INTRODUCTION
Theileria parasites belong to the subphylum Apicomplexa of protozoa, which includes a number of other important pathogens such as Plasmodium, Babesia, Toxoplasma and Eimeria species. They are predominantly parasites of ruminants, although one species, Theileria equi, is found in horses. Theileria are transmitted trans-stadially by ticks, with infections acquired by larval and nymphal stages being transmitted to new mammalian hosts by nymphal and adult stages respectively. The parasite has a haploid genome throughout much of its life cycle, with diploid forms being generated as part of a sexual phase of development within the tick vector. This can result in recombinant progeny if the ticks are infected with mixed parasite strains. Infection in the mammalian host is characterized by successive developmental stages in leukocytes and erythrocytes. The extent to which multiplication of the parasite occurs within either of these two cell types varies according to the Theileria species. In general, the most pathogenic species are those that undergo extensive replication during development in leukocytes. These include T. parva and T. annulata, which cause economically important diseases in cattle known as east coast fever and tropical theileriosis, respectively. T. parva is transmitted by Rhipicephalus appendiculatus ticks whereas T. annulata is transmitted by several species of Hyalomma ticks. The distinct geographical distributions of the parasites reflect the different ranges of the tick vectors. Thus T. parva occurs throughout a large area of east, central and southern Africa, whereas T. annulata is found within a subtropical zone in the northern hemisphere extending from southern Europe and northern Africa through the Middle East to India and the far east. Overlap in the distribution of the diseases is confined to a small region of southern Sudan.
Efforts to develop vaccines for theileria parasites have concentrated on T. parva and T. annulata because of their impact on livestock production. Accordingly, this review will focus on these two parasites.
THE NEED FOR EFFECTIVE VACCINES
Infection of cattle with T. parva or T. annulata results in an acute lymphoproliferative disease characterized by fever and lymph node enlargement (Irvin and Morrison, 1987). Anaemia, as a consequence of infection of erythrocytes, is also a feature of infections with T. annulata. Death can occur within 3–4 weeks of infection and high levels of mortality are often observed in fully susceptible stock, particularly in the case of T. parva, for which mortality levels of greater than 90% have been recorded. In general, indigenous cattle (usually Bos indicus) develop less severe disease and suffer lower levels of mortality than introduced European breeds (B. taurus) which are highly susceptible to both diseases. Although the basis of breed differences in susceptibility have not been investigated in detail, experiments in which the progeny of zebu cattle (Bos indicus) from western Kenyan and Friesian cattle (B. taurus) were compared for susceptibility to infection with T. parva confirmed the greater susceptibility of the latter (Ndungu, Brown and Dolan, 2005). Nevertheless, the zebu suffered severe disease when infected with higher doses of parasites. Despite their vulnerability to disease, there has been extensive introduction of European breeds into Theileria-endemic regions to satisfy increasing demands for milk production. Maintenance of these animals requires effective control of theileriosis.
Two closely related theilericidal compounds, parvaquone and buparvaquone, are marketed for treatment of theileriosis (McHardy et al. 1985; Dolan et al. 1988). However treatment needs to be applied in the early stages of clinical disease in order to achieve rapid resolution of infection. This is because of the widespread destruction of the immune system that occurs during the advanced stages of the disease. During these later stages, several doses of the drug are required to effect cure and affected animals may not return to full productivity for several weeks. Both compounds are also relatively expensive and are often not affordable by, or available to poor small-holder farmers. Thus, although chemotherapy can clearly contribute to control of theileriosis, its use as a primary control measure has major limitations.
Traditionally, prevention of tick infestation by application of acaricides has been used to control these diseases. Parasite challenge often occurs during many months of the year, and therefore acaricides need to be applied regularly over prolonged periods to be effective. Because of the expense and organisation involved in their application, tick control protocols are difficult to sustain. It is also well established that intensive use of acaricides can result in the emergence drug resistant tick populations. An additional disadvantage of intensive tick control is that the cattle populations remain susceptible to tick-borne pathogens and are therefore vulnerable to major disease outbreaks in the event that acaricides become inaccessible due to changes in local political or economic circumstances.
The shortcomings of the disease control measures discussed above highlight the need for effective vaccines to provide a sustainable means of controlling these diseases.
IMPORTANT BIOLOGICAL PROPERTIES OF THEILERIA PARASITES
A number of features of the development of Theileria parasites within their mammalian hosts are particularly relevant to considerations of vaccine development. Firstly, unlike other apicomplexan protozoa, which typically reside within endocytic vacuoles, Theileria parasites develop within the cytoplasm of host cells (Fawcett et al. 1982). This is achieved by dissolution of the host endosomal cell membrane shortly after invasion of the host leukocyte/erythrocyte by the infective sporozoite stage, which occurs by receptor-mediated endocytosis.
A second important property of these parasites is their ability to remain in an intracellular location throughout the replication phase within leukocytes. This is a consequence of the unique relationship that the parasite has evolved with host leukocytes, whereby development of the schizont stage from invading sporozoites results in induction of proliferation of the host cell (Fawcett, Musoke and Voight, 1984; Dobbelaere, Fernandez and Heussler, 2000; Dobbelaere and Rottenberg, 2003). Division of the parasite is synchronised with that of the host cell, so that parasite multiplication occurs by clonal expansion of the infected cells (Hulliger et al. 1964). This process appears to be dependent on the ability of the parasite to associate with the host cell mitotic spindle. Available evidence indicates that the parasite up-regulates a number of signalling pathways promoting cell proliferation and simultaneously down-regulates apoptotic pathways (Dobbelaere et al. 2000). Recent studies with T. annulata have identified a family of DNA-binding molecules, some members of which are exported from the schizont into the host cell nucleus (Swan et al. 1999, 2001; Dobbelaere et al. 2000). Observations on cells transfected with genes encoding these molecules indicate that they have a regulatory role in host cell activation (Shiels et al. 2004). However, it is likely that transformation of the host cell by Theileria parasites is a complex process involving multiple parasite genes. Although this process appears fundamentally similar in T. parva and T. annulata, the two parasites have distinct cell tropisms, the former infecting T and B lymphocytes (Baldwin et al. 1988) and the latter infecting monocytes, dendritic cells and B lymphocytes (Spooner et al. 1989; Stephens and Howard, 2002). Infection with both parasites results in rapid multiplication of parasitised cells, which disseminate throughout the lymphoid system causing marked hyperplasia and eventually extensive destruction of lymphoid tissues (Morrison et al. 1981). Parasites within a proportion of the infected leukocytes differentiate to merozoites which upon release invade erythrocytes giving rise to the tick-infective piroplasm stage of the parasite. Animals that recover from infection continue to carry small numbers of parasites (both schizonts and piroplasms) for months or years (Geysen et al. 1999; Skilton et al. 2002). Although this persistent infection, referred to as the carrier state, is difficult to detect microscopically, parasites can be transmitted by ticks. It has been estimated that 90% of T. parva infections transmitted by ticks in endemic areas are acquired by feeding on carrier animals (Medley, Perry and Young, 1993).
The mode of replication of Theileria within leukocytes, coupled with the apparent absence of expression of parasite proteins on the surface of infected cells means that the parasite in not accessible to antibodies during this critical phase of infection, implying that cell-mediated immune responses are likely to have an important role. Moreover, the intracytoplasmic location of the parasite should favour immune recognition by CD8+ T cells as a consequence of parasite molecules released into the host cell entering the class I MHC antigen processing pathway.
An additional property of Theileria-infected leukocytes, of direct practical value for vaccination, is the ease with which they can be propagated in vitro as continuously growing cell lines (Brown et al. 1973). Such cell lines can be established either with parasitised cells collected from infected cattle or by infection of leukocytes in vitro with sporozoites. Once established, they grow to densities of 1–2 million cells per ml, have a cell doubling time of 18–24 hours and apparently have the capacity to grow indefinitely in vitro. Parasitised cell lines subjected to prolonged passage in vitro have been used successfully to vaccinate cattle against T. annulata (see below). Infected cell lines have also proved invaluable for immunological studies to dissect the nature and specificity cellular immune responses induced by both T. parva and T. annulata.
LIVE VACCINES
Broadly speaking, two main approaches have been pursued in attempts to develop anti-Theileria vaccines, namely administration of live parasites and the use of defined antigens believed to represent targets of protective immune responses.
All early attempts to vaccinate cattle against theileriosis using crude antigen extracts from parasitized leukocytes in adjuvants were unsuccessful. By contrast, it has long been recognised that animals that recover from clinical disease are immune to subsequent challenge. Almost 100 years ago Theiler (1911) demonstrated that cattle could be infected with T. parva by transfer of infected cells from tissues of clinically affected cases and that recovered animals were immune to subsequent challenge. On the basis of this and other observations Spreull (1914) undertook a field trial involving immunisation of 283000 cattle with spleen and lymph node cell suspensions from T. parva-infected cattle. Unfortunately approximately 25% of the animals died as a result of infection established by the immunisation procedure, but about 70% of the survivors were immune to subsequent challenge. Based on subsequent studies, it is now known that the clinical outcome following immunisation with parasitised leukocytes is influenced by the dose of cells administered (Pirie, Jarrett and Crighton, 1970) and also the similarity between of MHC genotypes of the donor cells and the recipient animal (Buscher, Morrison and Nelson, 1984).
Early studies of T. annulata identified naturally occurring parasite strains that were relatively avirulent. Unlike T. parva, cattle can readily be infected with T. annulata by transfer of small quantities of blood from an infected animal, thus allowing parasites to be propagated in vivo without a requirement for tick passage (Pipano, 1974). This method was used in Israel during the 1950s to maintain a mild Algerian strain of T. annulata, in order to provide parasites for vaccination of cattle using infected blood (Pipano, 1974). However, the parasite was not completely attenuated and vaccination resulted in disease and mortality in a small proportion of animals.
The development of methods for culturing parasitized cells (Tsur and Adler, 1965; Brown et al. 1973) and for cryopreserving parasites (Cunningham et al. 1973) (both sporozoites and parasitised cell lines), in the 1960s and 1970s, provided a new impetus to work on development of live vaccines. Long-term culture of T. annulata-infected cells was shown to result in attenuation of their virulence and animals inoculated with such cells were found to be immune to subsequent challenge (Pipano and Tsur, 1966). Around the same time, an infection and treatment protocol involving simultaneous administration of long-acting tetracycline and defined doses of cryopreserved sporozoites was found to induce immunity against T. parva (Radley et al. 1975a). While this protocol (termed infection and treatment) resulted in solid immunity to challenge with the homologous parasite isolate, there was variable protection against heterologous isolates. This problem was overcome by using a mixture of 3 parasite isolates for immunisation (Radley et al. 1975b). Despite the undoubted efficacy of these two methods of vaccination, they have a number of practical disadvantages that have curtailed their widespread use. Their merits and shortcomings will, therefore, be considered in further detail.
Vaccination against T. parva by infection and treatment
Treatment with tetracyclines has little effect on the course of infection with T. parva when applied after the onset of clinical signs. However, administration of oxytetracycline at the time of infection, as a slow release formulation providing 5–6 days of activity, delays parasite development resulting in a mild transient infection with recovery (Brocklesby and Bailey, 1965; Radley et al. 1975a). Experimental immunisation of cattle with the Muguga isolate of T. parva, using this method, resulted in solid immunity to challenge with a thousand-fold lethal dose of sporozoites of the same parasite isolate (Morzaria et al. 1987b). Immunity was also found to persist for up to 3·5 years in the absence of further parasite challenge (Burridge et al. 1972). However, early studies revealed that immunisation with a single parasite isolate did not usually provide complete protection against challenge with heterologous parasite isolates (Radley et al. 1975a); typically, a proportion of the immunised animals were immune whereas the others remained susceptible to challenge. Nevertheless, subsequent experiments demonstrated that immunisation with a mixture of 3 selected parasite isolates (Muguga, Kiambu 5 and Serengeti) resulted in immunity against a range of heterologous isolates (Radley et al. 1975b). This mixture of isolates (referred to as the Muguga cocktail) has been used successfully for field vaccination of cattle by infection and treatment at a local level in several east African countries (Uilenberg et al. 1976). More recently, experimental immunisation with a single parasite isolate (Marikebuni) provided protection against challenge with a number of heterologous isolates (Morzaria et al. 1987a). This parasite stock has also been used successfully for vaccination of cattle in the field in Kenya. Molecular and immunological studies of the Marikebuni parasite have shown that it is genetically and antigenically heterogeneous (Goddeeris et al. 1990; Toye et al. 1991), which almost certainly accounts for its broad protective activity.
The main practical constraints to the widespread use of the infection and treatment method of vaccination relate to the infrastructure required to produce and distribute the vaccine. Each batch of sporozoites (of each of the constituent parasite isolates) used in the vaccine needs to be produced by passage through ticks fed on infected cattle and the appropriate dose of sporozoites needs to be determined by titration in cattle. The latter is necessary because long-acting tetracycline fails to control infections initiated with higher doses of parasites. The use of an in vivo vaccine production system in cattle also introduces significant risk of contamination with extraneous bovine pathogens, necessitating rigorous quality control of each vaccine batch. The stocks of parasites are stored as cryopreserved stabilates in liquid nitrogen and, since the organisms need to be administered shortly after thawing, a cold chain is required for distributing vaccine to regions distant from the site of production. Ideally, vaccine users should have access to therapeutic theilericidal drugs to allow treatment of any animals in which the tetracycline does not effectively control the immunising infection. Thus, vaccine production and distribution is a complex procedure requiring considerable infrastructure and specialised expertise, which are not available in some countries where the disease is a problem.
An additional reason why infection and treatment has not been adopted in some regions is the perceived risks associated with establishment of carrier infections with the ‘foreign’ parasite strains present in the vaccine and hence their introduction into local tick populations. Specifically, the main concern is that the introduced parasites may be more virulent or differ antigenically from the indigenous parasites. However, the observation that immunisation with the Muguga cocktail provides protection in geographically distant regions suggests that the parasites within the vaccine are unlikely to differ radically in their antigenic composition from local parasites. In any event, vaccination should be most effective against the parasites in the vaccine. Of potentially greater concern, relating to vaccine efficacy, is whether sustained vaccination might result in selection of minor antigenically distinct populations that are not covered by the vaccine (reviewed in Morrison, 1996; McKeever and Morrison, 2002).
Vaccination against T. annulata with parasitised cell lines
In the 1960s, the development of methods to establish continuously growing parasitised cell lines provided, for the first time, a reliable in vitro system for producing Theileria for vaccination. In the case of T. annulata, inoculation of calves with as few as 103 cells from such cultures was shown to result in establishment of infection in a proportion of animals, although doses of at least 105 cells were required to achieve reproducible infection in all animals (Pipano, 1981). Animals receiving 1–5×106 cells from recently established cultures of virulent isolates developed patent infections, a proportion of which resulted in clinical disease and mortality (Pipano and Tsur, 1966). However, prolonged passage of these cultures resulted in attenuation of virulence, such that doses of up to 107 cells resulted in only mild transient infection with minimal clinical signs (Pipano, 1981; Pipano, 1989). Importantly, animals immunised with these cultured cells (usually with doses of 1–5×106 cells) were immune to subsequent challenge with potentially lethal doses of sporozoites (Pipano, 1989; Pipano and Tsur, 1966).
A key feature of infections established by inoculation with parasitised cell lines is that the parasite transfers from the donor cells into the cells of the recipient animals. Thus, there is an initial transient febrile response between days 5 and 8, during which the inoculated cells may be re-isolated from the regional lymph node; this is followed by a more prolonged reaction starting about day 12, during which parasitised cells displaying the recipient animal's MHC genotype can be isolated from blood and lymphoid tissues (Nichani et al. 1999; Graham et al. 2001). Given the evidence that immunity is mediated by T cell responses and is thought to require presentation of parasite antigens on self MHC molecules (see below), this transfer of infection into the recipient animals' cells is believed to be essential for induction of immunity. In this regard, transfer of infection from T. parva-infected cells appears to occur at a much lower frequency, and immunity can only be achieved reliably by administering large doses (~108 or more) of parasitised cells (Pirie et al. 1970; Emery et al. 1982). To date there are no reproducible systems for studying parasite transfer in vitro and therefore the mechanisms by which transfer occurs and the reason why the efficiency of transfer differs for T. annulata and T. parva are not understood.
As with the immunity induced against T. parva by infection and treatment, there is some evidence of antigenic heterogeneity between isolates of T. annulata, as judged by the immune status of animals immunised with attenuated parasitised cell lines. Thus, there are reports that such animals exhibit more severe reactions following challenge with heterologous parasite isolates compared to that observed following homologous challenge (Dargouth et al. 1996; Pipano, 1974). These observations are consistent with earlier reports that animals recovered from infection with one T. annulata isolate sometimes developed severe reactions when subsequently challenged with a different parasite isolate (reviewed in Pipano, 1974). Recent experiments involving immunisation of cattle with cell lines infected with Tunisian T. annulata isolates have indicated that the passage history of the cell line can influence the level of protection against heterologous isolates (Dargouth et al. 1996); cattle immunised with cell lines that had undergone prolonged passage suffered more severe reactions following challenge than those immunised with the same cell lines at an earlier level of passage. Analyses of these cell lines for parasite isoenzymes and polymorphic genotypic markers indicated that prolonged passage resulted in reduced genetic diversity in the parasites within the cell lines and these authors suggested that this loss in diversity may account for the reduced capacity to provide protection against challenge with heterologous parasite isolates.
Despite these potential limitations, attenuated parasitised cell lines have been used successfully in a number of countries to vaccinate cattle against T. annulata. This method was first developed in Israel, where an attenuated vaccine line was developed by in vitro passage of a locally derived parasite isolate; culture of the cells for more than 10 months was required to achieve reliable attenuation (Pipano and Tsur, 1966; Pipano, 1981). The vaccine, which utilised a dose of 2×106 cells, was effective at controlling the disease, although occasional cases of disease occurred in vaccinated dairy cows. Subsequently, similar vaccines have been developed and used successfully in other countries, including India, Iran, Turkey and the Soviet Union.
The use of cell lines for vaccination overcomes some of the quality control problems associated with the infection and treatment method of immunisation. Large batches of cells can be produced and cryopreserved prior to use. The main practical disadvantage of the vaccine is the requirement for a cold chain for distribution to remote sites. However, unlike sporozoites, parasitised cells reconstituted in appropriate culture medium remain viable for several days at 4 °C and therefore can be distributed under refrigeration. As with T. parva, there is a reluctance to use cell line vaccines that have been produced in another country because of the perceived risk of introducing parasite strains that are different from those already present. The parasites within some attenuated cell lines have been reported to lose the ability to differentiate to piroplasms in vivo, following prolonged in vitro passage, implying that they would be unable to infect ticks (Pipano, 1974). However, since this claim was based on the failure to detect infected erythrocytes microscopically in vaccinated animals, the possibility that small numbers of piroplasms are generated, sufficient to enable tick transmission, cannot be excluded. In any event, since experimental work on cell line vaccines has focused mainly on testing their ability to protect against local parasite populations, relatively little information is available on the capacity of parasites isolated from different countries to cross-protect.
SUBUNIT VACCINES
The practical limitations imposed by using live parasites for vaccination against theileriosis have led to efforts to develop alternative vaccines based on defined parasite antigens. This work has largely focused on understanding the immune responses that mediate immunity, with a view to using immune probes to identify candidate antigens for vaccination. These studies have provided strong evidence that, in animals immunised with live parasites, cellular immune responses directed towards parasitised leukocytes play an important role in immunity. Induction of antibody responses capable of neutralising the infectivity of sporozoites has also been demonstrated. Antigens expressed in the different stages of parasite development have been identified and tested for immunogenicity.
Use of sporozoite antigens for vaccination
Cattle that recover from a primary infection with T. parva or T. annulata, with or without the aid of chemotherapy, show little evidence of immunological control of infection at the level of the sporozoite stage following subsequent challenge with sporozoites. Thus, parasitised leukocytes are detected transiently in such animals around the same time as, or slightly later than, in challenge controls, implying that the challenge infection is controlled by responses against the infected leukocytes. Moreover, cattle exposed to a single infective dose of sporozoites, such as that used for infection and treatment, show only low levels of anti-sporozoite antibodies (Musoke et al. 1984). This probably reflects the quantities of sporozoite antigen to which the animals are exposed, since severe infections can be initiated with relatively small numbers of sporozoites (Cunningham et al. 1974). However, repeated challenge of cattle with T. parva sporozoites results in induction of antibodies capable of neutralising the infectivity of sporozoites for lymphocytes in vitro (Musoke et al. 1982). Murine monoclonal antibodies with similar neutralising activity have also been generated against T. parva (Dobbelaere et al. 1984; Dobbelaere, Shapiro and Webster, 1985; Musoke et al. 1984) or T. annulata sporozoites (Williamson et al. 1989) and were shown, in each case, to recognise a dominant protein on the surface of the sporozoite. Immunoelectron microscopic observations on T. parva indicated that the protein was present within a loosely arranges proteinaceous surface coat on the sporozoite (Webster, Dobbelaere and Fawcett, 1985). These findings prompted a series of studies to investigate the possibility of vaccinating cattle against this stage of the parasite.
Identification of sporozoite surface antigens
The genes encoding the respective sporozoite proteins in T. parva and T. annulata were identified by using specific antibodies to screen parasite cDNA expression libraries. The T. parva product is a 709 amino acid polypeptide with a relative molecular mass of 67 kDa (Nene et al. 1992) and is commonly known as p67. The gene that encodes it is present as a single copy and is entirely conserved in all cattle-derived isolates examined to date, although some diversity is observed in parasites derived from buffalo (Nene et al. 1996).
The major surface antigen identified for T. annulata, SPAG-1, has 907 amino acid residues (Hall et al. 1992) and significant homology to p67, particularly at the amino- and carboxy-terminals (Knight et al. 1996). The SPAG-1 gene is also present as a single copy but, unlike p67, is polymorphic between stocks of T. annulata, with most diversity being observed in the central region of the molecule (Katzer et al. 1994).
Experimental immunisation with sporozoite antigens
Several recombinant forms of p67 have been generated in E. coli and these have been evaluated extensively in laboratory immunisation and challenge experiments (Musoke et al. 1992; Nene et al. 1996; Bishop et al. 2003). These have generally taken the form of three to five monthly immunisations using proprietary adjuvant formulations, followed by sub-cutaneous needle challenge with an LD70 of cryopreserved sporozoites two to three weeks after the final boost. Almost invariably, these experiments have resulted in 70% of immunised animals being protected from severe disease, which, given the challenge dose, is roughly equivalent to 50% protection (Ballingall et al. 2004). Approximately 30% show no signs of clinical disease and the remainder undergo infections of intermediate severity. Although the underlying concept behind the vaccine is neutralisation of infection, no correlation has been found between the level of protection and antibody levels; ELISA titres of the order of 104–5 are equally common in animals that develop severe disease and those that remain clinically normal. Similarly, isotype and avidity analyses of these responses have revealed no significant differences between the two reaction phenotypes; in all cases, isotype profiles have been characteristic of a mature, T cell-dependent response (Musoke, Nene and Mckeever, 1995). In spite of this, in vitro specific CD4+ T cell responses are detected only intermittently and with low intensity in immunised animals (Musoke et al. 1995; Ballingall et al. 2004). Finally, when the epitope specificity of the antibody response in protected animals was compared with that of animals undergoing severe disease, using overlapping peptides, no clear differences were observed (Musoke et al. 1995; Nene et al. 1999). Epitopes locate to the amino- and carboxy-terminals of the protein in both cases. Interestingly, those epitopes at the carboxy-terminal are sufficient to induce protection (Bishop et al. 2003).
One interpretation of these observations is that the immune phenotype is associated with greater recognition of conformational epitopes. At least one determinant present on the native molecule (defined by the monoclonal antibody TpM12) is absent from E. coli recombinant forms of p67 (Nene et al. 1995). In addition, comparison of data on the titres of antibody detected by assaying binding to recombinant protein or the sporozoite surface (Ballingall et al. 2004) or by measuring neutralising activity (Musoke et al. 1992) suggest that neutralisation is mediated by only a minor proportion of elicited antibody. However, no differences are apparent between severe reactors and those protected against challenge in either sporozoite-binding (Ballingall et al. 2004) or neutralising antibody titres (Musoke et al. 1992). In addition, cattle immunised with recombinant p67 of eukaryotic origin, such as that expressed by baculovirus (which does incorporate the TpM12 determinant), or recombinant vaccinia virus, show levels of protection similar to those observed with the E. coli-derived antigen and the same spectrum of reactions to challenge (Nene et al. 1995; Honda et al. 1998; Kaba et al. 2005). A recent trial with baculovirus-derived p67 provided evidence for an association between serum neutralising activity and the outcome of challenge (Kaba et al. 2004). However, this was based on only a single serum dilution and definitive evidence for enhanced conformational fidelity of these constructs will require further serological analysis.
It has been argued that the spectrum of reactions observed in p67-immunised animals following challenge relates to individual variation in intrinsic resistance mechanisms (Ballingall et al. 2004). Individual variation has been observed in susceptibility to infection and it is known that the severity of ECF is dose dependent, with a threshold being observed above which all animals react severely, but below which a range of reactions occur (Dolan et al. 1984). It is possible therefore that all animals immunised with p67 antigen preparations achieve similar levels of neutralisation, but have variable success in controlling the residual infection. In this regard, Ballingall and others, in a study of 201 vaccinates, observed significant associations between individual BoLA DRB3 MHC class II alleles and vaccine success or failure (Ballingall et al. 2004). It is possible that these alleles are markers for linked genes involved in innate mechanisms associated with the control of infection.
Live antigen delivery systems offer potential advantages over conventional adjuvant formulations in that they abrogate the need for large quantities of purified antigen and may only require one or two doses of vaccine. Laboratory immunisation and challenge experiments have been conducted in cattle using various p67 expression constructs delivered in recombinant vaccinia and Salmonella typhimurium (Gentschev et al. 1998; Heussler et al. 1998; Honda et al. 1998). More recently, an inactivated baculovirus incorporating the carboxy-terminal of the antigen in its envelope has been evaluated (Kaba et al. 2004). These trials yielded results similar to those seen previously. However, protection in the vaccinia system required co-inoculation of a recombinant vaccinia virus incorporating the bovine IL-2 gene (Honda et al. 1998), and did not appear to be associated with antibody titre (although antibodies were evaluated using an ELISA based on the E. coli recombinant). In the case of Salmonella, there was evidence from small numbers of animals that protection was enhanced by secretion of the antigen into the extracellular space (Gentschev et al. 1998).
In spite of the failure to identify an immunological parameter that correlates with the outcome of challenge, the laboratory immunisation experiments undoubtedly yielded promising results regarding the utility of p67 as a neutralising vaccine. Nonetheless, the laboratory challenge conditions used differ in two important aspects from those that can be expected in the field. First, animals were challenged with a sub-lethal dose at only two-three weeks after the final vaccine boost, which is at the height of the active antibody response. Second, inoculation of sporozoites by an infected tick differs in several aspects from needle delivery. For example, the parasite is delivered in a trickle fashion over a period of days, during which a substantial inflammatory reaction is present at the attachment site (Walker and Fletcher, 1986). The performance of the p67 vaccine in the face of tick-delivered challenge in the field was therefore evaluated at three sites in endemic areas of Kenya (Musoke et al. 2005). The immunisation protocol essentially mirrored that of the laboratory trials, with animals receiving three inoculations of recombinant p67 in adjuvant at monthly intervals, followed by field exposure two weeks after the final boost along with age-matched unimmunised controls. To avoid exposure during the immunisation process, this was conducted either remotely in an ECF-free location or locally under stringent tick control. Field exposure was allowed to continue until all control animals had developed patent infection, which resulted in variation between trials in the duration of challenge. The intensity of challenge also varied between the sites, as indicated by the incidence of severe disease in the control animals. The outcomes of the trials were somewhat ambivalent, with severe disease being observed in both control and immunised groups in each case. Although no significant differences were observed between the groups in the incidence of severe disease at two of the sites, when data from the three trials were combined, a significant reduction of 50% was apparent in the overall incidence of severe disease in immunised animals. Nonetheless, when the effective challenge at each site is taken into account, this equates to a level of protection that is somewhat lower than that observed in the laboratory trials. Significantly, specific antibody titres were observed to decline by an order of magnitude in immunised animals during the challenge period, indicating that the responses to p67 were not boosted by natural challenge.
Evaluation of the vaccine potential of the T. annulata SPAG-1 molecule has focused on recombinant forms of both the full length product and a carboxy-terminal fragment known as SR1. The latter incorporates the terminal 108 residues of the protein and is known to contain neutralising epitopes (Williamson et al. 1989; Boulter et al. 1994). As with the p67 trials, immunisation has generally comprised 3–5 inoculations using a variety of adjuvant formulations, followed by needle challenge with an LD50 dose of stabilated sporozoites (Boulter et al. 1999). The SR1 fragment has been evaluated in separate experiments as a β-galactosidase fusion protein (Williamson et al. 1989) and a chimaera with the hepatitis B core antigen (Boulter et al. 1995). Similarly, two distinct full-length constructs have been tested (Knight, 1993; Hall et al. 2000). In general, these antigen preparations have given rise to variable levels of protection, which is manifest as a delay in the onset of disease and a reduction in its duration. As with p67, a proportion of animals remain fully susceptible to challenge. Neutralising antibodies are produced in response to immunisation, but their presence is not correlated with protection. Similarly, although specific CD4+ T cell responses are detected in SPAG-1 immunised animals, they do not correlate with protection (Boulter et al. 1999; Hall et al. 2000). Interestingly, neutralising determinants in SPAG-1 and p67 appear to be cross-reactive, as revealed by immunoblotting (Knight et al. 1996). One such determinant has been defined at the peptide level and lies at the carboxy-terminal of both molecules (Boulter et al. 1994; Nene et al. 1999), which is the region with highest homology. The significance of this cross-reactivity has been confirmed in cross immunisation experiments. Inoculation with full-length E. coli-recombinant forms of either protein provides a degree of protection against the reciprocal parasite.
Data from a large number of cattle subjected to immunisation with the p67 antigen and subsequent challenge with T. parva (Ballingall et al. 2004), indicate that levels of up to 50% protection against severe disease can be achieved, with protected animals displaying a range of clinical manifestations. Broadly speaking, SPAG-1 immunisation appears to provide a similar degree of protection against T. annulata, although data are available from fewer animals. Given that all available data are based on sub-lethal challenges at the height of the vaccine response, and that p67 antibody responses gradually decline after immunisation, it is likely that protection under natural field conditions will be lower than that observed to date. It is therefore difficult at present to envisage significant uptake of vaccines based on these antigens in endemic areas.
Use of schizont antigens for vaccination
Over the last 20 years, a series of studies of immune responses against the intra-leukocytic stages of T. parva and T. annulata have provided clear evidence that T cell responses against parasitised cells play an important role in immunity. The responses to T. parva have, in particular, been subjected to detailed investigation, which has yielded information on the nature and specificity of protective responses and provided immunological tools that, recently, have been used to identify candidate antigens for vaccination.
CELLULAR IMMUNE RESPONSES TO T. PARVA
In experiments carried out by Emery. in 1981, transfer of thoracic duct leukocytes from immune to naïve twin calves was found to confer protection against challenge with T. parva (Emery, 1981), whereas earlier attempts to transfer immunity with serum from immune animals had been unsuccessful (Muhammed, Laverman and Johnson, 1975). The ability to generate parasitised cell lines by infection of lymphocytes in vitro with sporozoites allowed the development of sensitive assays for parasite-specific T cell responses as well as systems to propagate and clone the specific T cells in vitro. Using these systems, cattle immunised with T. parva by infection and treatment were shown to generate CD4+ and CD8+ T cell responses specific for parasitised cells (Pearson et al. 1979; Eugui and Emery, 1981; Emery et al. 1981; Goddeeris et al. 1986b; Baldwin, Goddeeris and Morrison, 1987; Morrison et al. 1987; Brown, Sugimoto and Grab, 1989). The latter involved classical class I MHC-restricted CD8+ T cells, which exhibited strong cytotoxic activity against parasitised cells of the appropriate MHC genotypes (Goddeeris et al. 1986a,b; Morrison et al. 1987). Potent cytotoxic T lymphocyte (CTL) responses were detected directly in CD8+ T cells obtained ex vivo from animals undergoing immunisation or challenge, coinciding with remission of infection; quantitative analyses using limiting dilution assays indicated that, at the peak of the response following challenge, the specific CD8+ T cells represented at least 3% of the cells in efferent lymph from the lymph node draining the site of challenge (McKeever et al. 1994). Specific CD8+ memory cells could be detected in blood, at frequencies ranging from 1[ratio ]2500 to 1[ratio ]10000, for many months following recovery from immunisation or challenge infections (Taracha et al. 1992).
The role of CD8+ T cells in protection
The protective activity of T. parva-specific CD8+ T cells was demonstrated directly by adoptive transfer of CD8+ T cell-enriched populations between immune and naive identical twin calves (McKeever et al. 1994). These experiments involved harvesting cells from efferent lymph during the active response to challenge of a previously immunised calf, depletion of CD4+ T cells, γ/δ T cells and B cells by antibody and complement-mediated lysis and transfer of the remaining cells into the naïve recipient 2–6 days after infection with sporozoites. Transfer of 8–10×109 cells resulted in effective control of the infection, whereas an animal that received a population of cells from which CD8+ T cells were also depleted remained fully susceptible to infection. While these experiments provided unambiguous evidence that CD8+ T cells are important mediators of immunity they do not exclude the possibility that other cells can also act as immune effectors and/or are required for induction of the CD8+ T cell response. With regard to the latter point, in vitro experiments involving mixing of different combinations of purified CD4+ and CD8+ T cells from genetically identical naïve and immune calves (Taracha, Awino and Mckeever, 1997) have indicated that antigen-primed CD4+ cells are required for effective activation of CD8+ T cells by parasitised cells.
Although many of the studies of CD8+ T cell responses to T. parva have relied on measurement of the ability of the cells to kill parasitised cells, there is no direct evidence that this cytotoxic activity is required for immunity. While studies in mice have provided evidence that perforin-mediated killing of infected cells by CD8+ T cells is an important effector mechanism against a number of intracellular pathogens (Kagi et al. 1994; Walsh et al. 1994; Nickell and Sharma, 2000), there is also evidence that such killing on its own is insufficient and that it acts in concert with other effector mechanisms (White and Harty, 1998; Badovinac and Harty, 2000; Nickell and Sharma, 2000). Production of IFN-γ by CD8+ T cells is important in effecting immunity against many intracellular pathogens (Sher and Coffman, 1992; Huang et al. 1993), including a number of protozoa (Schofield et al. 1987; Suzuki et al. 1988; Rose, Wakelin and Hesketh, 1991). T. parva-specific CD8+ T cells produce large quantities of IFN-γ (Ballingall et al. 2000). However, DeMartini and Baldwin found that addition of IFN-γ to cultures of blood mononuclear cells immediately following infection with T. parva sporozoites had no inhibitory effects on development of the parasite or survival of the infected cells, but rather resulted in some enhancement in the growth of the early parasitised cell cultures (DeMartini and Baldwin, 1991). By contrast, addition of TNF-α had a modest inhibitory effect (33%) on the numbers of cells in which infection established. In the same study, addition of IFN-γ or TNF-α to established parasitised cell lines had no inhibitory effect on the viability or growth of the parasites or host cells. Although another study (Preston, Brown and Richardson, 1992) reported that IFN-γ partially inhibited establishment of T. parva-infected cell lines, the findings are open to question as they were based on measuring the percentage of infected cells in cultures seeded at a single cell concentration, whereas DeMartini and Baldwin (1991) measured numbers and proliferation of infected cells in cultures seeded at limiting dilution. Indeed, many parasitised cell lines express IFN-γ mRNA (McKeever et al. 1997) and produce biologically active IFN-γ protein (DeMartini and Baldwin, 1991). Moreover, we have observed production of large quantities of IFN-γ by uninfected T lymphoblasts in vivo during the early stages of primary infections with T. parva, coinciding with rapid growth of parasitised cells (W. I. Morrison, unpublished data). Since the kinetics of infection following challenge of immune animals indicate that the immune response targets the fully developed schizonts, these findings suggest that IFN-γ does not have a direct effector role in immunity to T. parva, although the possibility that it is required to provide an accessory signal for the activity of other effector molecules cannot be ruled out. Nevertheless, it is likely that the protection against T. parva provided by CD8+ T cells involves other, as yet unidentified mechanisms.
Parasite strain specificity of the CD8+ T cell response to T. parva
A logical extension of the argument that CD8+ T cells play a prominent role in immunity to T. parva, is that parasite strains that fail to cross-protect should be differentially recognised by CD8+ T cells. Indeed, parasite CD8+ T cell lines derived from animals immunised with one parasite strain often recognise cells infected with some parasite strains but not other strains (Goddeeris et al. 1986a,b). As mentioned above, experiments involving immunisation with one parasite strain and subsequent challenge with a second heterologous strain have invariably shown that some animals are protected whereas others remain susceptible to the second strain (Radley et al. 1975a). This feature has been exploited to investigate the relationship between parasite strain specificity of the CD8+ T cell response and immunity to challenge. The strain specificity of the CTL response in 22 cattle immunised either with the Muguga strain, which appears to be a clonal population, or a cloned derivative of the Marikebuni stock (Marikebuni 3219), was found to correlate closely with the susceptibility of the animals to challenge with the reciprocal parasite (Taracha et al. 1995a,b). This finding not only provided further evidence for the involvement of CD8+ T cell responses in immunity but also, importantly, indicated that the antigens recognised by the detectable CTL response are relevant to protection.
Given that T cells can theoretically recognise a large number of epitopes in complex pathogens, the notion that one strain of Theileria can generate a CD8+ T cell response lacking any cross-reactivity with a second parasite strains seems counter-intuitive. However, the explanation for the restricted specificity of the response appears to be that the response in individual animals is focused on a limited number of antigens, most of which exhibit polymorphism between parasite strains and that animals of different MHC genotypes respond to different antigenic determinants (Morrison, 1996; Morrison and McKeever, 1998). This interpretation, which was initially suggested on the basis of the observations that the responses in individual animals were often focused on antigens presented by one of the expressed class I MHC molecules (heterozygous animals may have up to 6 class I genes) and exhibited different patterns of parasite strain specificity in animals of different MHC genotypes (Goddeeris et al. 1990; Taracha et al. 1995b), is supported by data on the responses to recently identified target antigens (see below). This phenomenon of immunodominance, whereby the response is focused on a few of the antigens to which an animal is capable of responding, is a recognised feature of CD8+ T cell responses to many virus infections (Goulder et al. 1997; Butz & Bevan, 1998; Crotzer et al. 2000), most notably HIV-1, influenza and a number of herpes viruses, but had not been reported previously for responses to parasites. Several observations from the studies of strain specificity of CTL responses to T. parva indicate that the restricted specificity of the response reflects a true dominance hierarchy rather than a limited capacity to respond to only a few antigens (Taracha et al. 1995b). First, immunisation of genetically identical twin calves with two different parasites (Muguga and Marikebuni 3219) was shown to result in a CTL response specific for the immunising parasite (Muguga) in one twin, whereas the response in the other twin was cross-reactive between the two parasites. Second, some animals that mounted a strain-specific CTL response to immunisation with Muguga were found to generate cross-reactive CTL following recovery from challenge with Marikebuni 3219. These findings have important implications for vaccine development and will be discussed further below.
CELLULAR IMMUNE RESPONSES TO T. ANNULATA
Although the specific cellular immune responses to T. annulata have not been studied in the same detail, available data indicate that they are similar to those generated by T. parva. The kinetics of cellular changes in the regional lymph node following primary and challenge infections are remarkably similar for both parasites and, as with T. parva, animals immunised with T. annulata exhibit strong parasite-specific CD4+ and CD8+ T cell responses against parasitised leukocytes (Ahmed and Mehlhorn, 1999; Nichani et al. 2003). Class I MHC-restricted CTL activity can also be detected transiently in blood mononuclear cells in animals undergoing immunisation or challenge with T. annulata (Innes et al. 1989; Ahmed et al. 2000) and in one study the CTL were found to exhibit restricted parasite strain specificity (Conze et al. 1998). However, propagation of these cells in vitro as cell lines has proved more difficult than for T. parva.
Induction of TNF-α and nitric oxide (NO) production by macrophages in response to infection with T. annulata have been reported (Preston et al. 1993; Visser et al. 1995). Generation of macrophages with cytostatic activity for T. annulata-infected cells in vitro has also been observed in animals recovering from infection (Preston and Brown, 1988). However, the role, if any, of these responses in control of the parasite remains unclear. Where inducible NO is known to be involved in killing of other intracellular pathogens, it operates primarily as a consequence of induction within the infected cells themselves. To date there is no evidence that this pathway can be activated in T. annulata-infected cells. Although, the same authors have reported that TNF-α and IFN-γ both partially inhibit the establishment of infection with T. annulata in in vitro cultures (Preston et al. 1992), as discussed above (for similar experiments conducted with T. parva in the same study), this finding may partly reflect the way in which infection was quantified in the cultures. As with T. parva, these cytokines had no inhibitory effects on established T. annulata cell lines. Thus, the suggestion that the mechanisms of immunity to T. annulata differ substantially from those operative against T. parva (Preston et al. 1999) is largely unsubstantiated.
Nevertheless cells infected with the 2 parasites differ in a number of properties that might influence the nature of the immune responses that they induce. Most T. annulata-infected cell lines express transcripts for pro-inflammatory cytokines (IL-1, IL-6 and TNF-α) whereas T. parva-infected cell lines show variable patterns of cytokine expression, including T cell associated cytokines such as IL-2 and IL-4 (Brown et al. 1995; McKeever, Nyanjui and Ballingall, 1997). Interestingly, despite the strong immunostimulatory properties of parasitised cells in vitro, all T. parva-infected cells and most T. annulata-infected cells express the anti-inflammatory cytokine IL-10. The most notable difference between cells infected with the 2 parasites is in expression of interferons. Thus, whereas T. annulata-infected cells produce type I interferon, specifically IFN-β, most T. parva-infected cells produce IFN-γ (McKeever et al. 1997; Sager, Brunschwiler and Jungi, 1998). Stimulation of T. annulata-infected cells with the interferon inducer poly I[ratio ]C results in marked upregulation of IFN-β secretion (Sager et al. 1998). Both types of IFN are known to have a regulatory role in induction of immune responses but differ in some of the signalling pathways that they activate (Boehm et al. 1997; Stark et al. 1998; Jefferies and Fitzgerald, 2005).
Identification of T cell target antigens in T. parva
Two recent developments have been exploited by workers at the International Livestock Research Institute (ILRI) in Kenya to identify T. parva antigens recognised by parasite-specific CD8+ T cells. First, a complete genome sequence has been generated for the Muguga strain of T. parva (Gardner et al. 2005). Application of bioinformatics to the genome sequence allowed the identification of genes encoding candidate proteins, based on the presence of recognisable signal peptides in the translated sequences (i.e. proteins that potentially could be exported into the cytoplasm of the host cell). Cloned cDNAs encoding these proteins were then transfected into cells expressing appropriate bovine class I MHC molecules and tested for recognition by CD8+ T cells. Second, a high throughput screening system developed and used successfully for identification of antigens recognised by human tumour cell-specific CD8+ T cells (DePlaen et al. 1997), has been adapted for screening Theileria cDNA libraries. This involved construction of a cDNA library in a eukaryotic expression vector, using RNA from purified T. parva schizonts, and allocation of the library into a series of pools of 50 cDNA clones, each of which was co-transfected along with appropriate bovine class I genes into Cos cells and screened for recognition by CD8+ T cell lines by measuring IFN-γ production. Positive pools were then further resolved to identify the cDNA clone encoding the recognised antigen.
Using these methods, Graham et al. (2006) identified 6 T. parva antigens recognised by CD8+ T cells from immune cattle of different MHC genotypes. Analysis of the predicted amino acid sequences of the genes did not reveal any obvious functional relationship between the proteins. One of the antigens, Tp7, was identified as a heat shock protein (hsp90) and 2 further antigens, Tp4 and Tp5, exhibited homologies with elongation translation initiation factor 1A and the ε subunit of T complex protein 1 respectively. Interestingly, 3 of the antigens did not contain recognisable signal peptides and would therefore not have been identified by the candidate antigen approach using the genome sequence. CD8+ T cell lines specific for the antigens were shown to kill cells transfected with the respective parasite genes and produced IFN-γ in response to stimulation with such cells. The relevance of responses to these antigens to control of infections in vivo was examined by analysing CD8+ T cell responses following sporozoite challenge of immune cattle shown to have memory CD8+ T cells specific for the antigens. Ex vivo responses with kinetics similar to that documented previously for total CD8+ T cell responses were observed for 4 of the antigens, whereas a delayed response was observed for one antigen and no response was observed for another antigen. Thus, at least for 4 of these antigens, the in vivo responses indicate that they are likely to be involved in the protective response.
IMMUNISATION WITH SCHIZONT ANTIGENS
Five of the 6 reported T. parva schizont antigens have also been tested for their ability to induce protective immune responses (Graham et al. 2006). These experiments involved the use of prime-boost protocols involving priming with plasmid DNA or recombinant canarypox viruses followed by a single boost with recombinant vaccinia viruses (the replication defective Ankara strain). Animals were immunised simultaneously with 5 of the antigens in separate DNA or viral constructs and challenged 3 weeks later with a lethal dose of sporozoites. Most of the immunised animals (19/24) exhibited readily detectable antigen-specific CD8+ T cell IFN-γ responses following immunisation but cytotoxic activity for parasitised cells was only detected in 4 of 18 immunised animals. Moreover, in contrast to conventionally immunised animals in which specific cytotoxic CD8+ T cells can readily be detected following a single in vitro stimulation with parasitised cells, detection of the CTL response in these immunised animals apparently required 3 rounds of in vitro stimulation.
Based on the summary data presented by Graham et al. (2006), all of the immunised animals appeared to develop severe clinical reactions following challenge, which were not significantly different from those of the challenge controls. Nevertheless, 10 of the 24 immunised animals survived the challenge, suggesting some protective effect. Since only 4 challenge controls were used, one of which survived challenge, this overall difference in survival was not statistically significant. Those immunised animals that exhibited a CTL response to the antigens used for immunisation, either prior to challenge or 14 days after challenge, showed a significantly increased survival rate compared to those that showed no CTL response. Thus, although the immunisation protocols used in this study clearly do not induce a level of immunity that is of practical value for vaccination, they appeared to result in some reduction in severity of disease, which was possibly associated with induction of cytotoxic activity in the specific CD8+ T cell response.
The results of these experiments raise a number of questions concerning the choice of antigens and the antigen delivery systems employed: Are these the correct antigens for induction of protective CD8+ T cell responses? Are the functional properties of the CD8+ T cells induced by the prime-boost protocol appropriate to provide protection? Are other responses, in particular CD4+ T cell responses, required for protection?
The antigens
As discussed above, the close correlation observed between the strain specificity of CD8+ T cell mediated CTL responses and the ability of parasite stains to cross-protect (Taracha et al. 1995a) indicates that parasite antigens recognised by CTL are relevant to protection. Characterization of the antigens used by Graham et al. (2006) for immunisation included the demonstration that they were recognised by CTL from immune cattle; moreover, active CTL responses against 4 of the antigens were also detected following homologous challenge of immune animals coinciding with remission of the challenge infection. In unpublished studies with two of the antigens (Tp1 and Tp2) used in the immunisation experiment (N. MacHugh, T. Connelley, S. Graham, E. Taracha, D. McKeever, I. Morrison, unpublished data), we have also obtained evidence that over 50% of the CTL response is directed to these antigens, indicating that they are dominant antigens with respect to induction of CTL responses. Thus, at least some of these antigens appear to be appropriate candidates for use in immunisation.
An appropriate CD8+ T cell response
The poor protection provided by the CD8+ T cell responses induced by the prime-boost protocol may have been due to lack of functional properties required for protection. The relatively weak CTL activity of the CD8+ T cells induced by prime-boost immunisation certainly indicated that they differ functionally from those induced by infection and treatment. Although, as discussed above, cytotoxic activity may or may not be required for protection, this difference could reflect other important functional differences in the cells. Recent progress in understanding how innate immune responses contribute to acquisition of immunity has revealed that innate responses not only help to promote the induction of adaptive immune responses but also influence the nature of the responses that are induced (Werling and Jungi, 2003). The latter is largely determined by signalling via a series of receptor molecules, including the Toll-like receptors (Takeda and Akira, 2005), that recognise different foreign molecular patterns in invading pathogens. Studies of the nature of the signals generated by infections with protozoan parasites are at an early stage (Gazzinelli, Ropert and Campos, 2004; Yarovinsky and Sher, 2006) and the innate responses elicited by Theileria have not been studied in any detail. Nevertheless, such signals are likely to be different from those triggered by viruses, including the poxviruses used in prime-boost protocols, and may be important in generating an immune response with appropriate functional activity. This is an area that requires further investigation.
Requirement for CD4+ T cell responses
Infection with T. parva is known to result in strong CD4+ T cell responses against parasitised leukocytes (Baldwin et al. 1987; Brown et al. 1989). Such cells may play an important role in immunity either by providing help for induction of CD8+ T cell responses or by acting as effectors in concert with CD8+ T cells. In the immunisation experiments reported by Graham et al. (2006), the authors stated that only weak CD4+ responses against the T. parva antigens were detected; CD4+ T cell responses to poxvirus antigens may have provided help for induction of CD8+ T cells during the boost phase of the immunisation protocol. However, if indeed effective recall of the memory CD8+ T cells is dependent on CD4+ T cell help, then priming of parasite-specific CD4+ T cells would have been required. This notion is supported by findings from a series of in vitro experiments reported by Taracha and others (Taracha et al. 1997), which indicated that optimal activation of memory CD8+ T cells from T. parva-immune cattle required CD4+ help and that the antigens recognised by the two T cell populations needed to be presented on the same cell. Generation of primary CD8+ T cell responses is now generally considered to require cross-priming, i.e. transfer of pathogen antigens from infected cells into dendritic cells, for efficient presentation to CD8+ T cells (Ackerman and Cresswell, 2004). However, since T. parva-infected cells express high levels of MHC molecules, as well as a number of co-stimulatory molecules, and have been shown to maintain proliferation of Theileria-specific T cells in vitro in the absence of accessory cells, they may be capable of directly activating parasite-specific memory CD8+ T cells in vivo following challenge of immunised animals. If so, optimal recall of CD8+ T cell responses may well require CD4+ T cells specific for parasite antigens presented on the surface of the same parasitised cells.
USE OF MEROZOITE ANTIGENS FOR VACCINATION
Since anaemia is a feature of infections with T. annulata, there has been some interest in the possibility of immunising against the intra-erythrocytic stage of this parasite. An antigen of 30–32 kDa expressed on the surface of T. annulata merozoites, Tams-1, induces strong antibody responses in infected cattle; however, this antigen shows marked allelic variation (Dickson and Shiels, 1993; Shiels et al. 1995). Nevertheless, two alleles of Tams-2 have been used in preliminary immunisation experiments specifically designed to test for protection against the intra-erythrocytic stage (d'Oliveira et al. 1997). Groups of 3 animals were immunised with the antigens, in the form of recombinant proteins in ISCOMs or delivered in recombinant Salmonella typhimurium or plasmid DNA, and challenged with blood from a T. annulata-infected animal with 30% piroplasm parasitaemia. Such blood will also have contained schizont-infected leukocytes. The 3 animals immunised with protein in ISCOMs and 2 of the 3 animals immunised with plasmid DNA were protected, based on substantial reduction in the levels of piroplasms and severity of anaemia. Although animals immunised with protein in ISCOMs developed antibody responses to Tams-1, those immunised with DNA had no detectable antibody. The authors suggested that the immunity in the latter animals may have been mediated by cellular immune responses against infected leukocytes, since Tams-1 is expressed upon differentiation of schizonts to merozoites. While these results are encouraging, it will be important to determine whether or not animals immunised in this way show similar protection against other parasite strains and also when challenged with sporozoites as opposed to infected blood.
PROSPECTS FOR DEVELOPING IMPROVED VACCINES
Live vaccines
In the short to medium term, the use of live parasites still offers the best option for vaccination against east coast fever and tropical theileriosis. Despite the fact that vaccination methods using live parasites have been available for almost 3 decades and have been shown to be effective under field conditions, uptake of such vaccines has been limited by lack of infrastructure and expertise for vaccine production and distribution, and also by concerns about the introduction of vaccine parasite strains into field tick populations. In the case of T. annulata cell line vaccines, the latter concern has led to individual countries developing vaccines for their own use. This in turn has been a limiting factor in commercialisation of these vaccines. Difficulties in standardising the parasites within the vaccines, with respect to antigenic composition and (in the case of T. annulata) the level of attenuation, have also been a limitation in this regard. Thus, most vaccines for T. parva and T. annulata have been produced with limited knowledge of whether the parasite stocks employed were antigenically homogeneous or heterogeneous and whether the content of the stocks changed following passage. Recent genotypic analyses of the Marikebuni stock of T. parva (F. Katzer, D. McKeever, unpublished) provide evidence that the composition of this stock has changed following tick passage, resulting in some components of the stock becoming progressively more abundant. Although attempts have been made to minimise variability in vaccine parasite constituents by laying down large parent and grandparent stocks of parasites from which batches of vaccine are prepared, such stocks are inevitably limited and eventually need to be replenished by further passage. The availability of new methods for genotypic and antigenic typing of parasite populations now provides tools that can be used to improve the standardisation of parasites used for vaccination. In the case of T. parva, consideration should also be given to re-evaluating the parasites included in current vaccines with a view to modifying the content to maximise the diversity of antigenic alleles represented in the vaccines.
The current strategy for developing T. annulata cell line vaccines involves a long and tedious process, involving in vitro passage and testing in animals, which (as currently deployed) needs to be duplicated in each country. A better understanding of the properties that determine the virulence of parasitised cell lines could provide markers that would allow more rapid selection of attenuated lines. Studies involving immunisation with cloned lines derived from recently established T. annulata cell cultures (Graham et al. 2001) have demonstrated variation in virulence between different clones. Although these authors presented evidence that this was linked to the levels of pro-inflammatory cytokines produced by the lines, the number of lines examined was insufficient to be confident that this property was directly related to virulence. Furthermore, the possibility that the differences were associated with parasite genotype could not be excluded since the infected cell clones harboured different parasite genotypes. Nevertheless, these observations indicate the feasibility of using alternative approaches to select attenuated lines.
The potential for vaccine parasites to transmit through ticks is a major issue that constrains the widespread use of these vaccines. The production of parasites with a proven deficiency in tick transmissibility should overcome this problem and allow any vaccine to be used throughout the affected region, assuming that it conferred broad protection against different parasite strains. Cloned T. annulata-infected cell lines have been identified in which the schizonts have a markedly reduced capacity to differentiate to merozoites in vitro in response to increased temperature (Shiels, 1999). However, the capacity of these parasites to differentiate in vivo and transmit to ticks has not been examined. Ultimately, genetic manipulation of the parasite would be the most reliable means of producing transmission-defective parasites, but methods for genetic transformation of theileria parasites have not yet been developed.
Subunit vaccines
Given the evidence that the pathogenesis of infections with T. parva and T. annulata is largely attributable to the intra-leukocytic stages of the parasites and that immunity acquired following recovery from infection is mediated by T cell responses against these stages, it is clear that antigens recognised by T lymphocytes on infected leukocytes represent the best candidates for subunit vaccines. Unlike Plasmodium and Babesia species, the intra-erythrocytic stages of T. parva and T. annulata have a limited capacity to replicate; therefore, immune responses that limit schizont development should also reduce the levels of piroplasm parasitaemia and (in the case of T. annulata) anaemia during the acute stage of infection.
The development and successful use of methods for identifying T. parva antigens recognised by CD8+ T cells is a major step forward. This now needs to be extended to identification of CD4+ T cell target antigens. The next major challenge is to develop an antigen delivery system that can generate protective T cell responses. Initial data from immunisation experiments with recently identified T. parva antigens, as well as findings from vaccination trials for a number of other pathogens, indicate that attainment of immunity is not merely dependent on the antigenic specificity of T cells that are induced but also on their functional properties and the speed with which memory cells can be recalled upon challenge with the pathogen. Thus, further investigation of antigen delivery systems for vaccination need to proceed hand-in-hand with studies to better understand the properties of parasite-specific T cells that are critical for protection.
Assuming that protective immune responses can be generated using defined parasite antigens, such protection will need to be effective against antigenically different strains of the parasite. This is a particularly important issue for T. parva. As discussed above, the observed focusing of the CD8+ T cell response on a few, mainly polymorphic antigenic determinants in animals immunised with live T. parva appears to account for the strain restricted immunity. However, although different parasite strains show considerable antigenic polymorphism, we have previously presented evidence that the patterns of polymorphism overlap between strains, possibly reflecting previous genetic recombination events generated during the sexual phase of development in the tick (McKeever and Morrison, 1998; Morrison, 1996). If indeed these experimental findings are representative of field populations of parasites, we would predict that induction of T cell responses to a larger number of epitopes, even though they may be polymorphic, would be sufficient to provide broad protection. Achievement of such protection with subunit vaccines would thus require induction of T cell responses to an extended but perhaps limited set of parasite antigens.
