Chicken IFN-gamma Research Articles

Marek's disease virus-specific T cells proliferate, express antiviral cytokines but have impaired degranulation response.

The major histocompatibility complex (MHC) haplotype is one of the major determinants of genetic resistance and susceptibility of chickens to Marek’s disease (MD) which is caused by an oncogenic herpesvirus; Marek’s disease virus (MDV). To determine differential functional abilities of T cells associated with resistance and susceptibility to MD, we identified immunodominant CD4+TCRvβ1 T cell epitopes within the pp38 antigen of MDV in B19 and B21 MHC haplotype chickens using an ex vivo ELISPOT assay for chicken IFN-gamma. These novel pp38 peptides were used to characterize differential functional abilities of T cells as associated with resistance and susceptibility to MD. The results demonstrated an upregulation of cytokines (IL-2, IL-4, IL-10) and lymphocyte lysis-related genes (perforin and granzyme B) in an antigen specific manner using RT-PCR. In the MD-resistant chickens (B21 MHC haplotype), antigen-specific and non-specific response was highly skewed towards Th2 response as defined by higher levels of IL-4 expression as well as lymphocyte lysis-related genes compared to that in the MD-susceptible chicken line (B19 MHC haplotype). Using CD107a degranulation assay, the results showed that MDV infection impairs cytotoxic function of T cells regardless of their genetic background. Taken together, the data demonstrate an association between type of T cell response to pp38 and resistance to the disease and will shed light on our understanding of immune response to this oncogenic herpesvirus and failure to induce sterile immunity.

Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-γ

The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-γ were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-γ (ChIFN-γ) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killed-vaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-γ did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV.

IFN-γ Priming of Chicken Heterophils Upregulates the Expression of Proinflammatory and Th1 Cytokine mRNA Following Receptor-Mediated Phagocytosis ofSalmonella entericaserovarenteritidis

Responsiveness to invasive pathogens, clearance via the inflammatory response, and activation of appropriate acquired responses are all coordinated by innate host defenses. Polymorphonuclear leukocytes (PMNs) are cellular components of innate response, with the primary PMN in poultry being the heterophil. Priming is the potentiation of the phagocyte activation process. Interferon-gamma (IFN-gamma) is a pleiotropic cytokine involved in basically all phases of immune and inflammatory responses that has been shown to prime heterophil functional activities. In the present experiments, using real-time quantitative RT-PCR, we evaluated the role of recombinant chicken IFN-gamma (rChIFN-gamma) as a priming mediator to control heterophil responses at the level of gene transcription and expression of the mRNA for proinflammatory (interleukin-1beta [IL-1beta], IL-6, IL-8) and Th1 (IL-18 and IFN-gamma) cytokine genes following stimulation with phagocytosis agonists, opsonized and nonopsonized Salmonella enteritidis. rChIFN-gamma primed the heterophils for an increase in transcription of proinflammatory cytokines induced by phagocytic agonists but also upregulated expression of Th1 cytokine (IL-18 and IFN-gamma) mRNA and stimulated an increased production of IFN-gamma. Although rChIFN-gamma priming modulated the expression of cytokine mRNA in heterophils stimulated by different phagocytic agonists, rChIFN-gamma by itself did not directly induce gene expression of either proinflammatory or Th1 cytokines. The enhanced expression of cytokine mRNA does not appear to be differentially expressed depending on the receptor activated during phagocytosis. The results from the present experiments suggest that rChIFN-gamma may play a significant role in avian innate immunity against Salmonella infection and may offer an adjunct use in the prevention and treatment of salmonellae infections in newly hatched chickens.

An Interferon-γ-binding Protein of Novel Structure Encoded by the Fowlpox Virus

Poxviruses have evolved various strategies to counteract the host immune response, one of which is based on the expression of soluble cytokine receptors. Using various biological assays, we detected a chicken interferon-gamma (chIFN-gamma)-neutralizing activity in supernatants of fowlpox virus (FPV)-infected cells that could be destroyed by trypsin treatment. Secreted viral proteins were purified by affinity chromatography using matrix-immobilized chIFN-gamma, followed by two-dimensional gel electrophoresis. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis indicated that the viral IFN-gamma-binding protein in question was encoded by the FPV gene 016. The chicken IFN-gamma binding and neutralizing activity of the recombinant FPV016 protein was confirmed using supernatants of cells infected with a recombinant vaccinia virus that lacked its own IFN-gamma-binding protein but instead expressed the FPV016 gene. The FPV016 gene product also neutralized the activity of duck and human IFN-gamma but failed to neutralize the activity of mouse and rat IFN-gamma. Unlike previously known cellular and poxviral IFN-gamma receptors, which all contain fibronectin type III domains, the IFN-gamma-binding protein of FPV contains an immunoglobulin domain. Remarkably, it exhibits no significant homology to any known viral or cellular protein. Because IFN-gamma receptors of birds have not yet been characterized at the molecular level, the possibility remains that FPV016 represents a hijacked chicken gene and that avian and mammalian IFN-gamma receptors have fundamentally different primary structures.

Modulation of functional activities of chicken heterophils by recombinant chicken IFN-gamma.

The objective of the present studies was to examine the in vitro effects of recombinant chicken interferon-gamma (rChIFN-gamma) on shape change, phagocytosis, and the oxidative/nonoxidative killing activities of day-old chicken heterophils. Heterophils (4 x 10(6)/ml) were incubated with various concentrations of recombinant ChIFN-gamma from both Escherichia coli and transfected Cos cells for 2 h at 39 degrees C. The incubation of the neonatal heterophils with rChIFN-gamma resulted in significantly greater numbers of cells with membrane shape change when compared with the mock-treated heterophils. Both Cos cell-derived and E. coli-derived ChIFN-gamma significantly increased (p < 0.01) the phagocytosis of opsonized or nonopsonized Salmonella enteritidis by the neonatal heterophils in a concentration-dependent manner. Incubation with ChIFN-gamma induced no direct stimulation of the respiratory burst by the chicken heterophils but did prime the heterophils for a significantly strengthened respiratory burst to subsequent stimulation with opsonized zymosan (OZ). Lastly, incubation of the heterophils with ChIFN-gamma primed the cells for a significant increase in the release of beta-D-glucuronidase following stimulation with OZ. These results show that neonatal avian heterophils can respond to cytokine modulation with enhanced functional competence, suggesting that ChIFN-gamma can enhance the immune competence of the innate defenses of chickens during the first week of life.

A Recombinant Eimeria Protein Inducing Interferon-g Production: Comparison of Different Gene Expression Systems and Immunization Strategies for Vaccination against Coccidiosis

A rabbit antiserum against an 18- to 27-kD native protein fraction (F3) from Eimeria acervulina merozoites identified a cDNA (3-1E) containing a 1086-base pair insertion with an open reading frame of 170 amino acids (predicted molecular weight, 18,523). The recombinant 3-1E cDNA expressed in Escherichia coli produced a 60-kD fusion protein and a 23-kD protein after factor Xa treatment of the fusion protein. Both proteins were reactive with the F3 antiserum by western blot analysis. A rabbit antiserum against a synthetic peptide deduced from the amino acid sequence of the 3-1E cDNA reacted with a 27-kD recombinant 3-1E protein expressed in Sf9 insect cells and a 20-kD native protein expressed by E. acervulina sporozoites and Eimeria tenella sporozoites and merozoites. By immunofluorescence staining, a monoclonal antibody produced against the recombinant 3-1E protein reacted with sporozoites and merozoites of E. acervulina, E. tenella, and Eimeria maxima. Spleen lymphocytes from E. acervulina-immune chickens showed antigen-specific proliferation and interferon (IFN)-gamma production upon stimulation with the recombinant 3-1E protein, indicating that the protein activates cell-mediated immunity during coccidiosis. Immunization of chickens with either the E. coli- or Sf9-expressed recombinant 3-1E protein with adjuvant, or direct injection of the 3-1E cDNA, induced protective immunity against live E. acervulina. Simultaneous injection of the recombinant 3-1E protein, or the 3-1E cDNA, with cDNAs encoding chicken IFN-gamma or interleukin (IL)-2/15 further enhanced protective immunity. These results indicate that the recombinant E. acervulina 3-1E cDNA or its polypeptide product may prove useful as vaccines against avian coccidiosis.

Recombinant duck interferon gamma inhibits duck hepatitis B virus replication in primary hepatocytes.

Interferon gamma (IFN-gamma), which has been cloned in several mammalian species and recently in birds, plays a critical role in modulating immune system function. IFN-gamma and tumor necrosis factor alpha (TNF-alpha) have been shown to be crucial in the pathogenesis of viral hepatitis and in the transient disappearance of hepatitis B virus (HBV) from the liver after adoptive transfer of HBV-specific cytotoxic T lymphocytes into HBV-transgenic mice. Similar studies in the natural animal hosts of related hepadnaviruses have been limited because the corresponding probes and recombinant cytokines were not available. For this reason, we initiated studies to clone and characterize cytokines from the duck, the natural host of the duck hepatitis B virus (DHBV). We describe here the cDNA cloning and initial characterization of the IFN-gamma homologue of ducks (DuIFN-gamma). The DuIFN-gamma cDNA codes for a predicted mature protein of 145 amino acids with a molecular mass of 16.6 kDa. The precursor protein has 67% identity with the previously cloned chicken IFN-gamma and 21 to 34% identity with mammalian IFN-gamma. Recombinant DuIFN-gamma induces the transcription of several IFN-inducible genes including IFN regulatory factor 1 and guanylate-binding protein, and it exhibits antiviral activity that protects duck cells from vesicular stomatitis virus-mediated lysis. Importantly, treatment of primary duck hepatocytes with recombinant DuIFN-gamma inhibits DHBV replication in a dose-dependent fashion. Time course analysis revealed that IFN-gamma treatment does not affect initial covalently closed circular DNA (cccDNA) conversion but inhibits the synthesis of progeny cccDNA by amplification.

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%.

Avian IFN-gamma genes: sequence analysis suggests probable cross-species reactivity among galliforms.

Little is known about the evolution of cytokines in non-mammalian systems. To address this problem, we attempted to clone the gene for interferon-gamma (IFN-gamma) from a variety of avian species using oligonucleotide primers based on the sequence of the chicken IFN-gamma gene. The coding sequence and partial intron sequences were determined for four species, namely guinea fowl, ring-necked pheasant, Japanese quail, and turkey. To obtain sequence information on the gene extremities, a modified 5' and 3' RACE protocol was used. The sequence information showed that the coding regions of the IFN-gamma gene are highly conserved among the species studied (93.5%-96.7% and 87.8%-97.6% at the nucleotide and peptide levels, respectively) and are more conserved at the amino-terminal region (exons 1 and 2) than the carboxyl-terminal (exons 3 and 4). This high degree of overall identity at the predicted primary amino acid sequence level of the protein, including the deduced IFN-gamma receptor binding motifs, suggests that IFN-gamma may be cross-reactive among these species. Phylogenetic analysis shows that the similarity of the avian IFN-gamma sequences parallels the presumed evolutionary relationships between the species.