Physiological effects and adjuvanticity of recombinant brushtail possum TNF‐α

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.