¹ AgResearch, Wallaceville Animal Research Centre, Upper Hutt, New Zealand

Correspondence: DN Wedlock, AgResearch, Wallaceville Animal Research Centre, PO Box 40063, Upper Hutt, New Zealand

Received 28 July 1998; Accepted 6 October 1998.

Abstract

The present paper describes the physiological properties of recombinant possum TNF- and an adjuvant effect on antibody responses to the model protein antigen, keyhole limpet haemocyanin (KLH). For these studies recombinant possum TNF- was produced in the yeast Pichia pastoris. The recombinant cytokine was secreted into the culture medium and purified by gel filtration. Possum TNF- produced in this expression system was N-glycosylated and bioactive in two different assays. In a murine fibroblast L929 cytotoxicity assay, the possum TNF- had lower specific activity compared to human TNF-, while in a possum-specific assay, possum TNF- enhanced the proliferation of PHA-stimulated possum thymocytes and was more active than human TNF-. The physiological effect of the recombinant possum TNF- was investigated in groups of possums administered doses of 6, 30 or 150 g of cytokine. For each dose, TNF- 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- as an immunological adjuvant was determined in possums administered KLH (125 g) in an aqueous or Al(OH)₃-based formulation with or without added recombinant TNF- (150 g). Serum antibody responses to KLH were monitored by ELISA. The TNF- 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-.