Use of "one-pot, mix-and-read" peptide-MHC class I tetramers and predictive algorithms to improve detection of cytotoxic T lymphocyte responses in cattle

International audiencePeptide-major histocompatibility complex (p-MHC) class I tetramer complexes have facilitated the early detection and functional characterisation of epitope specific CD8+ cytotoxic T lymphocytes (CTL). Here, we report on the generation of seven recombinant bovine leukocyte antigens (BoLA) and recombinant bovine β2-microglobulin from which p-MHC class I tetramers can be derived in ~48 h. We validated a set of p-MHC class I tetramers against a panel of CTL lines specific to seven epitopes on five different antigens of Theileria parva, a protozoan pathogen causing the lethal bovine disease East Coast fever. One of the p-MHC class I tetramers was tested in ex vivo assays and we detected T. parva specific CTL in peripheral blood of cattle at day 15-17 post-immunization with a live parasite vaccine. The algorithm NetMHCpan predicted alternative epitope sequences for some of the T. parva CTL epitopes. Using an ELISA assay to measure peptide-BoLA monomer formation and p-MHC class I tetramers of new specificity, we demonstrate that a predicted alternative epitope Tp229-37 rather than the previously reported Tp227-37 epitope is the correct Tp2 epitope presented by BoLA-6*04101. We also verified the prediction by NetMHCpan that the Tp587-95 epitope reported as BoLA-T5 restricted can also be presented by BoLA-1*02301, a molecule similar in sequence to BoLA-T5. In addition, Tp587-95 specific bovine CTL were simultaneously stained by Tp5-BoLA-1*02301 and Tp5-BoLA-T5 tetramers suggesting that one T cell receptor can bind to two different BoLA MHC class I molecules presenting the Tp587-95 epitope and that these BoLA molecules fall into a single functional supertype

Synergistic Effect of Two Nanotechnologies Enhances the Protective Capacity of the Theileria parva Sporozoite p67C Antigen in Cattle

Multimerization of p67C Ag as nanoparticle increases its immunogenicity.Vaccine efficacy of p67C Ag increases delivered as nanoparticles.East Coast fever (ECF), caused by Theileria parva, is the most important tick-borne disease of cattle in sub-Saharan Africa. Practical disadvantages associated with the currently used live-parasite vaccine could be overcome by subunit vaccines. An 80-aa polypeptide derived from the C-terminal portion of p67, a sporozoite surface Ag and target of neutralizing Abs, was the focus of the efforts on subunit vaccines against ECF and subjected to several vaccine trials with very promising results. However, the vaccination regimen was far from optimized, involving three inoculations of 450 μg of soluble p67C (s-p67C) Ag formulated in the Seppic adjuvant Montanide ISA 206 VG. Hence, an improved formulation of this polypeptide Ag is needed. In this study, we report on two nanotechnologies that enhance the bovine immune responses to p67C. Individually, HBcAg-p67C (chimeric hepatitis B core Ag virus-like particles displaying p67C) and silica vesicle (SV)–p67C (s-p67C adsorbed to SV-140-C18, octadecyl-modified SVs) adjuvanted with ISA 206 VG primed strong Ab and T cell responses to p67C in cattle, respectively. Coimmunization of cattle (Bos taurus) with HBcAg-p67C and SV-p67C resulted in stimulation of both high Ab titers and CD4 T cell response to p67C, leading to the highest subunit vaccine efficacy we have achieved to date with the p67C immunogen. These results offer the much-needed research depth on the innovative platforms for developing effective novel protein-based bovine vaccines to further the advancement

Capture-based enrichment of Theileria parva DNA enables full genome assembly of first buffalo-derived strain and reveals exceptional intra-specific genetic diversity

Theileria parva is an economically important, intracellular, tick-transmitted parasite of cattle. A live vaccine against the parasite is effective against challenge from cattle-transmissible T. parva but not against genotypes originating from the African Cape buffalo, a major wildlife reservoir, prompting the need to characterize genome-wide variation within and between cattle- and buffalo-associated T. parva populations. Here, we describe a capture-based target enrichment approach that enables, for the first time, de novo assembly of nearly complete T. parva genomes derived from infected host cell lines. This approach has exceptionally high specificity and sensitivity and is successful for both cattle- and buffalo-derived T. parva parasites. De novo genome assemblies generated for cattle genotypes differ from the reference by ~54K single nucleotide polymorphisms (SNPs) throughout the 8.31 Mb genome, an average of 6.5 SNPs/kb. We report the first buffalo-derived T. parva genome, which is ~20 kb larger than the genome from the reference, cattle-derived, Muguga strain, and contains 25 new potential genes. The average non-synonymous nucleotide diversity (πN) per gene, between buffalo-derived T. parva and the Muguga strain, was 1.3%. This remarkably high level of genetic divergence is supported by an average Wright’s fixation index (FST), genome-wide, of 0.44, reflecting a degree of genetic differentiation between cattle- and buffalo-derived T. parva parasites more commonly seen between, rather than within, species. These findings present clear implications for vaccine development, further demonstrated by the ability to assemble nearly all known antigens in the buffalo-derived strain, which will be critical in design of next generation vaccines. The DNA capture approach used provides a clear advantage in specificity over alternative T. parva DNA enrichment methods used previously, such as those that utilize schizont purification, is less labor intensive, and enables in-depth comparative genomics in this apicomplexan parasite

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

Abstract Background The 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). Results Four 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. Conclusion We 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

Theileria parva sporozoite invasion and development within bovine dendritic cells

Theileria parva, an obligate intracellular protozoan parasite of cattle, causes East Coast fever, a fatal, lymphoproliferative disease, endemic in large areas of East and Central Africa. The mammalian-infective sporozoites of T. parva mature within the salivary glands of the ixodid tick Rhipicephalus appendiculatus diculatus and are inoculated into the host during feeding. Sporozoites invade T and B cells and dendritic cells (DC). Mode of entry into these cell types appears very similar. However, studies of the subsequent parasite Development have been confined largely to lymphocytes. Dendritic cells are a feature of bovine dermis and are present in large numbers at the site of tick attachment in cattle. These cells are believed to migrate via the afferent lymphatics to the draining lymph node, where they differentiate into interdigitating dendritic cells within the paracortex. Dendritic cells collected from bovine afferent lymph have been shown to take up large numbers of sporozoites in vitro. Since proliferation of infected lymphoblasts is first observed in the lymph node that drains the site of inoculation, we have characterised sporozoite invasion and differentiation in bovine dendritic cells with a view to evaluating their role in the establishment of infection. This paper summarises Theileria parva sporozoite invasion and Development within bovine dendritic cells. Sporozoites within the cytoplasm of a dendritic cell is illustrated

Distinct CD4 + T cell helper requirements in Theileria parva-immune and -naive bovine CTL precursors

There is strong evidence that class I MHC-restricted parasite-specific CD8+ CTL protect cattle against the protozoan parasite Theileria parva. As part of an effort to develop a subunit vaccine for the induction of these responses, we have investigated the factors involved in the generation of T. parva-specific CTL in cattle. Purified populations of bovine immune and naive CD8+ T cells were cocultured with autologous T. parva-infected lymphoblasts (TpL) in the presence or absence of immune CD4+ T cells or cytokine preparations. Neither population developed CTL activity when cultured with TpL alone, whereas incorporation of immune CD4+ T cells in the cultures supported the generation of parasite-specific CTL from both immune and naive CD8+ precursors. The helper function of parasite-specific CD4+ T cells for immune, but not naive, CTL precursors could be replaced by CD4+ T cells responding to an unrelated Ag or by the addition of T cell growth factors or recombinant bovine IL-2. In experiments with two-chamber culture plates, in which cocultures of CD4+ and CD8+ T cells with TpL were separated by a semipermeable membrane, CTL activity was observed to develop only in immune precursor populations. Hence, although bovine T. parva-specific CD8+ memory T cells need no helper signals other than IL-2 for activation, their naive counterparts require close contact with responding parasite-specific CD4+T cells. This may reflect essential receptor-ligand interactions, or alternatively, a requirement for more stringent microenvironmental cytokine conditions