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Generation and characterization of chicken bone marrow‐derived dendritic cells

Dendritic cells (DCs) are bone marrow‐derived professional antigen‐presenting cells. The in vitro generation of DCs from either bone marrow or blood is routine in mammals. Their distinct morphology and phenotype and their unique ability to stimulate naïve T cells are used to define DCs. In this study, chicken bone marrow cells were cultured in the presence of recombinant chicken granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and recombinant chicken interleukin‐4 (IL‐4) for 7 days. The cultured population showed the typical morphology of DCs, with the surface phenotype of major histocompatibility complex (MHC) class II+ (high), CD11c+ (high), CD40+ (moderate), CD1·1+ (moderate), CD86+ (low), CD83− and DEC‐205−. Upon maturation with lipopolysaccharide (LPS) or CD40L, surface expression of CD40, CD1·1, CD86, CD83 and DEC‐205 was greatly increased. Endocytosis and phagocytosis were assessed by fluorescein isothiocyanate (FITC)‐dextran uptake and fluorescent bead uptake, respectively, and both decreased after stimulation. Non‐stimulated chicken bone marrow‐derived DCs (chBM‐DCs) stimulated both allogeneic and syngeneic peripheral blood lymphocytes (PBLs) to proliferate in a mixed lymphocyte reaction (MLR). LPS‐ or CD40L‐stimulated chBM‐DCs were more effective T‐cell stimulators in MLR than non‐stimulated chBM‐DCs. Cultured chBM‐DCs could be matured to a T helper type 1 (Th1)‐promoting phenotype by LPS or CD40L stimulation, as determined by mRNA expression levels of Th1 and Th2 cytokines. We have therefore cultured functional chBM‐DCs in a non‐mammalian species for the first time.

Expression levels of MHC class I molecules are inversely correlated with promiscuity of peptide binding

Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Mareks disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation. DOI: http://dx.doi.org/10.7554/eLife.05345.001

Towards a universal vaccine for avian influenza: protective efficacy of modified Vaccinia virus Ankara and Adenovirus vaccines expressing conserved influenza antigens in chickens challenged with low pathogenic avian influenza virus.

Highlights ► Current influenza vaccines do not generate heterologous protection. ► Targeting internal influenza antigens may confer cross protection. ► We tested Adenovirus and MVA vectored NP and M1 in chickens. ► Heterologous prime-boost resulted in earlier cessation of viral shedding.

Improved adjuvanting of seasonal influenza vaccines: Preclinical studies of MVA‐NP+M1 coadministration with inactivated influenza vaccine

Licensed seasonal influenza vaccines induce antibody (Ab) responses against influenza hemagglutinin (HA) that are limited in their ability to protect against different strains of influenza. Cytotoxic T lymphocytes recognizing the conserved internal nucleoprotein (NP) and matrix protein (M1) are capable of mediating a cross‐subtype immune response against influenza. Modified vaccinia Ankara (MVA) virus encoding NP and M1 (MVA‐NP+M1) is designed to boost preexisting T‐cell responses in adults in order to elicit a cross‐protective immune response. We examined the coadministration of HA protein formulations and candidate MVA‐NP+M1 influenza vaccines in murine, avian, and swine models. Ab responses postimmunization were measured by ELISA and pseudotype neutralization assays. Here, we demonstrate that MVA‐NP+M1 can act as an adjuvant enhancing Ab responses to HA while simultaneously inducing potent T‐cell responses to conserved internal Ags. We show that this regimen leads to the induction of cytophilic Ab isotypes that are capable of inhibiting hemagglutination and in the context of H5 exhibit cross‐clade neutralization. The simultaneous induction of T cells and Ab responses has the potential to improve seasonal vaccine performance and could be employed in pandemic situations.

Early replication in pulmonary B cells after infection with marek's disease herpesvirus by the respiratory route

Abstract Natural infection with Mareks disease virus occurs through the respiratory mucosa after chickens inhale dander shed from infected chickens. The early events in the lung following exposure to the feather and squamous epithelial cell debris containing the viral particles remain unclear. In order to elucidate the virological and immunological consequences of MDV infection for the respiratory tract, chickens were infected by intratracheal administration of infective dander. Differences between susceptible and resistant chickens were immediately apparent, with delayed viral replication and earlier onset of interferon (IFN)-gamma production in the latter. CD4(+) and CD8(+) T cells surrounded infected cells in the lung. Although viral replication was evident in macrophages, pulmonary B cells were the main target cell type in susceptible chickens following intratracheal infection with MDV. In accordance, depletion of B cells curtailed viremia and substantially affected pathogenesis in susceptible chickens. Together the data described here demonstrate the role of pulmonary B cells as the primary and predominant target cells and their importance for MDV pathogenesis.

Low frequency of the Mx allele for viral resistance predates recent intensive selection in domestic chickens

Avian influenza is a serious threat to the poultry industry and, as the potential source of a human pandemic virus, to public health. Different Mx alleles have been reported to confer resistance or susceptibility to influenza virus replication, and so knowledge of their frequencies is important when considering the potential for improvement of modern commercial flocks. We analysed a range of chicken lines and ancestral breeds for the relevant Mx codon that confers resistance or susceptibility to influenza virus replication. We confirmed the high frequency of the susceptibility allele in contemporary meat-type (broiler) birds compared to egg-laying strains and found this difference is present already in ancestral breeds. We sequenced full-length complementary DNA (cDNA) and noted additional substitutions, which may be associated with the resistance haplotypes. High frequencies of the susceptibility allele could be readily reduced by modern breeding techniques.

Protection from infectious bursal disease virus (IBDV)-induced immunosuppression by immunization with a fowlpox recombinant containing IBDV-VP2

Immunosuppression resulting from infectious bursal disease virus (IBDV) infection has critical health and welfare implications for birds, yet it is incompletely understood and largely overlooked as a measure of vaccine efficacy. The ability of a fowlpoxvirus recombinant (fpIBD1) containing the VP2 protein of IBDV to protect against IBDV-induced immunosuppression was investigated by measuring the convalescent chickens ability to mount antibody responses to IBDV infection, and to inactivated IBDV and salmonella vaccines. An immunoglobulin (Ig)M response, but no IgG response, occurred after IBDV infection. Uninfected chickens produced a sustained IgM response and some IgG response to inactivated IBDV vaccine, while in previously infected birds only a transient IgM response was detected. A moderate suppression of the response to a commercial salmonella vaccine was evident after IBDV infection, which was largely prevented by immunization with fpIBD1. These results indicate that measurement of immunosuppression could be a useful strategy for assessing the efficacy of vaccines to protect against the consequences of IBDV infection.

Expression of Chicken DEC205 Reflects the Unique Structure and Function of the Avian Immune System

The generation of appropriate adaptive immune responses relies critically on dendritic cells, about which relatively little is known in chickens, a vital livestock species, in comparison with man and mouse. We cloned and sequenced chicken DEC205 cDNA and used this knowledge to produce quantitative PCR assays and monoclonal antibodies to study expression of DEC205 as well as CD83. The gene structure of DEC205 was identical to those of other species. Transcripts of both genes were found at higher levels in lymphoid tissues and the expression of DEC205 in normal birds had a characteristic distribution in the primary lymphoid organs. In spleen, DEC205 was seen on cells ideally located to trap antigen. In thymus it was found on cells thought to participate in the education of T cells, and in the bursa on cells that may be involved in presentation of antigen to B cells and regulation of B cell migration. The expression of DEC205 on cells other than antigen presenting cells (APC) is also described. Isolated splenocytes strongly expressing DEC205 but not the KUL01 antigen have morphology similar to mammalian dendritic cells and the distinct expression of DEC205 within the avian-specific Bursa of Fabricius alludes to a unique function in this organ of B cell diversification.

Analysis of the Early Immune Response to Infection by Infectious Bursal Disease Virus in Chickens Differing in Their Resistance to the Disease

ABSTRACT Chicken whole-genome gene expression arrays were used to analyze the host response to infection by infectious bursal disease virus (IBDV). Spleen and bursal tissue were examined from control and infected birds at 2, 3, and 4 days postinfection from two lines that differ in their resistance to IBDV infection. The host response was evaluated over this period, and differences between susceptible and resistant chicken lines were examined. Antiviral genes, including IFNA, IFNG, MX1, IFITM1, IFITM3, and IFITM5, were upregulated in response to infection. Evaluation of this gene expression data allowed us to predict several genes as candidates for involvement in resistance to IBDV. IMPORTANCE Infectious bursal disease (IBD) is of economic importance to the poultry industry and thus is also important for food security. Vaccines are available, but field strains of the virus are of increasing virulence. There is thus an urgent need to explore new control solutions, one of which would be to breed birds with greater resistance to IBD. This goal is perhaps uniquely achievable with poultry, of all farm animal species, since the genetics of 85% of the 60 billion chickens produced worldwide each year is under the control of essentially two breeding companies. In a comprehensive study, we attempt here to identify global transcriptomic differences in the target organ of the virus between chicken lines that differ in resistance and to predict candidate resistance genes.

Route of challenge is critical in determining the clinical outcome of infection with a very virulent oncogenic herpesvirus, Marek?s disease virus.

The majority of experimental studies examining Mareks disease virus infection have used parenteral injection of cell-associated virus. The aim of this study was to examine whether the route of entry of virus was critical in determining the outcome of infection. Susceptible (L7) and resistant (L6) White Leghorn chickens were infected with a very virulent Mareks disease virus, RB1B, by either the intra-abdominal or intra-tracheal route. Birds infected by the intra-tracheal route had earlier, higher or more sustained blood, spleen and lung viral concentrations than those infected by the intra-abdominal route. L7 birds had higher viral loads than L6 birds infected by the same route. Clinical outcomes reflected these data. Resistant birds infected by the intra-tracheal route had an increased prevalence of tumours and shorter survival times compared with those infected by the intra-abdominal route. Susceptible birds infected by the intra-tracheal route became paralysed 10 days after infection. L7 birds had shorter survival times and increased prevalences of tumours than L6 birds. The pathology and viraemia seen with intra-tracheal infection could not be fully replicated by increasing the dose in intra-abdominal infections. We conclude that instillation of infective dust produces a more aggressive infection that depends on the route of entry and form of virus, and not just on the challenge dose.