Physiological effects and adjuvanticity of recombinant brushtail possum TNF-α

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Abstract
The present paper describes the physiological properties of recombinant possum TNF-α and an adjuvant effect on antibody responses to the model protein antigen, keyhole limpet haemocyanin (KLH). For these studies recombinant possum TNF-α was produced in the yeast Pichia pastoris. The recombinant cytokine was secreted into the culture medium and purified by gel filtration. Possum TNF-α produced in this expression system was N-glycosylated and bioactive in two different assays. In a murine fibroblast L929 cytotoxicity assay, the possum TNF-α had lower specific activity compared to human TNF-α, while in a possum-specific assay, possum TNF-α enhanced the proliferation of PHA-stimulated possum thymocytes and was more active than human TNF-α. The physiological effect of the recombinant possum TNF-α was investigated in groups of possums administered doses of 6, 30 or 150 μg of cytokine. For each dose, TNF-α caused profound effects on the numbers of circulating leucocytes characterized by a three-to-four-fold increase in neutrophil numbers at 6–24 h after injection and an initial sharp decrease in lymphocyte numbers. The efficacy of TNF-α as an immunological adjuvant was determined in possums administered KLH (125 μg) in an aqueous or Al(OH)3-based formulation with or without added recombinant TNF-α (150 μg). Serum antibody responses to KLH were monitored by ELISA. The TNF-α stimulated two-fold and four-fold increases in antibody levels in aqueous and Al(OH)3-based vaccine formulations, respectively. The strongest antibody responses were observed in the group of possums that received KLH formulated in Al(OH)3 with addition of TNF-α.

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