title: Physiological effects and adjuvanticity of recombinant brushtail possum TNF-alpha.
abstract: The present paper describes the physiological properties of recombinant possum TNF-alpha and an adjuvant effect on antibody responses to the model protein antigen, keyhole limpet haemocyanin (KLH). For these studies recombinant possum TNF-alpha was produced in the yeast Pichia pastoris. The recombinant cytokine was secreted into the culture medium and purified by gel filtration. Possum TNF-alpha produced in this expression system was N-glycosylated and bioactive in two different assays. In a murine fibroblast L929 cytotoxicity assay, the possum TNF-alpha had lower specific activity compared to human TNF-alpha, while in a possum-specific assay, possum TNF-alpha enhanced the proliferation of PHA-stimulated possum thymocytes and was more active than human TNF-alpha. The physiological effect of the recombinant possum TNF-alpha was investigated in groups of possums administered doses of 6, 30 or 150 micrograms of cytokine. For each dose, TNF-alpha 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-alpha as an immunological adjuvant was determined in possums administered KLH (125 micrograms) in an aqueous or Al(OH)3-based formulation with or without added recombinant TNF-alpha (150 micrograms). Serum antibody responses to KLH were monitored by ELISA. The TNF-alpha 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-alpha.
authors: AgResearch, Wallaceville Animal Research Centre, Upper Hutt, New Zealand.
authors: D N
authors: DN
authors: Wedlock
authors: L P
authors: LP
authors: Goh
authors: A R
authors: AR
authors: McCarthy
authors: R G
authors: RG
authors: Midwinter
authors: N A
authors: NA
authors: Parlane
authors: B M
authors: BM
authors: Buddle
mesh_terms: D000276:Adjuvants, Immunologic / Q000235:genetics / Q000494:pharmacology / Q000502:physiology; D000818:Animals; D000917:Antibody Formation; D017931:DNA Primers / Q000235:genetics; D006433:Hemocyanins / Q000276:immunology; D006801:Humans; D009893:Opossums / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology* / Q000502:physiology*
chemical_list: D000276:Adjuvants, Immunologic; D017931:DNA Primers; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin