia) in rabbits and a selfantigen (Myelin Basic Protein; MBP; 18,5 kDa), a synthetic peptide of 2,5 kDa and a particulate antigen (M. pneumoniae) in mice. Rabbits did not appear to be severely and chronically impaired by the experimental procedure. Lesions were most severe after injection of RIBI/antigen or FCA/antigen and minimal to mild after injection of TiterMax/antigen or Specol/antigen. Antibody responses were high after immunization with FCA/antigen or Specol/antigen and low after RIBI/antigen or TiterMax/antigen. From this study it may be concluded that Specol is a possible alternative to FCA for enhancement of the immune response in rabbits. However, more research is needed to confirm that it is an alternative to FCA for a wide range of antigens. In mice, no signs of prolonged pain or distress were demonstrated based on comprehensive behavioural studies, clinical findings and physiological state. Lesions were most severe after injection of antigen in combination with FCA or Quil A, moderate with Specol and minimal with Lactobacillus, ISCOMS or saline. After injection of synthetiic peptide in combination with FCA, Specol and ISCOMS specific antibody responses were high. Immunization with selfantigen resulted in high antibody titers when injected in ISCOMS, moderate antibody responses when injected intraperitoneally with FCA and low antibody titers when injected with Specol, Lactobacillus or saline. For selfantigens ISCOMS seem to be a suitable alternative while Specol is a possible alternative to FCA for the production of specific antibodies to synthetic peptide. However, depending on the nature of the antigen alternative adjuvants may induce severe pathological changes.