Identification of immediate early gene products of bovine herpes virus 1 (BHV-1) as dominant antigens recognized by CD8 T cells in immune cattle.

In common with other herpes viruses, bovine herpes virus 1 (BHV-1) induces strong virus-specific CD8 T-cell responses. However, there is a paucity of information on the antigenic specificity of the responding T-cells. The development of a system to generate virus-specific CD8 T-cell lines from BHV-1-immune cattle, employing Theileria-transformed cell lines for antigen presentation, has enabled us to address this issue. Use of this system allowed the study to screen for CD8 T-cell antigens that are efficiently presented on the surface of virus-infected cells. Screening of a panel of 16 candidate viral gene products with CD8 T-cell lines from 3 BHV-1-immune cattle of defined MHC genotypes identified 4 antigens, including 3 immediate early (IE) gene products (ICP4, ICP22 and Circ) and a tegument protein (UL49). Identification of the MHC restriction specificities revealed that the antigens were presented by two or three class I MHC alleles in each animal. Six CD8 T-cell epitopes were identified in the three IE proteins by screening of synthetic peptides. Use of an algorithm (NetMHCpan) that predicts the peptide-binding characteristics of restricting MHC alleles confirmed and, in some cases refined, the identity of the epitopes. Analyses of the epitope specificity of the CD8 T-cell lines showed that a large component of the response is directed against these IE epitopes. The results indicate that these IE gene products are dominant targets of the CD8 T-cell response in BHV-I-immune cattle and hence are prime-candidate antigens for the generation of a subunit vaccine.

Identification of Theileria lestoquardi Antigens Recognized by CD8+ T Cells.

As part of an international effort to develop vaccines for Theileria lestoquardi, we undertook a limited screen to test T. lestoquardi orthologues of antigens recognised by CD8+ T lymphocyte responses against T. annulata and T. parva in cattle. Five MHC defined sheep were immunized by live T. lestoquardi infection and their CD8+ T lymphocyte responses determined. Thirteen T. lestoquardi orthologues of T. parva and T. annulata genes, previously shown to be targets of CD8+ T lymphocyte responses of immune cattle, were expressed in autologous fibroblasts and screened for T cell recognition using an IFNγ assay. Genes encoding T. lestoquardi antigens Tl8 (putative cysteine proteinase, 349 aa) or Tl9 (hypothetical secreted protein, 293 aa) were recognise by T cells from one animal that displayed a unique MHC class I genotype. Antigenic 9-mer peptide epitopes of Tl8 and Tl9 were identified through peptide scans using CD8+ T cells from the responding animal. These experiments identify the first T. lestoquardi antigens recognised by CD8+ T cell responses linked to specific MHC class I alleles.

Limited genetic and antigenic diversity within parasite isolates used in a live vaccine against Theileria parva.

An infection and treatment protocol is used to vaccinate cattle against Theileria parva infection. Due to incomplete cross-protection between different parasite isolates, a mixture of three isolates, termed the Muguga cocktail, is used for vaccination. While vaccination of cattle in some regions provides high levels of protection, some animals are not protected against challenge with buffalo-derived T. parva. Knowledge of the genetic composition of the Muguga cocktail vaccine is required to understand how vaccination is able to protect against field challenge and to identify the potential limitations of the vaccine. The aim of the current study was to determine the extent of genetic and antigenic diversity within the parasite isolates that constitute the Muguga cocktail. High throughput multi-locus sequencing of antigen-encoding loci was performed in parallel with typing using a panel of micro- and mini-satellite loci. The former focused on genes encoding CD8(+) T cell antigens, believed to be relevant to protective immunity. The results demonstrate that each of the three component stocks of the cocktail contains limited parasite genotypic diversity, with single alleles detected at many gene/satellite loci and, moreover, that two of the components show a very high level of similarity. Thus, the vaccine incorporates very little of the genetic and antigenic diversity observed in field populations of T. parva. The presence of alleles at low frequency (<10%) within vaccine component populations also points to the possibility of variability in the content of vaccine doses and the potential for loss of allelic diversity during tick passage. The results demonstrate that there is scope to modify the content of the vaccine in order to enhance its diversity and thus its potential for providing broad protection. The ability to accurately quantify genetic diversity in vaccine component stocks will facilitate improved quality control procedures designed to ensure the long-term efficacy of the vaccine.

Haematopoietic depletion in vaccine-induced neonatal pancytopenia depends on both the titre and specificity of alloantibody and levels of MHC I expression.

Bovine Neonatal Pancytopenia (BNP) is a disease of calves characterised by haematopoietic depletion, mediated by ingestion of alloantibodies in colostrum. It has been linked epidemiologically to vaccination of the dams of affected calves with a particular vaccine (Pregsure) containing a novel adjuvant. Evidence suggests that BNP-alloantibodies are directed against MHC I molecules, induced by contaminant bovine cellular material from Madin-Darby Bovine Kidney (MDBK) cells used in the vaccine's production. We aimed to investigate the specificity of BNP-alloantibody for bovine MHC I alleles, particularly those expressed by MDBK cells, and whether depletion of particular cell types is due to differential MHC I expression levels. A complement-mediated cytotoxicity assay was used to assess functional serum alloantibody titres in BNP-dams, Pregsure-vaccinated dams with healthy calves, cows vaccinated with an alternative product and unvaccinated controls. Alloantibody specificity was investigated using transfected mouse lines expressing the individual MHC I alleles identified from MDBK cells and MHC I-defined bovine leukocyte lines. All BNP-dams and 50% of Pregsure-vaccinated cows were shown to have MDBK-MHC I specific alloantibodies, which cross-reacted to varying degrees with other MHC I genotypes. MHC I expression levels on different blood cell types, assessed by flow cytometry, were found to correlate with levels of alloantibody-mediated damage in vitro and in vivo. Alloantibody-killed bone marrow cells were shown to express higher levels of MHC I than undamaged cells. The results provide evidence that MHC I-specific alloantibodies play a dominant role in the pathogenesis of BNP. Haematopoietic depletion was shown to be dependent on the titre and specificity of alloantibody produced by individual cows and the density of surface MHC I expression by different cell types. Collectively, the results support the hypothesis that MHC I molecules originating from MDBK cells used in vaccine production, coupled with a powerful adjuvant, are responsible for the generation of pathogenic alloantibodies.

Understanding the basis of parasite strain-restricted immunity to Theileria parva.

Infection with Theileria parva is asymptomatic in African buffalo but results in severe disease in cattle. Currently, vaccination relies on infection and treatment, using a mixture of three parasite isolates to overcome the strain specificity of immunity. Genotypic analyses of field populations of T. parva indicate a panmictic population structure, reflecting frequent sexual recombination. Profound immunodominance of protective CD8 T cell responses, together with polymorphism of the target antigens and frequent genetic recombination, contribute to the strain-restricted immunity. The dominant CD8 target antigens are highly polymorphic, but the live vaccine appears to contain limited diversity. A model to explain the ability of the vaccine to confer immunity against highly diverse field parasite challenge is discussed. Parasites in cattle exhibit much more limited antigenic diversity than parasites in buffalo, consistent with other evidence that the cattle-maintained population represents a subset of T. parva recently adapted to cattle.

Theileria annulata-transformed cell lines are efficient antigen-presenting cells for in vitro analysis of CD8 T cell responses to bovine herpesvirus-1.

Continuously growing cell lines infected with the protozoan parasite Theileria annulata can readily be established by in vitro infection of leukocytes with the sporozoite stage of the parasite. The aim of the current study was to determine whether such transformed cell lines could be used as antigen presenting cells to analyse the antigenic specificity of bovine CD8 T cell responses to viral infections. Bovine herpes virus 1 (BHV-1), which is known to induce CD8 T cell responses, was used as a model. T. annulata- transformed cells were shown to express high levels of CD40 and CD80 and were susceptible to infection with BHV-1, vaccinia and canarypox viruses. The capacity of the cells to generate antigen-specific CD8 T cell lines was initially validated using a recombinant canarypox virus expressing a defined immunodominant T. parva antigen (Tp1). Autologous T. annulata-transformed cells infected with BHV-1 were then used successfully to generate specific CD8 T cell lines and clones from memory T cell populations of BHV-1-immune animals. These lines were BHV-1-specific and class I MHC-restricted. In contrast to previous studies, which reported recognition of the glycoproteins gB and gD, the CD8 T cell lines generated in this study did not recognise these glycoproteins. Given the ease with which T. annulata-transformed cell lines can be established and maintained in vitro and their susceptibility to infection with poxvirus vectors, these cell lines offer a convenient and efficient in vitro system to analyse the fine specificity of virus-specific CD8 T cell responses in cattle.

Escape from CD8+ T cell response by natural variants of an immunodominant epitope from Theileria parva is predominantly due to loss of TCR recognition.

Polymorphism of immunodominant CD8(+) T cell epitopes can facilitate escape from immune recognition of pathogens, leading to strain-specific immunity. In this study, we examined the TCR ß-chain (TRB) diversity of the CD8(+) T cell responses of cattle against two immunodominant epitopes from Theileria parva (Tp1(214-224) and Tp2(49-59)) and investigated the role of TCR recognition and MHC binding in determining differential recognition of a series of natural variants of the highly polymorphic Tp2(49-59) epitope by CD8(+) T cell clones of defined TRB genotype. Our results show that both Tp1(214-224) and Tp2(49-59) elicited CD8(+) T cell responses using diverse TRB repertoires that showed a high level of stability following repeated pathogenic challenge over a 3-y period. Analysis of single-alanine substituted versions of the Tp2(49-59) peptide demonstrated that Tp2(49-59)-specific clonotypes had a broad range of fine specificities for the epitope. Despite this diversity, all natural variants exhibited partial or total escape from immune recognition, which was predominantly due to abrogation of TCR recognition, with mutation resulting in loss of the lysine residue at P8, playing a particularly dominant role in escape. The levels of heterozygosity in individual Tp2(49-59) residues correlated closely with loss of immune recognition, suggesting that immune selection has contributed to epitope polymorphism.

Extensive polymorphism and evidence of immune selection in a highly dominant antigen recognized by bovine CD8 T cells specific for Theileria annulata.

Although parasite strain-restricted CD8 T cell responses have been described for several protozoa, the precise role of antigenic variability in immunity is poorly understood. The tick-borne protozoan parasite Theileria annulata infects leukocytes and causes an acute, often fatal lymphoproliferative disease in cattle. Building on previous evidence of strain-restricted CD8 T cell responses to T. annulata, this study set out to identify and characterize the variability of the target antigens. Three antigens were identified by screening expressed parasite cDNAs with specific CD8 T cell lines. In cattle expressing the A10 class I major histocompatibility complex haplotype, A10-restricted CD8 T cell responses were shown to be focused entirely on a single dominant epitope in one of these antigens (Ta9). Sequencing of the Ta9 gene from field isolates of T. annulata demonstrated extensive sequence divergence, resulting in amino acid polymorphism within the A10-restricted epitope and a second A14-restricted epitope. Statistical analysis of the allelic sequences revealed evidence of positive selection for amino acid substitutions within the region encoding the CD8 T cell epitopes. Sequence differences in the A10-restricted epitope were shown to result in differential recognition by individual CD8 T cell clones, while clones also differed in their ability to recognize different alleles. Moreover, the representation of these clonal specificities within the responding CD8 T cell populations differed between animals. As well as providing an explanation for incomplete protection observed after heterologous parasite challenge of vaccinated cattle, these results have important implications for the choice of antigens for the development of novel subunit vaccines.

MHC class I bound to an immunodominant Theileria parva epitope demonstrates unconventional presentation to T cell receptors.

T cell receptor (TCR) recognition of peptide-MHC class I (pMHC) complexes is a crucial event in the adaptive immune response to pathogens. Peptide epitopes often display a strong dominance hierarchy, resulting in focusing of the response on a limited number of the most dominant epitopes. Such T cell responses may be additionally restricted by particular MHC alleles in preference to others. We have studied this poorly understood phenomenon using Theileria parva, a protozoan parasite that causes an often fatal lymphoproliferative disease in cattle. Despite its antigenic complexity, CD8+ T cell responses induced by infection with the parasite show profound immunodominance, as exemplified by the Tp1(214-224) epitope presented by the common and functionally important MHC class I allele N*01301. We present a high-resolution crystal structure of this pMHC complex, demonstrating that the peptide is presented in a distinctive raised conformation. Functional studies using CD8+ T cell clones show that this impacts significantly on TCR recognition. The unconventional structure is generated by a hydrophobic ridge within the MHC peptide binding groove, found in a set of cattle MHC alleles. Extremely rare in all other species, this feature is seen in a small group of mouse MHC class I molecules. The data generated in this analysis contribute to our understanding of the structural basis for T cell-dependent immune responses, providing insight into what determines a highly immunogenic p-MHC complex, and hence can be of value in prediction of antigenic epitopes and vaccine design.

CD8+ T-cell responses to Theileria parva are preferentially directed to a single dominant antigen: Implications for parasite strain-specific immunity.

Although immunodominance of CD8(+) T-cell responses is a well-recognised feature of viral infections, its role in responses to more antigenically complex pathogens is less clear. In previous studies we have observed that CD8(+) T-cell responses to Theileria parva exhibit different patterns of parasite strain specificity in cattle of different MHC genotypes. In the current study, we demonstrated that animals homozygous for the A10 and A18 MHC haplotypes have detectable responses to only one of 5 T. parva antigens. Over 60% of the responding T cells from the A18(+) and A10(+) animals recognised defined epitopes in the Tp1 and Tp2 antigens, respectively. Comparison of T-cell receptor beta chain expression profiles of CD8(+) T-cell lines and CD8(+) T cells harvested ex vivo confirmed that the composition of the T-cell lines was representative of the in vivo memory CD8(+) T-cell populations. Analysis of the Tp1 and Tp2 antigens revealed sequence polymorphism, which was reflected by differential recognition by T-cell lines. In conclusion, we have demonstrated a profound immunodominance in the CD8(+) T-cell response to T. parva, which we propose is a major determinant of the parasite strain specificity of the response and hence immune protection.

Use of a Pan-Vbeta primer permits the amplification and sequencing of TCRbeta chains expressed by bovine T-cell clones following a single semi-nested PCR reaction.

We report in this study the design and validation of a Pan-Vbeta primer that in combination with Cbeta-specific primers enables the amplification, in a single semi-nested PCR, of TCRbeta chains expressed by bovine T-cell clones irrespective of the expressed Vbeta sequence. Using the Pan-Vbeta primer we examined the TCRbeta chains expressed by 16 Theileria parva-specific clones that had not been previously analysed. TCRbeta chain sequence was obtained from 15 of the clones following direct sequencing of the PCR product, whilst the other clone appeared to express 2 different TCRbeta chains which were characterised following sub-cloning of the PCR product. We have also successfully used the Pan-Vbeta primer to amplify the TCRbeta chains expressed by 19 T-cell clones, on which previous analysis using Vbeta-subfamily-specific primers had failed to do. Sequencing of these TCRbeta chains has identified members of 2 novel bovine Vbeta subfamilies-Vbeta5 and VbetaX. This method offers a simple and rapid method of analyzing the TCRbeta chains of bovine T-cell clones that has many potential applications in the investigation of bovine T-cell responses.

Dissection of the clonal composition of bovine alphabeta T cell responses using T cell receptor Vbeta subfamily-specific PCR and heteroduplex analysis.

Although techniques that permit analysis of the clonal composition of T cell populations have been used extensively to provide a better understanding of the mechanisms that influence efficacy of T cell responses in humans and mice, such methods are lacking for other animal species. In this paper we report the establishment and validation of a panel of Vbeta subfamily-specific semi-nested PCR assays, and a CDR3beta heteroduplex technique for analysing the clonal diversity of bovine alphabeta T cell responses. Development of these methods was based on available sequence data for 48 functional Vbeta genes classified within 17 subfamilies. These techniques were used to determine the clonal composition of parasite-reactive CD8(+) T cells obtained from two animals immunised with the protozoan parasite Theileria parva. Analyses of uncloned T cell lines as well as large panels of cloned T cells derived from each of these lines confirmed the specificity and sensitivity of the assays. Specific PCR products were obtained from 96% of the T cell clones examined, indicating that the currently identified Vbeta genes represent most of the functional Vbeta subfamilies in cattle. Heteroduplex analyses, coupled with sequencing of PCR products, identified over 20 clonal expansions within each of the T cell lines, distributed over a large number of Vbeta subfamilies, although a limited number of clonotypes numerically dominated the response in both animals. The development and validation of these methods provides for the first time a generic set of molecular tools that can be used to perform detailed analysis of the TCR diversity and clonal composition of bovine T cell responses.

Characterization of the fine specificity of bovine CD8 T-cell responses to defined antigens from the protozoan parasite Theileria parva.

Immunity against the bovine intracellular protozoan parasite Theileria parva has been shown to be mediated by CD8 T cells. Six antigens targeted by CD8 T cells from T. parva-immune cattle of different major histocompatibility complex (MHC) genotypes have been identified, raising the prospect of developing a subunit vaccine. To facilitate further dissection of the specificity of protective CD8 T-cell responses and to assist in the assessment of responses to vaccination, we set out to identify the epitopes recognized in these T. parva antigens and their MHC restriction elements. Nine epitopes in six T. parva antigens, together with their respective MHC restriction elements, were successfully identified. Five of the cytotoxic-T-lymphocyte epitopes were found to be restricted by products of previously described alleles, and four were restricted by four novel restriction elements. Analyses of CD8 T-cell responses to five of the epitopes in groups of cattle carrying the defined restriction elements and immunized with live parasites demonstrated that, with one exception, the epitopes were consistently recognized by animals of the respective genotypes. The analysis of responses was extended to animals immunized with multiple antigens delivered in separate vaccine constructs. Specific CD8 T-cell responses were detected in 19 of 24 immunized cattle. All responder cattle mounted responses specific for antigens for which they carried an identified restriction element. By contrast, only 8 of 19 responder cattle displayed a response to antigens for which they did not carry an identified restriction element. These data demonstrate that the identified antigens are inherently dominant in animals with the corresponding MHC genotypes.

Generation and maintenance of diversity in the cattle MHC class I region.

Major histocompatibility complex (MHC) class I genes play a crucial role in the immune defence against intracellular pathogens. An important evolutionary strategy is to generate and maintain a high level of diversity in these genes. Humans express three highly polymorphic classical MHC class I genes (HLA-A, HLA-B and HLA-C). In contrast, some species, for example rat and rhesus macaque, maintain diversity by generation of haplotypes that vary considerably with regard to the number and combination of transcribed genes. Cattle appear to use both strategies. We show that various combinations of six apparently classical genes, three of which are highly polymorphic, are transcribed on different haplotypes. Although additional sequences were identified in both cDNA and gDNA, it was not possible to assign them to any of these defined genes. Most were highly divergent or were non-classical class I genes. Thus, we found little evidence for frequent duplication and deletion of classical class I genes as reported in some other species. However, the maintenance of class I diversity in cattle may involve limited gene shuffling and deletion, possibly as a result of unequal crossing-over within the class I region.

The kinetics of Theileria parva infection and lymphocyte transformation in vitro.

Theileriaparva is an intracellular protozoan parasite that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. The parasite infects host lymphocytes causing their transformation and uncontrolled proliferation. Infiltration of major organs with parasitized lymphoblasts results in most cases in death within 3 weeks. Although both T and B lymphocytes are susceptible to infection, the majority of cell lines arising from infection of peripheral blood mononuclear cells in vitro are of T cell lineage. To explore the basis of this phenotypic bias we have followed the very early stages of parasite development in vitro at the single cell level. Peripheral blood mononuclear cells were infected and stained for both surface phenotype and intracellular parasite antigen and analysed by flow cytometry. Although the parasite antigen was detected intracellularly as early as 6h p.i., our data indicate that parasite infection does not lead to cell transformation in all instances. Rather, specific cell types appear to undergo selection very early after infection and expansion of particular cell subsets results in survival and growth of only a small proportion of the cells originally parasitized.

Serological and molecular diversity in the cattle MHC class I region.

Information on major histocompatibility complex (MHC) diversity in cattle is important to aid our understanding of immune responses and may contribute to maintenance of healthy cattle populations. Equally, understanding the mechanisms involved in generating this diversity may shed light on the complex nature of mammalian MHC evolution. The aim of this study was to assess molecular and serological variation within cattle MHC class I molecules and to study the mechanisms generating diversity. To address this aim, sequence variation was examined in 12 serologically assigned alleles from three putative loci and correlated with monoclonal antibody (mAb) binding data. The results demonstrate that both alloantisera and mAbs often fail to distinguish gene products that differ by a significant number of amino acids. Conversely, some mAbs could distinguish alleles differing by only one or two amino acids. Examination of the sequences demonstrates sharing of motifs between alleles, some encoded at distinct loci, supporting the occurrence of interlocus recombination within the cattle MHC class I region. The implications of this for MHC sequence diversity, and functional capability, are discussed.

The DY genes of the cattle MHC: expression and comparative analysis of an unusual class II MHC gene pair.

The major histocompatibility complex of cattle (BoLA) contains the class II genes DYA and DIB which are transcribed with a dendritic cell restricted distribution. As part of the process to determine whether these genes have any functional significance, we demonstrate that they form a closely linked pair characteristic of other expressed class II MHC molecules. Accepted nomenclature convention suggests that BoLA-DIB should therefore be renamed BoLA-DYB. Analysis of the first full-length DYA and DYB transcripts revealed open reading frames with potential to translate 253 and 259 amino acid proteins, respectively. Comparative sequence analysis between the DY polypeptides and classical cattle, human and mouse class II MHC alpha and beta polypeptide chains revealed 16 unique amino acid residues at positions predicted to form and line the putative peptide-binding region. Expression of tagged constructs demonstrates for the first time that the DY genes of cattle are capable of translating distinctive class II MHC alpha and beta polypeptide chains.