Adjuvant effects of IL-1beta, IL-2, IL-8, IL-15, IFN-alpha, IFN-gamma TGF-beta4 and lymphotactin on DNA vaccination against Eimeria acervulina.

Eight chicken cytokine genes (IL-1beta, IL-2, IL-8, IL-15, IFN-alpha, IFN-gamma, TGF-beta4, lymphotactin) were evaluated for their adjuvant effect on a suboptimal dose of an Eimeria DNA vaccine carrying the 3-1E parasite gene (pcDNA3-1E). Chickens were given two subcutaneous injections with 50 microg of the pcDNA3-1E vaccine plus a cytokine expression plasmid 2 weeks apart and challenged with Eimeria acervulina 1 week later. IFN-alpha (1 microg) or 10 microg of lymphotactin expressing plasmids, when given simultaneously with the pcDNA3-1E vaccine, significantly protected against body weight loss induced by E. acervulina. Parasite replication was significantly reduced in chickens given the pcDNA3-1E vaccine along with 10 microg of the IL-8, lymphotactin, IFN-gamma, IL-15, TGF-beta4, or IL-1beta plasmids compared with chickens given the pcDNA3-1E vaccine alone. Flow cytometric analysis of duodenum intraepithelial lymphocytes showed chickens that received the pcDNA3-1E vaccine simultaneously with the IL-8 or IL-15 genes had significantly increased CD3+ cells compared with vaccination using pcDNA3-1E alone or in combination with the other cytokine genes tested. These results indicate that the type and the dose of cytokine genes injected into chickens influence the quality of the local immune response to DNA vaccination against coccidiosis.

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.

cDNA cloning of biologically active chicken interleukin-18.

By searching a chicken EST database, we identified a cDNA clone that appeared to contain the entire open reading frame (ORF) of chicken interleukin-18 (ChIL-18). The encoded protein consists of 198 amino acids and exhibits approximately 30% sequence identity to IL-18 of humans and various others mammals. Sequence comparisons reveals a putative caspase-1 cleavage site at aspartic acid 29 of the primary translation product, indicating that mature ChIL-18 might consist of 169 amino acids. Bacterially expressed ChIL-18 in which the N-terminal 29 amino acids of the putative precursor molecule were replaced by a histidine tag induced the synthesis of interferon-gamma (IFN-gamma) in cultured primary chicken spleen cells, indicating that the recombinant protein is biologically active.

Novel chicken CXC and CC chemokines.

Upon stimulation with lipopolysaccharide (LPS) the chicken macrophage cell line HD-11 secretes factors with cytokine activity. To characterize these molecules, representational difference analysis with RNA of LPS-induced and uninduced HD-11 cells was performed. Two cDNA clones were isolated that code for polypeptides with structural features of chemokines. cDNA K60 codes for a novel CXC chemokine of 104 residues including a putative signal peptide of 20 amino acids at the N-terminus. It is 67% identical to the previously cloned chicken chemokine 9E3/CEF4. K60 exhibits a similar degree of sequence identity to human interleukin 8 and other related CXC chemokines (about 50%), rendering straight-forward predictions of its biological properties difficult. cDNA K203 codes for a novel CC chemokine of 89 amino acids including a putative N-terminal signal peptide of 21 residues. It is 43% identical to a previously characterized chicken protein with homology to mammalian macrophage inflammatory protein 1beta (MIP-1beta). K203 exhibits about 50% sequence identity to human MIP-1beta and other related CC chemokines.

Immunoadjuvant activities of E. coli- and plasmid-expressed recombinant chicken IFN-alpha/beta, IFN-gamma and IL-1beta in 1-day- and 3-week-old chickens.

In the present study we assessed the capacity of recombinant E. coli- or plasmid-expressed chicken interferons (IFN) and chicken IL-1beta, to exert immunostimulatory activities for humoral immune responses, in day-old and adult chickens. We observed that both recombinant E. coli-expressed chicken IFN-alpha/beta and IFN-gamma facilitated the induction of a primary and also a secondary antibody response, using tetanus toxoid (TT) as a bacterial model antigen, in immunologically mature 3-week-old chickens. In contrast, no improvement of antibody either type of chicken IFN was co-injected with inactivated Infectious Bursal Disease Virus (IBDV) antigen. TT-specific antibody formation was marginally increased by co-injection of recombinant E. coli-expressed chicken IL-1beta. Combined administration of IFN-alpha/beta plus IFN-gamma or IL-beta increased responses to TT in an additive, but not synergistic fashion. Remarkably, no augmentation of antibody responses specific for TT, nor IBDV, was noted in day-old birds, receiving IFN-alpha/beta or IFN-gamma as adjuvant. Also, intramuscular immunization of 3-week-old birds, using plasmids encoding IFN-alpha/beta together with TT protein antigen, significantly increased the speed and magnitude of TT-specific antibody responses. Plasmids encoding chicken IL-beta or IFN-gamma had a minimal or inhibitory effect, respectively. These data indicate a potential for chicken cytokines as immunoadjuvant for particular types of chicken vaccine antigens.

Protective effects of type I and type II interferons toward Rous sarcoma virus-induced tumors in chickens.

Growth of tumors induced by Rous sarcoma virus (RSV) is controlled by alleles at the major histocompatibility complex locus in chickens, indicating that immunological host defense mechanisms play a major role. We show here that the resistance phenotype of CB regressor chickens can be partially reverted by treating the animals with a monoclonal antibody that neutralizes the major serotype of chicken type I interferon, ChIFN-alpha. Injection of recombinant ChIFN-alpha into susceptible CC progressor chickens resulted in a dose-dependent inhibition of RSV-induced tumor development. This treatment was not effective, however, in CC chickens challenged with a DNA construct expressing the v-src oncogene, suggesting that the beneficial effect of type I interferon in this system resulted from its intrinsic antiviral activity and probably not from indirect immunmodulatory effects. By contrast, recombinant chicken interferon-gamma strongly inhibited tumor growth when given to CC chickens that were challenged with the v-src oncogene, indicating that the two cytokines target different steps of tumor development.

Vaccinia virus-encoded cytokine receptor binds and neutralizes chicken interferon-gamma.

To counteract the host immune response, poxviruses have evolved secreted factors that bind cytokines and thereby neutralize their biological activities. The vaccinia virus B8R gene encodes a protein that neutralizes interferon-gamma (IFN-gamma) from several mammals including man, cow, rat, and rabbit but not mice. We now report that the activity of the B8R gene product is not restricted to cytokines of mammals: it also efficiently neutralized chicken IFN-gamma. B8R blocked chicken IFN-gamma-mediated induction of guanylate binding protein RNA in the chicken fibroblast cell line CEC-32 and secretion of nitric oxide in HD-11 cells. Radiolabeled baculovirus-expressed B8R efficiently bound to immobilized recombinant chicken IFN-gamma. Scatchard analysis revealed a binding constant of chicken IFN-gamma to B8R of approximately 0.5 nM. A mutant form of chicken IFN-gamma which lacks the 18 C-terminal amino acids and which has lost more than 99% of its biological activity was able to block the IFN-gamma-neutralizing effect of B8R. Binding studies showed that the mutant protein bound radiolabeled B8R only about threefold less well than wild-type chicken IFN-gamma but failed to compete with wild-type chicken IFN-gamma for binding to the cellular receptor. These results suggest that the extreme C terminus of chicken IFN-gamma is crucial for binding to its cellular receptor but less important for recognition by the viral cytokine receptor.

A chicken homolog of mammalian interleukin-1 beta: cDNA cloning and purification of active recombinant protein.

Upon induction with lipopolysaccharide (LPS) the chicken macrophage cell line HD-11 secretes an activity that stimulates the synthesis of a CXC chemokine in the chicken fibroblast cell line CEC-32. We used a cDNA expression cloning strategy in COS cells to characterize this activity. The isolated cDNA clone codes for a polypeptide of 267 amino acids which lacks a hydrophobic N-terminal domain that could serve as secretory signal. Sequence homology and structural features indicate that this protein is the chicken homolog of mammalian interleukin-1 beta (ChIL-1 beta). Northern blot analysis showed that ChIL-1 beta RNA is quickly induced in blood monocyte-derived macrophages reaching maximal levels within one hour after onset of LPS treatment. To test for biological activity of putative mature ChIL-1 beta, a cDNA fragment comprising amino acids 106 to 267 of the open reading frame was expressed in Escherichia coli so that the resulting polypeptide carried a histidine tag at its N-terminus for easy purification by nickel chelate affinity chromatography. Purified His-ChIL-1 beta potently induced CXC chemokine RNA synthesis in CEC-32 cells. When injected intravenously into adult chickens, it quickly induced a transient increase in serum corticosterone levels.

Biological properties of recombinant chicken interferon-gamma.

Supernatants of the chicken T cell line 855 contain antiviral and macrophage activating factor activity and strongly activate transcription of the guanylate binding protein (GBP) gene in chicken cells. To characterize the cytokine responsible for the GBP-inducing activity, we chose a cDNA expression cloning strategy in COS cells. Sequencing a positive clone revealed that it encode chicken interferon-gamma (ChIFN-gamma). Histidine-tagged ChIFN-gamma was expressed in Escherichia coli and purified by nickel chelate affinity chromatography. ChIFN-gamma from COS cells and E. coli both potently induced GBP RNA synthesis but were rather poor antiviral agents. In macrophages, recombinant ChIFN-strongly stimulated secretion of nitric oxide and enhanced expression of major histocompatibility complex class II antigen. A rabbit antiserum to E. coli derived ChIFN-gamma effectively neutralized the macrophage-activating factor activity secreted by concanavalin A-induced spleen cells and various T cell lines, suggesting that IFN-gamma is the major macrophage-activating factor of the chicken.

Vesicular stomatitis virus transcription inhibited by purified MxA protein.

MxA is a GTPase encoded by an interferon-inducible human gene. Its constitutive expression renders transfected mammalian cells resistant to infections with several different RNA viruses, including vesicular stomatitis virus (VSV). Differences in viral RNA levels of VSV-infected cells either expressing or lacking MxA indicated that VSV mRNA synthesis is the principal target of MxA action. We now used purified histidine-tagged MxA (His-MxA) that we produced in Escherichia coli to successfully inhibit VSV in vitro transcription, a reaction catalyzed by VSV ribonucleoprotein complexes isolated from virus-infected cells or from purified virions. MxA was inactive when added to preformed VSV mRNAs, arguing against the possibility that it has a negative effect on viral RNA stability. MxA inhibited both leader RNA and mRNA synthesis of VSV, suggesting that it interfered with transcription initiation. The degree of VSV inhibition correlated directly with the specific GTPase activities of the various wild-type MxA preparations. No inhibition of viral mRNA synthesis was observed when a C-terminally truncated, GTPase-inactive variant of His-MxA was added to the transcription reactions. Purified His-MxA-E645R, a mutant of MxA with normal GTPase activity whose range of antiviral activity in vivo is altered so that it no longer inhibits VSV, showed no inhibitory effect on VSV in vitro transcription. Since MxA inhibited VSV RNA synthesis in the presence of GMP-PNP or GTP gamma S, GTP analogs that are readily accepted by the viral polymerase but cannot be hydrolyzed by MxA, the possibility was excluded that MxA acts by depleting the viral polymerase for its nucleotide substrates. Thus, binding of GTP rather than its hydrolysis seems of importance for the anti-VSV activity of MxA.