Karen Staines

Variation in the number of expressed MHC genes in different cattle class I haplotypes.

Abstract Analysis of cattle major histocompatibility complex (MHC) (BoLA) class I gene expression using serological and biochemical methods has demonstrated a high level of polymorphism. However, analysis of class I cDNA sequences has failed to produce conclusive evidence concerning the number and nature of expressed genes. Such information is essential for detailed studies of cattle immune responses, and to increase our understanding of the mechanisms of MHC evolution. In this study a selective breeding programme has been used to generate a number of MHC homozygous cattle expressing common serologically defined class I specificities. Detailed analysis of five class I haplotypes was carried out, with transcribed class I genes identified and characterized by cDNA cloning, sequence analysis, and transfection/expression studies. Surface expression of the gene products (on lymphocytes) was confirmed using monoclonal antibodies of defined BoLA specificity. Phylogenetic analysis of available transcribed cattle MHC class I sequences revealed complex evolutionary relationships including possible evidence for recombination. The study of individual haplotypes suggests that certain groupings of related sequences may correlate with loci, but overall it was not possible to define the origin of individual alleles using this approach. The most striking finding of this study is that none of the cattle class I genes is consistently expressed, and that in contrast to human, haplotypes differ from one another in both the number and composition of expressed classical class I genes.

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.

CDNA SEQUENCE OF CATTLE MHC CLASS I GENES TRANSCRIBED IN SEROLOGICALLY DEFINED HAPLOTYPES A18 AND A31

The influence of major histocompatibility complex (MHC) phenotype on cattle immune responses and susceptibility to disease is at present poorly understood, due to a lack of detailed information regarding specific MHC genes and products. Since 1988, eleven BoLA class I nucleotide sequences have been published (Ennis et al. 1988; Brown et al. 1989; Bensaid et al. 1991; Garber et al. 1994; Sawhney et al. 1995). Despite this, it still remains unclear how many classical class I loci are present in the cattle genome, whether the number and nature of genes transcribed is consistent between haplotypes and breeds, and whether the products of the different genes carry out exactly the same function. Our aim in this study was to carry out a detailed analysis of well-defined class I haplotypes, using cDNA cloning, sequence analysis, and transfection/expression studies in order to identify the genes coding for the ubiquitously expressed, antigen presenting class I molecules. Alternative approaches have been described, for example polymerase chain reaction amplification of class I sequences from cDNA (Garber et al. 1994), and identification of expressed genomic clones (Sawhney et al. 1995) but neither of these methods provides information regarding the level of transcription of individual genes. Limited data suggest that there are at least ten class I genes in the cattle MHC (Lindberg et al. 1988), some of which are not transcribed in lymphocytes but do encode molecules which can be expressed and recognized by monoclonal antibodies (mAbs) to cattle class I, and may represent non-classical genes (N. Barker, personal communication). Identification of expressed class I molecules by transfection of genomic clones could therefore be mislead-

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.

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.

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.

Sequence of a complete chicken BG haplotype shows dynamic expansion and contraction of two gene lineages with particular expression patterns.

Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility complex (MHC), and show striking association with particular autoimmune diseases. In chickens, BG genes encode homologues with somewhat different domain organisation. Only a few BG genes have been characterised, one involved in actin-myosin interaction in the intestinal brush border, and another implicated in resistance to viral diseases. We characterise all BG genes in B12 chickens, finding a multigene family organised as tandem repeats in the BG region outside the MHC, a single gene in the MHC (the BF-BL region), and another single gene on a different chromosome. There is a precise cell and tissue expression for each gene, but overall there are two kinds, those expressed by haemopoietic cells and those expressed in tissues (presumably non-haemopoietic cells), correlating with two different kinds of promoters and 5′ untranslated regions (5′UTR). However, the multigene family in the BG region contains many hybrid genes, suggesting recombination and/or deletion as major evolutionary forces. We identify BG genes in the chicken whole genome shotgun sequence, as well as by comparison to other haplotypes by fibre fluorescence in situ hybridisation, confirming dynamic expansion and contraction within the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood.

The BAFF-Interacting receptors of chickens

The TNF superfamily cytokine BAFF has crucial roles in homoeostatic regulation of B cell populations in mammals. Similar effects on peripheral B cells have been reported for chicken as for mammalian BAFF. Unlike mammalian BAFF, chicken BAFF is produced by B cells, implying an autocrine loop and consequent differences in regulation of B cell homoeostasis. Understanding of these mechanisms requires investigation of BAFF-binding receptors in chickens. We identified and characterised chicken receptors BAFFR and TACI, but found that the gene encoding the third BAFF-binding receptor, BCMA, was disrupted, implying differences in mechanisms for maintenance of long-lived antibody responses. A BAFFR-Ig fusion protein expressed in vivo lowered B cell numbers, showing that it was functional under physiological conditions. We found changes in the ratio of BAFFR and TACI mRNAs in the bursa after hatch that may account for the altered requirements for B cell survival at this stage of development.

The peptide motif of the single dominantly expressed class I molecule of the chicken MHC can explain the response to a molecular defined vaccine of infectious bursal disease virus (IBDV).

In contrast to typical mammals, the chicken MHC (the BF-BL region of the B locus) has strong genetic associations with resistance and susceptibility to infectious pathogens as well as responses to vaccines. We have shown that the chicken MHC encodes a single dominantly expressed class I molecule whose peptide-binding motifs can determine resistance to viral pathogens, such as Rous sarcoma virus and Marek’s disease virus. In this report, we examine the response to a molecular defined vaccine, fp-IBD1, which consists of a fowlpox virus vector carrying the VP2 gene of infectious bursal disease virus (IBDV) fused with β-galactosidase. We vaccinated parental lines and two backcross families with fp-IBD1, challenged with the virulent IBDV strain F52/70, and measured damage to the bursa. We found that the MHC haplotype B15 from line 15I confers no protection, whereas B2 from line 61 and B12 from line C determine protection, although another locus from line 61 was also important. Using our peptide motifs, we found that many more peptides from VP2 were predicted to bind to the dominantly expressed class I molecule BF2*1201 than BF2*1501. Moreover, most of the peptides predicted to bind BF2*1201 did in fact bind, while none bound BF2*1501. Using peptide vaccination, we identified one B12 peptide that conferred protection to challenge, as assessed by bursal damage and viremia. Thus, we show the strong genetic association of the chicken MHC to a T cell vaccine can be explained by peptide presentation by the single dominantly expressed class I molecule.