Rosemary Saya

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.

Two Theileria parva CD8 T cell antigen genes are more variable in buffalo than cattle parasites, but differ in pattern of sequence diversity

Background Theileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8+ T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8+ T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8+ T-cell epitopes, and to analyse the sequences for evidence of selection. Methodology/Principal Findings Partial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (∼12%) in Tp1 and in 320 positions (∼61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle. Conclusions/Significance The results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point.

Analysis of the Cellular Immune Responses to Vaccines

Flow cytometry, enzyme-linked immunospot (ELISpot) and cellular cytotoxicity assays are powerful tools for studying the cellular immune response towards intracellular pathogens and vaccines in livestock species. Lymphocytes from immunized animals can be purified using Ficoll-Paque density gradient centrifugation and evaluated for their antigen specificity or reactivity towards a vaccine. Here, we describe staining of bovine lymphocytes with peptide (p)-MHC class I tetramers and antibodies specific towards cellular activation markers for evaluation by multiparametric flow cytometry, as well as interferon (IFN)-γ ELISpot and cytotoxicity using chromium ((51)Cr) release assays. A small component on the use of immunoinformatics for fine-tuning the identification of a minimal CTL epitope is included.

An Ad/MVA vectored Theileria parva antigen induces schizont-specific CD8+ central memory T cells and confers partial protection against a lethal challenge

The parasite Theileria parva is the causative agent of East Coast fever (ECF), one of the most serious cattle diseases in sub-Saharan Africa, and directly impacts smallholder farmers’ livelihoods. There is an efficient live-parasite vaccine, but issues with transmission of vaccine strains, need of a cold chain, and antibiotics limit its utilization. This has fostered research towards subunit vaccination. Cytotoxic T lymphocytes (CTL) are crucial in combating the infection by lysing T. parva-infected cells. Tp1 is an immunodominant CTL antigen, which induces Tp1-specific responses in 70–80% of cattle of the A18 or A18v haplotype during vaccination with the live vaccine. In this study, human adenovirus serotype 5 (HAd5) and modified vaccinia Ankara (MVA) were assessed for their ability to induce Tp1-specific immunity. Both viral vectors expressing the Tp1 antigen were inoculated in cattle by a heterologous prime-boost vaccination regimen. All 15 animals responded to Tp1 as determined by ELISpot. Of these, 14 reacted to the known Tp1 epitope, assayed by ELISpot and tetramer analyses, with CTL peaking 1-week post-MVA boost. Eleven animals developed CTL with specific cytotoxic activity towards peripheral blood mononuclear cells (PBMC) pulsed with the Tp1 epitope. Moreover, 36% of the animals with a Tp1 epitope-specific response survived a lethal challenge with T. parva 5 weeks post-MVA boost. Reduction of the parasitemia correlated with increased percentages of central memory lymphocytes in the Tp1 epitope-specific CD8+ populations. These results indicate that Tp1 is a promising antigen to include in a subunit vaccine and central memory cells are crucial for clearing the parasite.East Coast fever: Developing an accessible vaccine for African farmersA vaccine expressing parasitic proteins offers more convenient East Coast fever prophylaxis. Current vaccination for the cattle disease, caused by the parasite Theileria parva and a detriment to sub-Saharan African farmers, involves inconvenient injection with live parasites before antibiotic treatment (ITM). A collaboration led by Nicholas Svitek, of the Kenyan International Livestock Research Institute, designed a candidate to provoke cellular immune responses against the parasitic antigen Tp1—an ITM vaccine candidate. In tests on cattle, 93% created Tp1-targeting T cells, and 33% survived a lethal dose of T. parva. The East Coast fever reduction seen in animals in this research outperformed a recent study and was able to generate the same immune memory cells that ITM inspires to provide long-lasting protection. Future research might integrate more antigens with this Tp1 vaccine to provide more comprehensive protection.

Immune parameters to p67C antigen adjuvanted with ISA206VG correlate with protection against East Coast fever

Highlights • Three doses of p67C antigen generated stronger immune responses than two doses.• Antibody titers and CD4+ T-cell proliferation correlated with protection against ECF.• The number of doses could not be reduced from three to two without compromising the protection.

Immunization with one Theileria parva strain results in similar level of CTL strain-specificity and protection compared to immunization with the three-component Muguga cocktail in MHC-matched animals

BackgroundThe tick-borne protozoan parasite Theileria parva causes a usually fatal cattle disease known as East Coast fever in sub-Saharan Africa, with devastating consequences for poor small-holder farmers. Immunity to T. parva, believed to be mediated by a cytotoxic T lymphocyte (CTL) response, is induced following natural infection and after vaccination with a live vaccine, known as the Infection and Treatment Method (ITM). The most commonly used version of ITM is a combination of parasites derived from three isolates (Muguga, Kiambu 5 and Serengeti-transformed), known as the “Muguga cocktail”. The use of a vaccine comprising several strains is believed to be required to induce a broad immune response effective against field challenge. In this study we investigated whether immunization with the Muguga cocktail induces a broader CTL response than immunization with a single strain (Muguga).ResultsFour MHC haplotype-matched pairs of cattle were immunized with either the trivalent Muguga cocktail or the single Muguga strain. CTL specificity was assessed on a panel of five different strains, and clonal responses to these strains were also assessed in one of the MHC-matched pairs. We did not find evidence for a broader CTL response in animals immunized with the Muguga cocktail compared to those immunized with the Muguga strain alone, in either the bulk or clonal CTL analyses. This was supported by an in vivo trial in which all vaccinated animals survived challenge with a lethal dose of the Muguga cocktail vaccine stabilate.ConclusionWe did not observe any substantial differences in the immunity generated from animals immunized with either Muguga alone or the Muguga cocktail in the animals tested here, corroborating earlier results showing limited antigenic diversity in the Muguga cocktail. These results may warrant further field studies using single T. parva strains as future vaccine candidates.

Cytotoxic T lymphocytes from cattle sharing the same MHC class I haplotype and immunized with live Theileria parva sporozoites differ in antigenic specificity

ObjectivesThe objective of this study was to assess whether cytotoxic T cells (CTL) generated by the live vaccine, known as “ITM Muguga cocktail”, which is used for the cattle disease East Cost fever (ECF) in Sub-Saharan Africa, showed a broad reactivity against many different strains of the causative parasite Theileria parva. We also assessed whether immune responses were similar in cattle expressing the same MHC class I haplotypes.ResultsThe antigenic specificity of CTL from MHC class I-matched cattle vaccinated with the Muguga cocktail were different. Three cattle of MHC class I haplotype A18, one A18/A19 and two haploidentical (A18v/A12) animals, showed differential recognition of autologous cells infected with a panel of T. parva isolates. This could have implications in the field where certain strains could break through the vaccine. Furthermore, neither of the haploidentical cattle recognized the CTL epitope (Tp1214–224), presented by the A18 haplotype, in contrast to the third animal, showing differences in immunodominance in animals of the same haplotype A18. This suggests that the CTL specificities following immunization with the Muguga cocktail can vary even between haploidentical individuals and that some parasite strains may break through immunity generated by the Muguga cocktail.