Expression of biologically active recombinant porcine interleukin-12 from Escherichia coli.

The control of viral infections is of critical importance to livestock industries worldwide and is highlighted by costly infection outbreaks, such as that seen with foot and mouth disease virus. To ameliorate the impact of increasing problems with viral infections, new vaccine and anti-viral strategies are required and a greater understanding of the anti-viral response is essential. Furthermore, in pigs, evidence is still being gathered on the components of a defined anti-viral immune response. However, this has been greatly improved by the recent cloning and expression of critical cytokines involved in the anti-viral response. To assess the use of recombinant porcine interleukin-12 (rPoIL-12) as an immunotherapeutic and immunomodulator of swine, we have cloned and expressed rPoIL-12 as a single-chain fusion protein from Esherichia coli (E. coli). The fusion encodes the p40 and p35 subunits, linked by a glycine-serine linker and expressed as a C-terminal 6xHis tagged protein. rPoIL-12 stimulated the proliferation of human lymphoblasts and its activity on porcine cells was demonstrated by the ability of rPoIL-12 to increase the mRNA expression of porcine interleukin-18 receptor-alpha (poIL-18Ralpha) from porcine peripheral blood mononuclear cells (PoPMBCs). This data supports the inclusion of E. coli produced rPoIL-12 in immunomodulation strategies in the pig.

Potential use of cytokine therapy in poultry.

Newly hatched chickens are highly susceptible to infection during the first 2 weeks of life. The utilisation of cytokines as therapeutic agents in livestock animals, in particular poultry, has become more feasible with the recent cloning of cytokine genes and the progression of new technologies such as live vectors. We have constructed a live recombinant fowlpox virus (FPV) that expresses chicken myelomonocytic growth factor (fp/cMGF). Administration of fp/cMGF to chicks resulted in a marked and sustained increase in the number of circulating blood monocytes as well as an increase in their state of activation, as measured by enhanced phagocytic activity and elevated production of nitric oxide. We have recently cloned the gene for chicken interferon-gamma (ChIFN-gamma). Recombinant ChIFN-gamma was capable of protecting chick fibroblasts from undergoing virus-mediated lysis and induced nitrite secretion from chicken macrophages in vitro. Preliminary vaccination trials have indicated that co-administration of ChIFN-gamma with antigen (sheep red blood cells) resulted in enhanced secondary (IgG) antibody responses and allowed a 10-fold lower dose of antigen to be used. Furthermore, administration of ChIFN-gamma resulted in enhanced weight gain in chicks and improved their resistance to disease challenge. The ability of cytokines to combat infection and enhance vaccine efficacy makes them excellent candidates as a therapeutic agents and adjuvants.

In vivo effects of chicken interferon-gamma during infection with Eimeria.

Newly hatched chickens are highly susceptible to infection by opportunistic pathogens during the first 1 or 2 weeks of life. The use of cytokines as therapeutic agents has been studied in animal models as well as in immunosuppressed patients. This approach has become more feasible in livestock animals, in particular poultry, with the recent cloning of cytokine genes and the development of new technologies, such as live delivery vectors. We have recently cloned the gene for chicken interferon-gamma (Ch-IFN-gamma). Poly-HIS-tagged recombinant Ch-IFN-gamma was expressed in Escherichia coli, was purified by Ni chromatography, and was found to be stable at 4 degrees C and an ambient temperature for at least several months and Several weeks, respectively. Ch-IFN-gamma was capable of protecting chick fibroblasts from undergoing virus-mediated lysis, induced nitrite secretion from chicken macrophages in vitro, and enhanced MHC class II expression on macrophages. Administration of recombinant Ch-IFN-gamma to chickens resulted in enhanced weight gain over a 12-day period. Furthermore, the therapeutic potential of Ch-IFN-gamma was assessed using a coccidial challenge model. Birds were treated with Ch-IFN-gamma or a diluent control and then infected with Eimeria acervulina. Infected birds treated with Ch-IFN-gamma showed improved weight gain relative to noninfected birds. The ability of Ch-IFN-gamma to enhance weight gain in the face of coccidial infection makes it an excellent candidate as a therapeutic agent.