Characterisation of the porcine cytokines which activate the CD131ßc common sub-unit, for potential immune-augmentation.

Early acting cytokines and growth factors such as those of the CD131 ßc subunit, may offer an alternative method to the current use of antibiotics and chemicals such as anthelmintics in maintaining Porcine (Po) health. Thus far, the recombinant Po (rPo) Granulocyte-macrophage colony-stimulating factor (GM-CSF), rPo interleukin-3 (IL-3) and rPo interleukin-5 (IL-5) proteins have been identified and cloned and the biological activity of each cytokine has been confirmed in vitro, however, in vivo immune system regulation and hematopoietic stem cell (HSC) augmentation are regulated by numerous cytokines and cellular signals within the bone marrow (BM) niche. In order to quantify the use of recombinant cytokines in augmenting the immune response, it is necessary to determine the stages of hematopoiesis induced by each cytokine and possible areas of synergy requiring further investigation. Here we used the chemotherapeutic agent 5-fluorouracil (5-FU), to chemically induce a state of myelosuppression in young pigs. This allowed for the monitoring of both the autologous BM reconstitution and recombinant cytokine induced BM repopulation, precursor cell proliferation and cellular differentiation. The recombinant cytokines PoGM-CSF, PoIL-3 and PoIL-5 were administered by intramuscular injections (i.m.) following confirmation of 5-FU induced leukocytopenia. Blood and BM samples were collected and then analysed for cell composition. Statistically significant results were observed in several blood cell populations including eosinophils for animals treated with rPoIL-5, rPoGM-CSF and basophils for animals treated with rPoIL-3. BM analysis of CD90+ and CD172a+ cells confirmed myelosuppression in week one with significant results observed between rPoIL-3 and the 5-FU control group in week two and for the rPoGM-CSF group in week three. These results have demonstrated the effects of each of these rPo cytokines within the hematopoietic processes of the pig and may demonstrate similar outcomes in other mammalian models including human.

High-level production of recombinant chicken interferon-gamma by Brevibacillus choshinensis.

Cytokines, such as interferon-gamma have been shown to have adjuvant and growth promoting activity in poultry and livestock and have the potential to be used as alternatives to antibiotics. We have developed an efficient system for commercial-scale synthesis of recombinant chicken interferon-gamma (ChIFN-gamma) using Brevibacillus choshinensis as the host for protein production. The ChIFN-gamma expression vector, pNCIFN, was constructed using the novel Escherichia coli-B. choshinensis shuttle vector, pNCMO2. ChIFN-gamma expression was optimized by investigating different culture conditions and different host B. choshinensis mutants. The highest level of production was observed using the B. choshinensis HPD31-MB2 strain grown at 30 degrees C, where ChIFN-gamma was produced at approximately 300-500 mg/L. ChIFN-gamma was also produced as a His-tagged fusion protein by using the pNCHis-IFN expression vector, a derivative of pNCMO2. The protein was constitutively secreted into the culture supernatant and could be partially purified in a single step using a Ni-nitrilotriacetic acid column. This recombinant His-ChIFN-gamma was shown to have the same biological activity as native ChIFN-gamma.

Cytokine therapy: a natural alternative for disease control.

Disease control in food production animals is normally mediated through the use of vaccines, chemicals and antibiotics. However, the extensive use of antibiotics and chemicals in livestock has resulted in environmental and human health concerns, particularly with regard to the emergence of drug-resistant bacteria in the food chain. In fact, the World Health Organisation (WHO) has now urged meat producers to use environmentally-friendly alternative methods to control disease. Cytokines, as natural mediators of the immune response, offer exciting alternatives to conventional therapeutics. The utilisation of cytokines is becoming more feasible with the recent cloning of a number of cytokine genes. Since the chicken's immune system is similar to that of mammals, they offer an attractive model system with which to study the effectiveness of cytokine therapy in the control of disease in intensive livestock. In this report we will review our recent studies on the therapeutic potential of chicken interferon gamma (ChIFN-gamma) as a vaccine adjuvant and a growth promoter.

Recombinant chicken IFN-gamma expressed in Escherichia coli: analysis of C-terminal truncation and effect on biologic activity.

Interferon-gamma (IFN-gamma) possesses potent immunostimulatory properties, and it has recently been shown to have potential therapeutic properties. Recombinant protein technology is frequently used for commercial production of therapeutics, such as IFN. Biologically active recombinant chicken IFN-gamma (rChIFN-gamma) constructs bearing an N-terminal poly-His tag were expressed in Escherichia coli. Preparations of rChIFN-gamma contained varying ratios of a full-length and a truncated protein species (18 and 16 kDa, respectively). Amino acid sequence analysis of the full-length protein corroborated the sequence previously predicted from the cDNA sequence. Full-length rChIFN-gamma contains two cysteine residues at the C-terminus, and these were labeled by reduction and subsequent specific alkylation with fluorescent tag (5-I-AEDANS) to distinguish between full-length and C-terminally truncated forms of rChIFN-gamma. Comparative peptide mapping, amino acid sequencing, and mass spectrometry revealed that the 16 kDa protein was truncated at Lys133. It was also observed that the 18 kDa rChIFN-gamma protein was infrequently contaminated with small quantities of protein truncated at Arg141. A truncated recombinant construct (His1-Lys133) was also expressed in E. coli and had biologic activity comparable with that of the full-length construct. The 3-D structure of rChIFN-gamma was deduced by comparative modeling with bovine and human IFN-gamma crystallographic structures. Analysis of sequences and comparison of structures have revealed that the 3-D structure of rChIFN-gamma is similar to those of bovine and human molecules despite an overall amino acid identity of only 32%.

Coadministration of IFN-gamma enhances antibody responses in chickens.

The development of new generation vaccines has focused on the use of natural immunologic adjuvants that are capable of enhancing a protective immune response. The use of cytokines as immunomodulators in livestock animals, particularly poultry, is becoming more feasible with the recent cloning of several cytokine genes and the progression of new delivery technologies, such as live vectors and DNA delivery. Given that chickens are reared under intensive conditions that are conducive to infection by opportunistic pathogens, the primary mechanism for disease control in poultry is early and effective vaccination. However, many poultry vaccines offer only short-term protection or give nonuniform responses within flocks. We have developed a model system with which to measure the adjuvant potential of cytokines in chickens. This involves measuring antibody levels following coadministration of chicken interferon-gamma (Ch-IFN-gamma) with sheep red blood cells (SRBC). Groups of SPF and commercial broiler birds were injected with two different doses of SRBC with and without coadministration of Ch-IFN-y. Three weeks later, all birds were boosted with SRBC alone. Sera were collected weekly and anti-SRBC antibody titers (total Ig and IgG) were determined by hemagglutination. Priming Ch-IFN-gamma resulted in enhanced primary and secondary (IgG) antibody responses that persisted at higher levels when compared with birds that received SRBC alone. Second, coadministration of Ch-IFN-y allowed a 10-fold lower dose of antigen to be as effective as a high dose of antigen that was given without Ch-IFN-gamma. Third, treatment with Ch-IFN-y resulted in an increase in the proportion of birds responding to antigen challenge. These results suggest the potential use for Ch-IFN-gamma as a vaccine adjuvant.

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.

Chicken interferon types I and II enhance synergistically the antiviral state and nitric oxide secretion.

This report shows that chicken type I and type II interferons (IFNs), like their mammalian counterparts, act synergistically such that a mixture of the two has much greater activity than that expected from the separate contribution of each type. The degree of antiviral synergy was measured by virus plaque reduction and cytopathic effect (CPE) inhibition in both primary and secondary chicken embryo cell cultures. Mixtures of the two Ch-IFNs produced antiviral effects 3-10 times greater than that expected from strict additivity of each IFN acting alone. At high concentrations of IFN mixtures there was a qualitative shift to an exponential IFN action does-response curve that revealed synergistic effects greater than 100-fold. Synergy resulted even with mixtures containing less than 1 U/ml of either type of Ch-IFN. The antiviral effects developed more rapidly with mixtures than when type I or II Ch-IFN was used alone. Mixtures of the two types of Ch-IFN synergistically potentiated nitric oxide secretion in cells of the HD11 chicken macrophage line. Molecular mechanisms are cited that may account for synergy between type I and type II IFNs, and speculation is offered on the epidemiologic and therapeutic implications of synergy in vivo.

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.