Evans Taracha

Characterization of the Fine Specificity of Bovine CD8 T-Cell Responses to Defined Antigens from the Protozoan Parasite Theileria parva

ABSTRACT 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.

Extensive Genotypic Diversity in a Recombining Population of the Apicomplexan Parasite Theileria parva

ABSTRACT We evaluated sexual recombination in the apicomplexan parasite Theileria parva using genome-wide marker analysis of haploid sporozoite populations obtained from infected Rhipicephalus appendiculatus ticks. Analysis of 231 parasite clones derived by in vitro infection of bovine lymphocytes revealed 48 distinct combinations of 64 polymorphic marker loci. One genotype accounted for more than 75% of the clones, and the population was highly inbred with respect to this. The occurrence of frequent recombination was evident from reassortment of contiguous markers in blocks, with some recombination occurring within blocks. Analysis of four polymorphic loci encoding antigens targeted by protective cytotoxic-T-lymphocyte responses confirmed that these loci reassort, both within and between chromosomes, suggesting that recombination may influence immune recognition. Marker analysis of a panel of 142 clones derived from the population after an additional passage through a calf and the same tick colony revealed 18 genotypes, with the original dominant genotype accounting for 75% of the population and a higher level of inbreeding with respect to it in the remaining clones. Selected marker analysis of genomic DNA from these stabilates and the two preceding generations of the isolate, each derived from distinct tick colonies, revealed shifts in population structure with each generation, suggesting that the tick vector may impose nonrandom selective pressure on the parasite.

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 β 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.

Analysis of the transcriptome of the protozoan Theileria parva using MPSS reveals that the majority of genes are transcriptionally active in the schizont stage

Massively parallel signature sequencing (MPSS) was used to analyze the transcriptome of the intracellular protozoan Theileria parva. In total 1 095 000, 20 bp sequences representing 4371 different signatures were generated from T.parva schizonts. Reproducible signatures were identified within 73% of potentially detectable predicted genes and 83% had signatures in at least one MPSS cycle. A predicted leader peptide was detected on 405 expressed genes. The quantitative range of signatures was 4–52 256 transcripts per million (t.p.m.). Rare transcripts (<50 t.p.m.) were detected from 36% of genes. Sequence signatures approximated a lognormal distribution, as in microarray. Transcripts were widely distributed throughout the genome, although only 47% of 138 telomere-associated open reading frames exhibited signatures. Antisense signatures comprised 13.8% of the total, comparable with Plasmodium. Eighty five predicted genes with antisense signatures lacked a sense signature. Antisense transcripts were independently amplified from schizont cDNA and verified by sequencing. The MPSS transcripts per million for seven genes encoding schizont antigens recognized by bovine CD8 T cells varied 1000-fold. There was concordance between transcription and protein expression for heat shock proteins that were very highly expressed according to MPSS and proteomics. The data suggests a low level of baseline transcription from the majority of protein-coding genes.

Protective Immune Mechanisms against Theileria parva: Evolution of Vaccine Development Strategies

Theileria parva is an intracellular sporozoan parasite that infects and transforms bovine lymphocytes, causing a severe lymphoproliferative disease known as East Coast fever in eastern, central and southern Africa. In this article, Declan McKeever and colleagues summarize the current understanding of immune mechanisms provoked by the parasite with regard to their role in both pathogenesis and protection. In particular, the influence of genomic polymorphism in parasite and host on the development of immunity is discussed, along with the evolution of current vaccine development strategies as a result of immunological research on the disease.

A novel strategy for the identification of antigens that are recognised by bovine MHC class I restricted cytotoxic T cells in a protozoan infection using reverse vaccinology.

BackgroundImmunity against the bovine protozoan parasite Theileria parva has previously been shown to be mediated through lysis of parasite-infected cells by MHC class I restricted CD8+ cytotoxic T lymphocytes. It is hypothesized that identification of CTL target schizont antigens will aid the development of a sub-unit vaccine. We exploited the availability of the complete genome sequence data and bioinformatics tools to identify genes encoding secreted or membrane anchored proteins that may be processed and presented by the MHC class I molecules of infected cells to CTL.ResultsOf the 986 predicted open reading frames (ORFs) encoded by chromosome 1 of the T. parva genome, 55 were selected based on the presence of a signal peptide and/or a transmembrane helix domain. Thirty six selected ORFs were successfully cloned into a eukaryotic expression vector, transiently transfected into immortalized bovine skin fibroblasts and screened in vitro using T. parva-specific CTL. Recognition of gene products by CTL was assessed using an IFN-γ ELISpot assay. A 525 base pair ORF encoding a 174 amino acid protein, designated Tp2, was identified by T. parva-specific CTL from 4 animals. These CTL recognized and lysed Tp2 transfected skin fibroblasts and recognized 4 distinct epitopes. Significantly, Tp2 specific CD8+ T cell responses were observed during the protective immune response against sporozoite challenge.ConclusionThe identification of an antigen containing multiple CTL epitopes and its apparent immunodominance during a protective anti-parasite response makes Tp2 an attractive candidate for evaluation of its vaccine potential.

Heterologous Priming-Boosting Immunization of Cattle with Mycobacterium tuberculosis 85A Induces Antigen-Specific T-Cell Responses

ABSTRACT Heterologous priming-boosting vaccination regimens involving priming with plasmid DNA antigen constructs and inoculating (boosting) with the same recombinant antigen expressed in replication-attenuated poxviruses have recently been demonstrated to induce immunity, based on CD4+- and CD8+-T-cell responses, against several diseases in both rodents and primates. We show that similar priming-boosting vaccination strategies using the 85A antigen of Mycobacterium tuberculosis are effective in inducing antigen-specific gamma interferon-secreting CD4+ and CD8+ T cells, detected by a bovine enzyme-linked immunospot assay, in Bos indicus cattle. T-cell responses induced by priming with either plasmid DNA or fowlpox virus 85A constructs were enhanced by boosting with modified vaccinia virus Ankara expressing the same antigen administered intradermally. On the basis of the data, it appears that intradermal priming was more effective than intramuscular delivery of the priming dose for boosting with the modified vaccinia virus Ankara strain in cattle. Using either fowlpox virus or DNA priming, there was a significant bias toward induction of CD4+- rather than CD8+-T-cell responses. These data illustrate the general applicability of priming-boosting vaccination strategies for induction of antigen-specific T-cell responses and suggest that the method may be useful for development of veterinary vaccines.

Delivery of the Theileria parva p67 antigen to cattle using recombinant vaccinia virus: IL-2 enhances protection

To evaluate vaccinia virus as a delivery system for recombinant antigen in cattle, calves were immunized with a recombinant vaccinia virus (rVV) expressing the sporozoite surface antigen (p67) of Theileria parva (V-67) combined with those expressing bovine IL-4 (V-IL4) or IL-2 (V-IL2). The anti-p67 antibody levels detected in calves inoculated with the combination of V-67 and V-IL4 were higher than those produced by animals injected with V-67 alone or V-67 and V-IL2. On challenge with cryopreserved sporozoites, 5 of 7 animals receiving V-67 combined with V-IL2 were protected, while those receiving V-67 in conjunction with V-IL4 behaved like unimmunized control calves. Vaccination with a recombinant virus expressing a chimaeric p67(p583)/IL2 product gave rise to a lower level of protection, whereas V-IL2 provided no immunity. The results of this study demonstrate the potential of rVV as a delivery system for use in vaccination of cattle against Theileria parva infection.

Extensive Polymorphism and Evidence of Immune Selection in a Highly Dominant Antigen Recognized by Bovine CD8 T Cells Specific for Theileria annulata

ABSTRACT 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.

6.1 Reactivity of workshop antibodies on L cell and COS cell transfectants expressing bovine CD antigens

Mouse L cells and COS cells were transfected with either genomic DNA or cDNAs encoding leukocyte differentiation antigens. Positive transfectants were isolated by FACS and cloned by limiting dilution. Transfectants expressing CD4, CD5, CD8, CD25, CD44, two different WC1 gene products and a transfectant expressing an unknown gene product were isolated using these techniques. Antibodies from the various preliminary clusters were analysed for reactivity on these transfectants. The results confirm the fine specificity of mAbs for two alleles of CD4 and CD5; they also subdivide mAbs recognizing CD8 into two groups based on their specificity for the CD8 alpha chain or a combination of both alpha and beta chains. The data also provides support for the clustering of mAbs recognizing CD25 and CD44 based on transfection of cDNAs encoding these specificities. These results reflect the ability of transfection technology to elucidate the fine specificity of mAbs recognizing bovine CD antigens.