No Abstract. Ethiopian Journal of Health Development Vol. 22 (special issue) 2008: pp. 109-116

Theileria parva causes an acute lympho-proliferative disease in cattle, which can result in death of susceptible animals within 2–3 weeks of infection. Analyses of the cellular response in the lymph node draining the site of infection demonstrated an early T cell response, with the appearance of large numbers of uninfected lymphoblasts between 6 and 9 days p.i., coinciding with initial detection of parasitised cells. There was a marked increase in the representation of CD8+ T cells and the emergence of a sizable sub-population of CD2− CD8+ α/β T cells during this period. Analysis of T cell receptor β chain variable (TCR BV) gene expression did not reveal any evidence for the involvement of a superantigen in stimulating the response. Responding lymph node cells were found to produce increased quantities of IFNγ and IL-10, and both the CD2+ CD8+ and CD2− CD8+ populations expressed IFNγ transcripts. Purified CD2+ CD8+ cells proliferated when stimulated in vitro with autologous parasitised cells or non-specific mitogens, whereas CD2− CD8+ cells were refractory to these stimuli. In contrast to the parasite-specific cytotoxic activity associated with T cell responses in immune cattle, the responses to primary infection exhibited variable levels of non-specific cytotoxic activity. Stimulation of purified CD2+ CD8+ T cells in vitro with autologous parasitised cells also failed to reveal evidence of specific cytotoxic activity. These findings indicate that primary infection with T. parva induces an aberrant T cell response that lacks appropriate effector activity.

East Coast fever, caused by the tick-borne intracellular apicomplexan parasite Theileria parva, is a highly fatal lymphoproliferative disease of cattle. The pathogenic schizont-induced lymphocyte transformation is a unique cancer-like condition that is reversible with parasite removal. Schizont-infected cell-directed CD8(+) cytotoxic T lymphocytes (CTL) constitute the dominant protective bovine immune response after a single exposure to infection. However, the schizont antigens targeted by T. parva-specific CTL are undefined. Here we show the identification of five candidate vaccine antigens that are the targets of MHC class I-restricted CD8(+) CTL from immune cattle. CD8(+) T cell responses to these antigens were boosted in T. parva-immune cattle resolving a challenge infection and, when used to immunize naïve cattle, induced CTL responses that significantly correlated with survival from a lethal parasite challenge. These data provide a basis for developing a CTL-targeted anti-East Coast fever subunit vaccine. In addition, orthologs of these antigens may be vaccine targets for other apicomplexan parasites.

We report the genome sequence of Theileria parva, an apicomplexan pathogen causing economic losses to smallholder farmers in Africa. The parasite chromosomes exhibit limited conservation of gene synteny with Plasmodium falciparum, and its plastid-like genome represents the first example where all apicoplast genes are encoded on one DNA strand. We tentatively identify proteins that facilitate parasite segregation during host cell cytokinesis and contribute to persistent infection of transformed host cells. Several biosynthetic pathways are incomplete or absent, suggesting substantial metabolic dependence on the host cell. One protein family that may generate parasite antigenic diversity is not telomere-associated.

ABSTRACTHeterologous 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 ofMycobacterium tuberculosisare effective in inducing antigen-specific gamma interferon-secreting CD4+and CD8+T cells, detected by a bovine enzyme-linked immunospot assay, inBos indicuscattle. 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.

Dendritic cells (DC) constitute the most effective immune cell population for priming and recalling T cell responses to foreign antigens. DC patrol the peripheral tissues collecting foreign antigen for subsequent presentation by classical class II MHC molecules to T cells in the draining lymph nodes. Since the description of the DYA and DIB class II MHC genes, which are unique to ruminants, no transcript or protein have been reported. Here we provide evidence that these genes are transcribed in cattle and that paired transcription is restricted in afferent lymph to a functionally distinct population of DC. Analysis of lymph node, lung and thymus suggests that tissue DC also transcribe both genes. Cytokine-induced differentiation of cultured monocytes to a DC phenotype is linked with induction of both DYA and DIB transcription. This is consistent with an association of their products with the potent antigen presenting capacity of these cells in cattle.

We describe a highly sensitive, non-radioactive assay for T cell activation, based on the rapid induction of class II MHC expression by constitutively negative bovine endothelial cells, when cultured in the presence of supernatants derived from activated bovine T cells. We demonstrate the effectiveness of this assay in detecting rBoIFNgamma and activation of immune CD4(+) and CD8(+) T cell lines and clones in response to specific antigen and transfected COS-7 cells, respectively. We also demonstrate its utility in identifying purified pathogen fractions that activate immune CD4(+) T cell clones.

T cells bearing the gammadelta antigen receptor (gammadelta T cells) can constitute up to 50% of T cells in the peripheral blood and lymphoid organs of young cattle. We present data showing that gammadelta T cells are involved in immune responses against Theileria parva. gammadelta T cells isolated from peripheral blood mononuclear cells (PBMC) of T. parva-naive and -immune cattle proliferated in the presence of fixed or unfixed autologous T. parva-infected lymphoblasts (TpL) and heat-stressed concanavalin A (ConA)-induced blasts (ConA blasts) but not untreated ConA blasts. The specificity of response was further evaluated with a panel of gammadelta T-cell lines and clones. T-cell reactivity was blocked by GB21A, a monoclonal antibody (MAb) specific for the gammadelta T-cell receptor, but not by MAbs specific for class I and class II major histocompatibility complex (MHC) molecules. In addition, TpL but not ConA blasts from a variety of MHC-mismatched animals induced proliferation of the gammadelta T-cell lines and clones. These gammadelta T cells were found to respond to TpL infected with several different parasite stocks and failed to recognize TpL after elimination of the parasite by the theilericidal drug BW 720C. Assays for cytotoxic activity of gammadelta T cells sorted from bulk cultures of immune PBMC restimulated several times with autologous TpL demonstrated that effector cells whose specificity is similar to that of proliferating cells are generated. These results suggest that bovine gammadelta T cells are activated by and lyse T. parva-infected cells by recognizing conserved parasite-induced or parasite-derived antigens in an MHC-unrestricted fashion.

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.

ABSTRACT Peripheral blood mononuclear cells (PBMC) from immune cattle proliferate in the presence of autologous Cowdria ruminantium-infected endothelial cells and monocytes. Endothelial cells required treatment with T-cell growth factors to induce class II major histocompatibility complex expression prior to infection and use as stimulators. Proliferative responses to both infected autologous endothelial cells and monocytes were characterized by expansion of a mixture of CD4+, CD8+, and γδ T cells. However, γδ T cells dominated following several restimulations. Reverse transcription-PCR analysis of cytokine expression by C. ruminantium-specific T-cell lines and immune PBMC revealed weak interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-γ) transcripts at 3 to 24 h after stimulation. Strong expression of IFN-γ, tumor necrosis factor alpha (TNF-α), TNF-β, and IL-2 receptor α-chain mRNA was detected in T-cell lines 48 h after antigen stimulation. Supernatants from these T-cell cultures contained IFN-γ protein. Our findings suggest that in immune cattle a C. ruminantium-specific T-cell response is induced and that infected endothelial cells and monocytes may present C. ruminantiumantigens to specific T lymphocytes in vivo during infection and thereby play a role in induction of protective immune responses to the pathogen.

Cattle immunised with a recombinant form of p67, the major surface antigen of Theileria parva sporozoites, have been shown to be protected against parasite challenge. In an attempt to simplify the immunisation procedure live attenuated Salmonella strains expressing p67 have been constructed and used to induce anti-p67 immune responses in cattle. All animals immunised with these strains developed strong antibody responses to p67. Specific T cell responses could be detected in the majority of immunised cattle. Challenge with T. parva sporozoites revealed a significant level of protection in immunised calves compared to naive control animals or animals inoculated with non-recombinant attenuated Salmonella.

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.

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.

Class I major histocompatibility complex-restricted parasite-specific cytotoxic T lymphocytes (CTL) are known to be a major component of the bovine immune response to the protozoan parasite Theileria parva, but formal proof for their role in protection of cattle against infection with T. parva has been lacking. Animals immunized with one stock of T. parva show variations in the degree of protection against heterologous challenge and also in the parasite strain specificity of their CTL responses. The present study investigated the relationship of strain specificity of CTL responses and cross-protection in an effort to verify the role of CTL in protection. The parasite strain specificity of the CTL responses generated in 23 cattle immunized with either of two immunologically distinct parasite populations was examined, and the susceptibility of individual cattle to challenge with the heterologous parasite population was determined. The frequency of stock-specific or cross-reactive CTL precursor cells (CTLp) in individual animals was measured by a limiting-dilution microassay. A proportion of animals immunized with either parasite exhibited cross-reactive CTLp, whereas CTLp detected in the remaining animals were specific for the homologous parasite. On challenge with the heterologous stock, those animals with cross-reactive CTLp were solidly protected while those with strain-specific CTLp showed moderate to severe reactions, although many of them recovered, The finding of a close association between strain specificity of the CTL response and protection against challenge provides strong evidence that CTL are important in mediating immunity;

The parasite strain specificity of CTL responses to Theileria parva varies among cattle immunized with the same parasite stock. We have investigated the influence of class I MHC on the strain specificity of CTL responses to T. parva in 19 cattle of defined class I phenotype immunized with either of two T. parva populations, in which protection to subsequent reciprocal challenge correlated with CTL strain specificity, In the majority of animals the response was restricted by the products of one MHC haplotype and there was a consistent bias to some haplotypes in preference to others. In 10 of 13 cattle expressing the molecularly defined MHC specificities A10 and KN104 on one haplotype, the CTL response was restricted entirely by this haplotype, thus allowing a precise analysis of the MHC restriction specificities. The MHC restriction specificity and the parasite population used for immunization both influenced the strain specificity of the response. By examining responses in identical twins immunized with different parasites or in animals before and after challenge with heterologous parasites, animals that mounted a strain-specific response to primary infection were shown to be capable of responding to Ags shared by the two parasite populations. These findings indicate that the strain specificity of CTL responses to T. parva is not determined primarily by immune response genes that define the inherent capacity to respond, but rather is a consequence of the response in individual animals being biased toward a limited number of immunodominant peptide-MHC determinants.

Studies of the immune responses of cattle to Theileria parva have provided evidence that immunity to the parasite can operate at two levels, namely the sporozoite and the schizont-infected lymphoblast. Antibodies that neutralize the infectivity of sporozoites have been detected in the serum of hyperimmunized cattle, and a recombinant sporozoite surface antigen has been shown to induce neutralizing antibodies and protection against experimental challenge. However, the immunity that develops following primary infection with T. parva is accompanied by only low levels of antibodies to sporozoites; there is overwhelming evidence that under these circumstances protection is mediated by T cell responses against infected lymphoblasts. Potent class I MHC-restricted cytotoxic T lymphocyte (CTL) responses are detected in animals recovering from infection and treatment or challenge infections. Two recent findings have provided direct evidence for the importance of these responses in immunity. First, the strain specificity of CTL in cattle immunized with one stock of the parasite was found to correlate with the subsequent susceptibility of individual animals to challenge with a heterologous cloned parasite population (in these circumstances some animals are protected whereas others are susceptible to the heterologous challenge). Second, the adoptive transfer of lymphocytes highly enriched for CD8+ T cells, from immune to naive identical twin calves, was found to protect against experimental challenge. The CTL response in individual animals appears to be directed towards a limited number of antigenic epitopes. The antigenic specificity is determined in part by class I MHC phenotype although there is evidence that other phenomena such as antigenic competition are also involved. Current efforts are directed towards identification of the parasite antigens recognized by CTL with the eventual aim of exploring their potential for vaccination.

Evidence that class I major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) are involved in immunity to malaria has highlighted the potential importance of these cells in protection against intracellular parasites. Parasite-specific CTL are a prominent feature of the immune response of cattle to Theileria parva, a related apicomplexan parasite. The relationship between the appearance of these cells in the blood of immune cattle under challenge and the clearance of infection suggests that they are involved in the control of infection, but direct evidence is lacking that CTL can mediate protection. We have made a quantitative kinetic study of CTL responses in lymph originating from infected lymph nodes in a number of immune cattle under challenge with T. parva. Direct killing activity and the frequency of CTL precursors (CTLp) within responding cell populations were evaluated. A substantial increase in the proportion of CD8+ CTL was observed between days 8 and 11 after challenge. Frequencies of CTLp as high as 1:32 were observed and activity was essentially confined to the large blasting cell fraction. The analogous response in peripheral blood was of lower magnitude and delayed by 1-2 days. The high frequency of CTLp in efferent lymph permitted the adoptive transfer of this activity between immune and naive monozygotic twin calves. In separate experiments, naive calves lethally infected with T. parva were protected by inoculation of up to 10(10) responding CD8+ T cells derived from their immune twins. Elimination of CD8+ T cells within the inoculum abrogated this effect. These findings provide direct evidence that CD8+ T cells can control T. parva infections in immune cattle.

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 CD8alpha 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.

Summary A limiting dilution microculture system was optimized to quantify the frequency of Theileria parva ‐specific cytotoxic T lymphocyte precursors (CTLp) in peripheral blood mononuclear cells (PBMC) from immune cattle. Optimal results were obtained with responder cell input levels ranging from 2 × 10 4 /well to 6.25 × 10 2 /well, along with 1–5 × 10 3 /well stimulator cells in standard supplemented RPMI 1640 medium containing 2.5–5% T cell growth factors. Thirty‐six microtitre wells were established at each responder input level. Cultures were incubated for 7 days at 38°C, at the end of which time individual wells were screened for cytotoxic activity in a 4‐h 111 indium oxine‐release assay. Analysis of the cytotoxicity data, by a computer‐programmed maximum likelihood estimation method indicated that they conformed to the Poisson model of single‐hit kinetics. Estimates of frequencies ranged from 1:3600 to 1:5275 CTLp in PBMC of eight cattle between 1 and 24 months after immunization with T. parva . By contrast, no CTLp were detected in six naïve animals analysed to a responder cell input of 10 5 /well. Split‐well analysis of individual microwells showed that the CTL clones generated under limiting dilution conditions displayed exquisite specificity for parasitized cells, were genetically restricted and in some animals were parasite strain‐specific.