Chicken Interferon-gamma Research Articles

Obtaining of transgenic barrelclover plants (Medicago truncatula) producing chicken interferon gamma for veterinary use

At the Laboratory of Plant Genetic and Cellular Engineering, Department of Genetics and Biotechnology, St. Petersburg State University, five transgenic Medicago truncatula plants were obtained through Agrobacterium-mediated transformation, carrying one of the variants of the heterologous chicken interferon-gamma gene under the control of the constitutive 35S CaMV promoter. Among these, one plant harbored an unmodified gene insertion, while four had a modified gene with a deletion at the protease recognition site, providing resistance to proteolytic degradation.
 We demonstrate the application of the SWPOP-PCR “genome walking” method to determine the integration sites of T-DNA into the plant genome, identify the number of insertion copies and their orientation. Analysis of the obtained sequences revealed that only one plant exhibited a single T-DNA insertion, which represents the most optimal structure for stable expression.
 Upon self-pollination of T0 plants, 39 offspring were obtained and subjected to testing for the presence and expression of the transgene. Among them, six homozygous plants were identified using molecular methods. Quantitative assessment of transgene expression levels showed significant differences among representatives of different lines and among the offspring derived from a single transformed plant. Among the T1 and T2 progeny, the presence of the heterologous interferon protein in plant tissues was confirmed through Western blot analysis.
 The engineered barrelclover plants hold potential as bioreactors for the production of chicken interferon-gamma for veterinary applications. The use of an edible plant allows eliminating protein extraction and purification procedures, thereby resulting in a noteworthy reduction in production expenses of up to 80%.

Transcriptome analysis of pre-immune state induced by interferon gamma inhibiting the replication of H9N2 avian influenza viruses in chicken embryo fibroblasts.

Interferon (IFN), a critical antiviral cytokine produced by pathogens-induced cells, plays an important role in host innate immune system. In this study, to investigate the inhibition effect of IFN on avian influenza virus (AIV), Chicken Embryo Fibroblasts (CEFs) was infected by H9N2 AIV. The pre-immune state and transcriptome analysis have been observed and performed. The result showed chicken interferon gamma (chIFN-γ) have the most inhibitory effect on H9N2 virus among three types of chicken interferons (chIFNs). Inhibition of chIFN-γ on H9N2 virus was verified by indirect immunofluorescence, RT-qPCR and western blot. The possible signaling pathways induced by chIFN-γ with or without virus were analyzed by transcriptome. The transcriptome data were compared among H9N2-infected, chIFN-γ-treated, chIFN-γ+H9N2-treated, and Control groups. In summary, RNA-sequencing (RNA-seq) data suggested that H9N2 virus infection resulted in corresponding response of certain defensive, inflammatory and metabolism pathways to the virus replication in CEFs. Furthermore, while CEFs were treated with chIFN-γ, many immune-related signaling pathways in cells are affected and altered. Antiviral genes involved in these immune pathways such as interferon regulatory factors, chemokines, interferon-stimulated genes (ISGs) and transcription factors were significantly up-regulated, and showed significant antiviral responses. Compared with virus infected CEFs alone, pretreatment with IFN induced the expression of antiviral genes and activated related antiviral pathways, inhibited the viral replication as result. Our study provided functional annotations for antiviral genes and the basis for studying the mechanism of chIFN-γ mediated response against H9N2 AIV.

Chicken interferon gamma fold expression level and tracheal lesion score differences between commercial broiler chickens vaccinated with live and vectored ILT vaccines

Infectious laryngotracheitis (ILT) is a respiratory tract disease affecting chickens around the world. The disease generates severe production losses due to increased mortality, decreased egg production, delayed body weight gain, and a predisposition to other respiratory pathogens causing enormous economic losses to the intensive poultry industry. Two licensed vaccines, comprising live or vectored ILT, are available to control the disease. The present trial was conducted to determine changes in chicken interferon gamma fold expression levels in chicken spleens and to evaluate tracheal lesion scores before and after vaccination with live and vectored infectious laryngotracheitis vaccines. Broilers were kept under commercial conditions until 35 days of age. Spleen and trachea samples were taken at 14, 28, and 35 days of age. Tracheas were stained with H&E, histopathology was performed, and the INF-γ fold expression level in spleen samples was analyzed. In the spleens of birds vaccinated with the live ILT vaccine, the increase in INF-γ expression levels was statistically significantly (p < 0.01) lower at 28 days of age, or 7 days post vaccination, (trial 1) and higher at 35 days of age, or 14 days post vaccination, (both trials). At 7 days post vaccination with the live ILT vaccine, the mean tracheal lesion score was significantly higher (p < 0.01) than it was in the control group (both trials). At 14 days post vaccination with the live ILT vaccine, the mean tracheal lesion score was significantly higher (p < 0.05) than it was in the control group 3 (trial 1). No significant differences were found between the control group 3 and group 2, which was vaccinated with the vectored HVT/LT vaccine (both trials). The results of the histology of trachea lesions indicate that vaccination with live ILT may have induced early local immunity. The INF-γ analysis results might indicate that the birds in group 2 (HVT/LT) did not develop local immunity until 35 days of age From the practical point of view, the vectored ILT vaccine is more beneficial because of its ease of administration in the hatchery, lower labor cost, and the absence of clinical signs post vaccination. Its potential drawback, however, is poor local immunity and the slow onset of optimal immunity in commercial broiler chickens

Influence of PDI Gene Overexpression on the Production of Heterologous Proteins in Yeast Pichia pastoris

The Pichia pastoris yeast is used for recombinant secretory proteins production. The overexpression of assistant genes, the coding proteins involved in secretion, is one of the approaches used to improve the production of target proteins. PpPDI gene encodes P. pastoris yeast protein disulfide isomerase (Pdi). The aim of our study is to evaluate the effect of Pdi overproduction on the production of recombinant interferons. We obtain the yeast Pichia pastoris strains expressing PpPDI gene under the control of alcohol oxidase-1 (AOX1) promoter. The effect of the overexpression of the PpPDI gene on the production of heterogious proteins—interferon-alfa16 and chicken interferon-gamma—and on the cell growth rate is shown.

Influence of PDI gene overexpression on heterological proteins production in yeast Pichia pastoris

Summary: Background. The yeast Pichia pastoris is used for synthesis of recombinant secretory proteins. Overexpression of assistant genes, coding proteins involved in secretion, is one of approaches to improve the production of target protein. PpPDI gene encodes P. pastoris yeast protein disulfide isomerase (Pdi). The aim of our study was to evaluate the effect of Pdi overproduction on recombinant interferons (human interferon-alfa16 and chicken interferon-gamma) production.
 Materials and Methods. PpPDI gene was cloned under the control of the AOX1 gene promoter in plasmid pPICZA. Primers for AJ302014.1 nucleotide sequence of NCBI data base were used for PpPDI gene cloning. The chromosomal DNA of the GS115 strain was used as a template. To generate strains with PpPDI gene overexpression we used a previously obtained strains producing human interferon-alfa16 and chicken interferon-gamma. Yeast transformation was performed by electroporation. Cultivation was performed using single and two-stage strategies in standard media containing methanol as the sole carbon source to induce the AOX1 gene promoter.
 Results. We obtained interferon-producing strains with PpPDI gene overexpression. Over-expression of the PpPDI gene in yeast P. pastoris increases the production of interferon-alfa16, a protein containing disulfide bonds, regardless of the mode of cultivation. Effect of PpPDI gene over-expression on the production of interferon-gamma the protein without disulfide bonds, depends on cultivation mode.
 Conclusion. PpPDI gene overexpression can be used to enhance the production of interferons and other proteins that contain disulfide bonds. Effect of PpPDI gene overexpression on recombinant proteins without disulfide bonds may depend on cultivation procedure.

Characterization of two recombinant HVT-IBD vaccines by VP2 insert detection and cell-mediated immunity after vaccination of specific pathogen-free chickens

ABSTRACTInfectious bursal disease (IBD) is an avian viral disease that causes severe economic losses in the poultry industry worldwide. The live IBD virus (IBDV) has a potential immunosuppressive effect. Currently available IBDV vaccines have shortcomings, prompting the development of safer and more effective vaccination approaches, including the use of the recombinant turkey herpesvirus vaccine expressing the immunogenic structural VP2 protein of IBDV (recombinant HVT (rHVT)-IBD). The objectives of this study were twofold: (i) to develop in vitro assays and molecular tools to detect the VP2 protein and gene and (ii) to evaluate cell-mediated immunity (CMI) induced by rHVT-IBD vaccination of day-old specific pathogen-free chickens. The VP2 protein expressed by rHVT-IBD-infected chicken embryo fibroblasts was detected using the enzyme-linked immunosorbent assay and immunofluorescence. Using molecular techniques, the VP2 gene was detected in various organs, providing a method to monitor vaccine uptake. rHVT-IBD vaccination induced CMI responses in specific pathogen-free chickens at 5 weeks. CMI was detected by measuring chicken interferon-gamma after ex vivo antigenic stimulation of splenocytes. Moreover, our results showed that the enzyme-linked immunospot approach is more sensitive in detecting chicken interferon-gamma than enzyme-linked immunosorbent assay. The tools developed in this study may be useful in the characterization of new-generation recombinant vaccines and the cellular immune response they induce.

Effects of Chicken Interferon Gamma on Newcastle Disease Virus Vaccine Immunogenicity.

More effective vaccines are needed to control avian diseases. The use of chicken interferon gamma (chIFNγ) during vaccination is a potentially important but controversial approach that may improve the immune response to antigens. In the present study, three different systems to co-deliver chIFNγ with Newcastle disease virus (NDV) antigens were evaluated for their ability to enhance the avian immune response and their protective capacity upon challenge with virulent NDV. These systems consisted of: 1) a DNA vaccine expressing the Newcastle disease virus fusion (F) protein co-administered with a vector expressing the chIFNγ gene for in ovo and booster vaccination, 2) a recombinant Newcastle disease virus expressing the chIFNγ gene (rZJ1*L/IFNγ) used as a live vaccine delivered in ovo and into juvenile chickens, and 3) the same rZJ1*L/IFNγ virus used as an inactivated vaccine for juvenile chickens. Co-administration of chIFNγ with a DNA vaccine expressing the F protein resulted in higher levels of morbidity and mortality, and higher amounts of virulent virus shed after challenge when compared to the group that did not receive chIFNγ. The live vaccine system co-delivering chIFNγ did not enhanced post-vaccination antibody response, nor improved survival after hatch, when administered in ovo, and did not affect survival after challenge when administered to juvenile chickens. The low dose of the inactivated vaccine co-delivering active chIFNγ induced lower antibody titers than the groups that did not receive the cytokine. The high dose of this vaccine did not increase the antibody titers or antigen-specific memory response, and did not reduce the amount of challenge virus shed or mortality after challenge. In summary, regardless of the delivery system, chIFNγ, when administered simultaneously with the vaccine antigen, did not enhance Newcastle disease virus vaccine immunogenicity.

Pre-immune state induced by chicken interferon gamma inhibits the replication of H1N1 human and H9N2 avian influenza viruses in chicken embryo fibroblasts.

BackgroundInterferon gamma (IFN-γ), an immunoregulatory cytokine, is known to control many microbial infections. In a previous study, chicken interferon gamma (chIFN-γ) was found to be up-regulated following avian influenza virus (AIV) infection in specific pathogen-free chickens. We aimed to investigate whether the pre-immune state induced by chIFN-γ could generate an antiviral response against influenza virus.MethodsWe generated a chIFN-γ-expressing plasmid and transfected it into chicken embryo fibroblasts (CEFs) and then infected the cells with human origin H1N1 or avian origin H9N2 influenza viruses. Viral titers of culture medium were evaluated in MDCK cell and the viral RNA and IFN-stimulated genes (ISGs) were then quantified by real-time reverse transcriptase polymerase. To further evaluate the role of the antiviral effect of chIFN-γ by using a backward approach, synthetic small interfering RNAs (siRNA) targeting chIFN-γ were used to suppress chIFN-γ.ResultsThe chIFN-γ-stimulated CEFs inhibited the replication of viral RNA (vRNA) and showed a mild decrease in the infectious virus load released in the culture medium. Compared to the mock-transfected control, the messenger RNA (mRNA) levels of type I IFNs and IFN-stimulated genes were up-regulated in the cells expressing chIFN-γ. After treatment with the siRNA, we detected a higher expression of viral genes than that observed in the mock-transfected control.ConclusionsOur results suggest that apart from the important role played by chIFN-γ in the antiviral state generated against influenza virus infection, the pre-immune state induced by chIFN-γ can be helpful in mitigating the propagation of influenza virus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0527-1) contains supplementary material, which is available to authorized users.

Immune response assessment of inactivated Newcastle disease virus liposomal-based vaccine

Liposomes were evaluated as an alternative carrier to deliver inactivated Newcastle disease virus (NDV). The ability of a liposomal-based NDV vaccine to activate both cell-mediated immunity (CMI) and humoral immunity against NDV was assessed and compared with conventional NDV vaccines. Birds were assigned to 4 groups and received either no vaccine (control), NDV entrapped into liposomes (LN-NDV), NDV with oil adjuvant (oil-NDV), or live attenuated NDV (live-NDV). All birds were stimulated after 40 days of vaccination i<em>n vivo</em> by an intravenous injection of inactivated NDV crude antigen, which is considered an <em>in vivo</em> NDV-specific stimulation of the chicken immune system. After vaccination and<em> in vivo</em> stimulation, serum samples were collected for NDV-specific antibody response evaluation by a hemagglutination inhibition test (HI) and an enzyme-linked immune sorbent assay (ELISA). The CMI and humoral immunity were evaluated by a measurement of the chicken interferon gamma and specific antibody response in the serum, respectively. Conventional NDV vaccines were able to stimulate a strong humoral and CMI response. Although the newly tested vaccine induced a weak NDV-specific antibody response after vaccination, the response was highly up-regulated, several folds above the protective level, after in vivo stimulation. All NDV vaccine formulas were able to induce a CMI response after vaccination at variable time points. This study revealed that a liposomal NDV-based vaccination in this experimental model tends to induce CMI and can only be beneficial in priming vaccinated birds to promote a strong anti-body response to later NDV exposure.

A novel impedimetric immunosensor for the detection of chicken interferon-gamma based on a polythionine and gold nanoparticle modified glassy carbon electrode

Currently, there is no effective detection method for chicken interferon-γ (ChIFN-γ), therefore it is of great significance to develop a new, accurate and sensitive method for the rapid detection of ChIFN-γ to solve the problems which are directly impeding the in-depth research of immune regulatory factors and poultry immune responses, and which are preventing the analysis and evaluation of the immunity of poultry in diseases or after vaccination. This paper is the first report of a novel label-free electrochemical impedimetric immunosensor for the sensitive detection of ChIFN-γ, based on a 3E3 monoclonal antibody against chicken interferon-γ (denoted as 3E3McAb) immobilized on a glassy carbon electrode modified with polythionine and gold nanoparticle self-assembled layers. The surface modification steps were characterized by scanning electron microscopy and electrochemical impedance spectroscopy (EIS). Detection was performed by measuring the relative change in impedance before and after the 3E3McAb–ChIFN-γ reaction by EIS. Under the optimized conditions, the relative change in impedance was proportional to the logarithmic value of ChIFN-γ concentrations in the range of 0.10 to 1.0 × 103 ng mL−1 (r = 0.9946) with a detection limit of 0.10 ng mL−1. Compared to the traditional method of enzyme-linked immunosorbent assay, the proposed method performed a low-cost, sensitive, quantitative detection of ChIFN-γ in real samples with satisfactory results, indicating that the developed immunoassay could be a promising alternative approach for the detection of ChIFN-γ.

Interferon-γ influences immunity elicited by vaccines against very virulent Marek’s disease virus

Vaccination of chickens with herpesvirus of turkey (HVT) confers only partial protection against challenge with a very virulent Marek’s disease virus (MDV). Here, we evaluated the ability of recombinant chicken interferon-gamma (rChIFN-γ) to enhance protective efficacy of HVT against the very virulent MDV strain, RB1B. The bioactivity of IFN-γ expressed by a plasmid expression vector was confirmed by its ability to stimulate a chicken macrophage cell line (HD11) to produce nitric oxide (NO) in vitro. The administration of HVT with 5 μg of pcDNA:chIFN-γ plasmid reduced the incidence of tumor development significantly when compared to vaccinated birds (77.7% in the HVT + empty vector group and 80% in HVT group versus 33.3% in the HVT + chIFN-γ group) and significantly increased IFN-γ expression in the splenocytes of the protected group, suggesting that rChIFN-γ increases the potency of HVT against MDV. Further analysis demonstrated that the protected birds that received HVT vaccine and/or plasmid had lower MDV genome load and lower amounts of transcripts for meq and vIL-8 than in the birds without lesions. Similarly, lower expression of IL-10, IL-18 and IL-6 was observed in the chickens without lesions compared to the chickens that had lesions, suggesting an inverse association between up-regulation of these cytokines and vaccine-induced immunity. In conclusion, IFN-γ can positively influence immunity conferred by HVT vaccination against challenge with a very virulent Marek’s disease virus (vvMDV) in chickens.

Assessment of Bioactivity of a Recombinant Chicken Interferon-Gamma Expressed Using a Baculovirus Expression System

The full-length coding sequence of chicken interferon-γ (ChIFN-γ) was cloned into a baculovirus nonfusion vector, pFastBacDual, and expressed in Sf21 insect cells. Recombinant ChIFN-γ (rChIFN-γ) protein was found to be expressed both intracellularly as well as in the culture supernatants. The affinity-purified rChIFN-γ contained 14, 17, and 28 kDa proteins, possibly representing both glycosylated and nonglycosylated protein forms of ChIFN-γ. The bioactivity of rChIFN-γ was confirmed in vitro by production of nitric oxide in a chicken macrophage cell line (HD11) and antiviral activity against vesicular stomatitis virus in primary chicken embryonic fibroblast cells. Further, HD11 cells stimulated with rChIFN-γ showed significant upregulation of inducible nitric oxide synthases, IFN-γ, interleukin-1β, interleukin-12p35, signal transducers and activators of transcription 1, class II, major histocompatibility complex, transactivator, and major histocompatibility complex II-β chain (BL-B) transcripts. In conclusion, the present study provides information on the ability of functionally active rChIFN-γ expressed in a baculovirus system in inducing significant transcriptional upregulation of various immune system-related genes, including those that encode cytokines, antigen-presenting molecules, and transcription factors involved in the major histocompatibility complex and IFN-signaling pathway.

Enhancement of immunogenicity of a virosome-based avian influenza vaccine in chickens by incorporating CpG-ODN

Influenza virosomes are virus-like particles, representing a platform for vaccine development. In this study, we examined the immunogenicity of avian influenza virosomes with or without inclusion of recombinant chicken interferon-gamma (rChIFN-γ) or CpG-ODN in chickens. Immunization with virosomes adjuvanted with CpG-ODN elicited the highest haemagglutination inhibition antibody titres, as well as IgG and IgA serum antibody responses. Moreover, Virosomes+CpG-ODN formulation induced an antigen-specific spleen cell proliferation and IFN-γ expression. In conclusion, our results demonstrated that virus-specific antibody- and cell-mediated responses may be induced in chickens immunized with virosomes and these responses can be enhanced by incorporating CpG-ODN in the virosome vaccine formulation.

The DNA-induced protective immunity with chicken interferon gamma against poultry coccidiosis

The immunogenicity of Eimeria acervulina cSZ-2 and chicken interferon gamma was observed against Eimeria tenella challenge. The chickens were randomly divided into six groups of 24 chicks each. Three groups of chickens were injected with DNA vaccines pVAX1-cSZ2, pVAX1-chIFN-gamma and pVAX1-cSZ2-chIFN-gamma two times (at days 14 and 21) at a dose of 100 microg intramuscularly. Three other groups were kept as control and injected with TE buffer (10 mM Tris-HCl pH 7.6 and 1 mM EDTA). One week following the booster dose, all chickens except the non-infected, non-vaccinated control group were inoculated orally with 5 x 10(4) sporulated oocysts of E. tenella. Seven days post challenge, all chickens were weighted and slaughtered for cecal lesion scoring and oocyst counts. The results demonstrated that cSZ-2 in combination with interferon gamma can protect chickens from coccidiosis by significantly decreasing body weight loss and oocyst excretion reflecting partial protection against E. tenella infection, and further studies are necessary to test for protection against other Eimeria species.

Cloning, Prokaryotic Expression, and Biological Analysis of Recombinant Chicken IFN-γ

The full-length chicken interferon-gamma (CHIFN-gamma) gene was amplified by reverse transcription-PCR using total RNA extracted from the spleen cells of white Leghorn chicken, a local Chinese breeding species. A truncated CHIFN-gamma gene without the N-terminal signal peptide sequence was cloned into prokaryotic expression vector pET30a, resulting in a recombinant plasmid pET-30a-CHIFN-gamma. After the recombinant plasmid was transformed into host cells BL21(DE3)pLysS, the expression of CHIFN-gamma was induced by isopropyl beta-D-thiogalactoside (IPTG). Rabbit antiserum was raised using the soluble CHIFN-gamma as immunogen. Immunoreactivities of the CHIFN-gamma and its antiserum were investigated using immunoblotting and ELISA. Moreover, the antiviral effect of the CHIFN-gamma was analyzed. Our data indicate that the CHIFN-gamma is biologically active.

Heterologous expression of synthetic chicken IFN-γ in transgenic tobacco plants

AbstractTo develop a plant expression system for the production of the chicken interferon gamma (ChIFN-γ) oral vaccine adjuvant, we investigated whether the ChIFN-γ protein can be expressed in tobacco plants. The coding sequence of the ChIFN-γ gene was optimized by modification of codon usage to that of tobacco plant genes. A synthetic ChIFN-γ gene was inserted into plasmid pSW-IFNG containing the CaMV35S promoter, NOS terminator, GUS (β-glucuronidase) reporter gene and the nptII resistance gene. The synthetic ChIFN-γ gene, along with an endoplasmic reticulum retention signal (SEKDEL) was introduced into tobacco plants via Agrobacterium-mediated transformation. PCR analysis confirmed the presence of the ChIFN-γ gene in transformants. Histochemical GUS assay of the tobacco leaves revealed stable integration of ChIFN-γ into the genome. RT-PCR analysis revealed the presence of ChIFN-γ-specific transcript. Bands of approximately 30 and 40 kDa were detected by Western analysis of transgenic tobacco leaves using an anti-chicken IFN-γ antibody. Furthermore, quantitative ELISA detected ChIFN-γ protein, suggesting that tobacco leaves expressed ChIFN-γ of up to 10 to 20 µg/g fresh leaf weight, or 0.02 to 0.04% of total soluble protein. ChIFN-γ present in tobacco extracts possessed antiviral activity (4.8×105 IU/mg in vitro). The present results indicate that the ChIFN-γ introduced into tobacco plant was correctly transcribed and translated in plant. High-level expression of biologically active ChIFN-γ will allow further studies of oral administration and therapeutic effects of this cytokine.

Immunomodulatory Effect of Recombinant Chicken Interferon-gamma (rchIFN-γ) on Specific and Non-specific Immune Responses in Chicken Vaccinated Against Newcastle Disease Virus (NDV)

Present study was undertaken to evaluate the immunomodulatory effect of recombinant chicken interferon -gamma (rchIFN-γ) on specific and non-specific immune responses in chicken vaccinated against Newcastle Disease Virus (NDV). A total of 180 day old layer chicks (BV-300) were divided into three groups. Test Group I (TG-I) (64 chicks) was injected with 5 microgram/chick subcutaneously of rchIFN-γ along with ND vaccinations (LaSota and R B strains), Test Group II (TG-II) (64 chicks) was injected with same dose and 2 route of rchIFN-γ 6 h after ND vaccinations on both occasions, whereas, third group remained as vaccinated control group (52 chicks). Haemagglutination Inhibition (HI) test and Migration Inhibition (MI) in percentages estimated by Leukocyte Migration Inhibition Test (LMIT) were conducted to evaluate humoral and cell mediated immune responses respectively. Nitroblue Tetrazolium (NBT) reduction assay was conducted to evaluate the non-specific immune response by estimating the Phagocytic Index (PI) in percentages. The test results revealed that, significantly (p< 0.05) increased specific (humoral and cell mediated) and non-specific immune responses were recorded in the groups treated with rchIFN-γ i.e., higher results when given along with the vaccine and highest results when given 6 h after vaccinations. The results advocated that, rchIFN-γ may be used as an immunopotentiator in chicken vaccinated against NDV.

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.

Molecular Cloning and Characterization of Chicken Interferon-γ Receptor α-Chain

In this study, a cDNA sequence of Huiyang chicken interferon-gamma (IFN-gamma) receptor alpha-chain (chIFNGR-1) gene wasgenerated using rapid amplification of cDNA ends (RACE) method for the first time. The predicted 422 amino acids showed approximately 25%-29% sequence identity and 53%-55% similarity to mammalian homologues. There are two fibronectin type-III (FN-III) domains of about 110 residues in the extracellular domain, and LPKS and YDKPH motifs in the intracellular domain, which are conserved in the mammalian IFNGR-1 as the binding sites of JAK1 and STAT1. Expression analysis by Northern blot revealed that the chIFNGR-1 was highly expressed in spleen, thymus, peripheral blood lymphocytes (PBLs), lung, cecum tonsil, and liver. The extracellular region of chIFNGR-1 (chIFNGR-1EC) was expressed in Escherichia coli and purified. The purified IFNGR-1EC was further characterized by mass spectroscopy and circular dichroism (CD) spectroscopy. The molecular weight of the recombinant chIFNGR-1EC (rchIFNGR-1EC) was measured as 24 364 Da, and its secondary structure contained 17.6% alpha-helix, 36.4% beta-sheet, 17.2% turn, and 28.8% random coil. Furthermore, three-dimensional modeling presented the most probable structure of chIFNGR-1EC. These * ndings show that the identified chicken cDNA sequence encodes an IFNGR1 homologue, and the chIFNGR-1EC resembles the similar structure with other IFN receptors.

ELISPOT and intracellular cytokine staining: Novel assays for quantifying T cell responses in the chicken

The measurement of T cell responses in chickens, not only for quantitative aspects but also for the qualitative nature of the responses, becomes increasingly important. However, there are very few assays available to measure T cell function. Therefore, we have developed enzyme-linked immunosorbent spot assay (ELISPOT) and an intracellular cytokine staining (ICCS) assay. ELISPOT assay for the detection of chicken interferon-gamma (ChIFN-gamma) production was set up and shown to be reproducible for both polyclonal and antigen-specific stimuli such as Newcastle disease virus (NDV). However, the ELISPOT assay lacks the ability to identify individual cytokine-producing cells. Separation of CD4+ and CD8+ T cell populations gave additional information, but appeared to have the disadvantage of a loss of cell interactions during stimulation. In a further refinement, individual cells were identifiable by ICCS, which gives the possibility to characterize for multiple characteristics, such as cytokine production and phenotype of the cell. Using ICCS, ChIFN-gamma production was evaluated. Although cells were detected at only low frequencies, polyclonal stimulation of peripheral blood mononuclear cell (PBMC) or spleen cells resulted in a significant increase in ChIFN-gamma production by CD4+ and CD8+ cells.