Physiological effects and adjuvanticity of recombinant brushtail possum TNF-alpha

The present paper describes the physiological properties of recombinant pos sum TNF-a and an adjuvant effect on antibody responses to the model protein antigen, keyhole limpet haemocyanin (KLH). For these studies recombinant p ossum TNF-alpha was produced in the yeast Pichia pastoris is. The recombina nt cytokine was secreted into the culture medium and purified by gel filtra tion. Possum TNF-alpha produced in this expression system was N-glycosylate d and bioactive in two different assays. In a murine fibroblast L929 cytoto xicity assay, the possum TNF-alpha had lower specific activity compared to human TNF-alpha. while in a possum-specific assay, possum TNF-alpha enhance d the proliferation of PHA-stimulated possum thymocytes and was more active than human TNF-alpha. The physiological effect of the recombinant possum T NF-alpha was investigated in groups of possums administered doses of 6.30 o r 150 mu g of cytokine. For each dose, TNF-alpha caused profound effects on the number of circulating leucocytes characterized by a three-to-four-fold increase in neutrophil numbers at 6 24 hr after injection and an initial s harp decrease in lymphocyte numbers. The efficacy of TNF-alpha as an immuno logical adjuvant was determined in possums administered KLH ( 125 mu g) in an aqueous or Al(OH)(3)-based formulation with or without added recombinant TNF-alpha(150 mu g). Serum antibody responses to KLH were monitored by ELI SA. The TNF-a stimulated two-fold and four-fold increases in antibody level s in aqueous and Al(OH)(3)-based vaccine formulations, respectively. The st rongest antibody responses were observed in the group of possums that recei ved KLH formulated in Al(OH)(3) with addition of TNF-alpha.