Veterinary Immunology and Immunopathology, 20 (1989) 213-237 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
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T h e i l e r i a p a r v a in Cattle: C h a r a c t e r i z a t i o n of
Infected L y m p h o c y t e s and the I m m u n e Responses they P r o v o k e I W.I. MORRISON, B.M. GODDEERIS 2, W.C. BROWN a, C.L. BALDWIN and A.J. TEALE
International Laboratory for Research on Animal Diseases (ILRAD) P.O. Box 30709, Nairobi (Kenya) ~This is ILRAD Publication No. 613. 2B.M. Goddeeris was supported by the A.B.O.S./A.G.C.D. (General Administration for Development-Cooperation), Belgium :~Present address: Department of Veterinary Microbiology and Parasitology, College of Veterinary Medicine, Texas A & M University, College Station, TX 778443 (U.S.A.) TABLEOFCONTENTS 1.0 INTRODUCTION 2.0 THE PARASITE AND THE DISEASE 3.0 TARGET CELL TYPES FOR INFECTION 3.1 General 3.2 Studies of cell types infected in vitro 3.3 Phenotypes of infected cells in vivo 3.4 Pathogenicity of different infected cell types 3.5 Possible influences of functional properties of infected cells on pathogenicity or immunogenicity 4.0 IMMUNITY TO T. PARVA 4.1 General features of immunity 4.2 Immune effector mechanisms 4.3 Class I MHC-restricted T cell responses to parasitized cells 4.4 Generation of Theileria-specific T cells in vitro 4.5 Parasite strain-specificity of the T cell responses 5.0 IDENTIFICATION OF PARASITE ANTIGENS RECOGNISED BY T CELLS 6.0 REFERENCES
1.0 INTRODUCTION
The protozoan parasite Theileria parva infects cattle and causes an acute lymphoproliferative disease called East Coast fever (ECF) (Morrison et al., 1986a; Irvin and Morrison, 1987). The parasite also infects African buffalo (Syncerus caller), but in this species it establishes low-grade persistent infections without obvious signs of clinical disease. The buffalo, therefore, acts as a reservoir of infection in endemic areas. T. parva is transmitted by the three0165-2427/89/$03.50
© 1989 Elsevier Science Publishers B.V.
214 host tick Rhipicephalus appendiculatus, and control of ECF relies principally on regular spraying or dipping of cattle with acaricides to prevent tick infestation. The disease is prevalent throughout a large area of East and Central Africa, where it causes major economic losses, not only as a result of animal mortality and reduced productivity but also due to the costs incurred in implementing and maintaining tick-control programmes. The development of alternative methods of controlling ECF, such as vaccination, would therefore have a major impact on cattle production in East and Central Africa. T. parva is an intracellular parasite which in the mammalian host utilises, successively, lymphocytes and erythrocytes for completion of its life cycle. Proliferation of the parasite occurs mainly within the lymphocytes, and it is this intralymphocytic stage which is responsible for much of the pathology of the disease. Moreover, evidence from experiments carried out over the last 7 years indicates that immune responses against parasitised lymphocytes are important in protective immunity (Morrison et al., 1986a). Herein, we will review current information on the cell types infected by T. parva and on the host immune responses against parasitised lymphocytes, and discuss the relevance of the data to the pathogenesis and immunology of the disease. 2.0 THE PARASITEAND THE DISEASE Based on clinical and epidemiological parameters, isolates of T. parva are often classified into one of three subtypes, namely T. p. parva, T.p.lawrencei and T. p. bovis (Uilenberg, 1981). The main distinguishing features of the three subtypes are that T. p. boris is much less virulent than the other two in cattle and that T. p. parva is transmitted mainly between cattle, whereas T. p. lawrencei is transmitted mainly from buffalo to cattle. However, the subtypes are morphologically and serologically indistinguishable. Furthermore, monoclonal antibodies (MAb) and DNA probes which detect heterogeneity between isolates of the parasite have so far not revealed a clear distinction between the subtypes (Conrad et al., 1987a,b). In this paper, the term T. parva is used to include all three subtypes of the parasite. In view of the possibility of heterogeneity in the parasite populations within individual isolates of T. parva, each isolate is referred to as a stock. The infective stage of T. parva, the sporozoite, develops in the salivary glands of the tick vector. Sporozoites are ejected in the saliva several days after the tick commences feeding on a suitable host. In cattle and buffalo, the sporozoites are rapidly taken up by lymphocytes by a process of receptor-mediated endocytosis (Fawcett et al., 1984). Within 24 h, the host cell membrane surrounding the parasite is destroyed and the parasite lies free within the cytoplasm of the cell, usually close to the Golgi apparatus. During the next few days, development of the parasite into the multinucleated schizont stage causes the host cell to undergo blastogenesis. During cell division the schizont asso-
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ciates with the mitotic spindle and divides at the same time as the host cell, the nuclei being randomly distributed to the two daughter cells (Hulliger et al., 1964). Thus there is clonal expansion of the cells initially infected by the parasite. This relationship of the parasite with host lymphocytes allows parasitised cells to be cultivated in vitro as continuously growing cell lines (Fig. 1 ). Cell lines can be established either with cells from the tissues of infected cattle (Malmquist et al., 1970) or by infecting lymphocytes in vitro with tickderived parasites (Brown et al., 1973). Following inoculation of a susceptible bovine host with sporozoites of T. p. parva, the first evidence of infection is the appearance, after several days, of schizont-infected lymphoblasts in the lymph node draining the site of inoculation. Two to three days later, parasitised cells are detectable in a range of other lymphoid tissues. Within a further 5-7 days, levels of parasitosis in excess of 30% can be attained in the cells of the lymph nodes, and most animals die 14-21 days after inoculation with sporozoites. As the infection progresses, the mean number of nuclei per schizont increases and a proportion of the schizonts undergo merogony. Merozoites thus formed are released upon disruption of the host cell and invade erythrocytes to give rise to piroplasms. The piroplasm is the tick-infective stage of the parasite. Unlike some other Theileria species, there is negligible proliferation of piroplasms. In cattle which die from ECF caused by T. p. parua, large numbers of parasitised cells are found throughout the lymphoid system. There is also infiltra-
Fig. 1. Lymphoblastoid cells infected with T. parva: schizonts can be seen as multinucleate bodies within the cytoplasm of the cells. Acid-hydrolysed Giemsa stain ( X 1000).
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tion of parasitised lymphoblasts into non-lymphoid tissues; this is particularly apparent in the lungs and gastrointestinal tract and in the former is commonly associated with severe pulmonary oedema (Irvin and Morrison, 1987). Lymphoid tissues become markedly depleted of lymphocytes (Jarrett et al., 1969; Morrison et al., 1981 ) and there is a profound reduction in the recirculating pool of lymphocytes (Emery, 1981a). These changes are due in part to the extensive lymphocytolysis which occurs during the later stages of the disease (Barnett, 1960; Irvin and Morrison, 1987). However, significant leukopaenia occurs prior to the onset of lymphocytolysis and is probably due to a direct effect of invading parasitised lymphoblasts on haemopoietic tissues. While the parasite itself results in destruction of some cells, the cytolysis appears to involve both infected and uninfected lymphocytes. Thus other, as yet undefined, mechanisms must be involved in the widespread cell death. 3.0 TARGET CELL TYPES FOR INFECTION
3.1 General The fact that the Theileria parasite infects the same cell system which generates protective immune responses against the parasite has potentially important implications in both the pathogenesis and immunology of the infection. Thus, on the one hand, functional changes induced in host lymphocytes by the parasite might influence the generation of parasite-specific immune responses and, on the other hand, soluble mediators elaborated during the induction of such immune responses may modulate the growth or behaviour of the parasitised cells. Initial studies of the phenotype of infected cell lines and parasitised cells from infected cattle, with reagents specific for bovine immunoglobulins (Ig), showed that the cells were negative for surface and intracellular Ig (Duffus et al., 1978; Emery 1981a). This characteristic, together with the lymphoblastoid morphology of the cells, prompted the suggestion that they were T lymphocytes. In a later study, Pinder et al. (1981) demonstrated that a series of parasitised cell lines reacted with the monoclonal antibody (MAb) BT3/8.12 and with the lectins peanut agglutinin (PNA) and soyabean agglutinin (SBA). On the basis of the observation that BT3/8.12 and SBA reacted with a cell surface determinant on a subpopulation of lymphocytes which were negative for surface immunoglobulin (sIg), these authors suggested that the parasite preferentially infects a subpopulation of T lymphocytes. 3.2 Studies of cell types infected in vitro The development of techniques for infecting lymphocytes in vitro with sporozoites enabled more critical analyses of the cells that become infected with
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T. parva to be carried out, by examining the susceptibility to infection of purified populations of leukocytes. Using such an approach, Lalor et al. (1986) showed that when peripheral blood mononuclear cells (PBM) were separated into B cells and non-B cells and exposed to T. parva sporozoites, both cell populations gave rise to parasitised cell lines. The cell populations were separated with a cell sorter following staining of PMB with MAb specific for bovine IgM or class II major histocompatibility complex (MHC) antigens, and were of greater than 96% purity. Following incubation with sporozoites, the cells were distributed at limiting dilution into 96-well microtitre plates containing a fibroblast feeder layer. This allowed an estimate to be made of the frequency of cells in each population which gave rise to cell lines; this was similar for the B cell and non-B cell populations. Cell lines growing at clonal dilution were then expanded and their phenotypes compared with a series of MAb. Cell lines derived from the B cell-enriched population could not readily be distinguished from those derived from non-B cells; in most instances, they did not express detectable levels of surface or cytoplasmic Ig and they all reacted with MAb which, in resting PBM, recognise T cells and monocytes but not B cells (Lalor et al., 1986). Thus, some doubt remained as to whether or not the cell lines were indeed derived from B lymphocytes. In such circumstances, there was little point in trying to phenotype the parasitised cells in infected cattle. Recently, with the generation of lineage-specific MAb, it has been possible to resolve these issues. Several MAb have been particularly useful in these studies; MAb CH128A, IL-A26 and IL-A43 recognise the sheep erythrocyte receptor on bovine T cells (designated BoT2) and thus can be used as pan T cell markers (Davis et al., 1988; Baldwin et al., 1988b); MAb I L - A l l and ILA12 recognise a molecule (designated BoT4) on a subpopulation of bovine T cells, analogous to the human CD4 marker (Baldwin et al., 1986); MAb ILA17 and IL-A51 recognise a molecule (designated BoT8) on another subpopulation of bovine T cells, analogous to the human CD8 marker (Ellis et al., 1986 ); MAb IL-A29 reacts specifically with a small population of lymphocytes which do not express Ig and are negative for the above T lymphocyte markers (Morrison et al., 1988); although there is some evidence that these IL-A29 + cells are derived from the thymus, herein they are referred to as null cells. These MAb have been used in further experiments to sort P B M into the various subpopulations for infection with sporozoites, in order to analyse the frequencies of establishment of parasitised cell lines. These experiments confirmed that cell lines could be established from both B cell and T cell populations and, in addition, showed that null cells could be infected with the parasite and that within the T cell population both BoT4 + and BoT8 + cells became infected (Baldwin et al., 1988a). By contrast, neither monocytes nor granulocytes gave rise to infected cell lines. A previous report that parasitised cell lines could be obtained by infecting blood monocytes in vitro (Moulton et al., 1984 )
218 is probably erroneous, as considerable numbers of contaminating lymphocytes were present in the starting monocyte population. Comparisons of the frequencies of establishment of cell lines from the different cell populations, in our studies, could not be made between different experiments, because the concentration of infective sporozoites was not standardised from one experiment to another. However, within individual experiments, the frequency of establishment of cell lines from T cells tended to be higher than that from null cells, and the BoT4 + subpopulations of T cells gave a higher frequency of cell line establishment than the BoT8 + cells. In some experiments, B cells and non-B cells gave similar results, whereas in others, higher frequencies of cell line establishment were obtained with non-B cells. The significance of these differences in frequencies is difficult to assess as they may merely reflect preferential suitability of the culture conditions used in the experiments for propagation of particular infected cell types. By the same reasoning, the possibility that other cell types, such as monocytes, may be infected and transformed in vivo cannot be discounted. In order to analyse the cells that are infected in vivo, it is necessary to be able to distinguish the different cell types after infection with the parasite. Therefore, a large number of parasitised cell lines obtained at clonal dilution from the different lymphocyte populations in vitro were phenotyped with the MAb (Baldwin et al., 1988a). All of the cell lines obtained from B lymphocytes were negative for T cell and null cell differentiation antigens and, in contrast to the initial observations by Lalor et al. (1986), over half of the lines were found to express sIg (W.I. Morrison, unpubl, data, 1987). In most instances, this was IgM, although a few IgG + lines were detected, and only a variable proportion of the cells within each cloned line expressed Ig at any given time. With passage, many of the lines stopped expressing Ig so that after 6 months of culture only a small percentage of the lines were still positive. Intracellular Ig was also detected in those cell lines which were sIg +, but to date we have not been able to detect any secreted Ig in the culture medium (D. Williams and W.I. Morrison, unpubl, data, 1988). Many of the cell lines derived from the BoT4 + BoT8- cells showed expression of BoT8 on a proportion of the cells, whereas, with a few exceptions, lines derived from the BoT4- BoT8 + cells remained negative for BoT4 (Baldwin et al., 1988a). Both the T cell types showed variable expression of the null cell marker. Moreover, infected null cell lines invariably acquired partial expression of BoT2 and BoT8 but not BoT4. Phenotypic analysis of a large number of uncloned parasitised cell lines obtained by infection of unfractionated PBM has revealed that, with one exception, all express one or more of the T cell markers. This observation indicates that, in in vitro cultures, parasitised B cells are invariably overgrown by other cell types. The results of these experiments clearly demonstrate that all of the presently definable subpopulations of bovine lymphocytes can be infected in vitro
219 with T. parva. Based on the phenotypes of the cells following infection, it is possible to distinguish parasitised B cells from T cells and null cells, whereas infected null cells cannot readily be distinguished from T cells.
3.3 Phenotypes of infected cells in vivo With this information at hand, it was then feasible to examine the phenotypes of parasitised cells in cattle infected with T. parva, in order to determine the cell types represented and their possible involvement in the pathogenesis of the disease. A group of cattle was inoculated with a lethal dose of T. parva (Muguga), and PBM, lymph node cells and efferent lymph lymphocytes, from the lymph node draining the site of inoculation, were examined during the course of infection (Emery et al., 1988). Phenotyping of parasitised cells was carried out in two ways; first, by staining of smears by two-colour fluorescence with reagents specific for cell surface markers and for Theileria schizonts, and second, by separating populations of cells of specific phenotypes from efferent lymph using a cell sorter and examining each population for parasitised cells. The results of these experiments have shown that more than 99% of infected cells in infected cattle during the period of patent parasitosis, express T lymphocyte markers. Only in the advanced stages of the disease were a few sIg + schizont-infected cells detected. Both BoT4 + and BoT8 + cells were represented in the BoT2 ÷-infected cell populations. A striking feature was the appearance during the second week of infection of a population of BoT4 + BoT8 + lymphocytes, usually representing at least 15% of efferent lymph lymphocytes. A large proportion of these cells was parasitised. On the basis of both the kinetics of appearance of double positive cells in vivo and the observation that parasitised BoT4 + cells in vitro commonly acquire expression of BoT8, these cells were considered to have arisen from BoT4 + T lymphocytes (Emery et al., 1988). The majority of parasitised cells in vivo, therefore, fall into three phenotypic categories, namely BoT4 + BoT8-, BoT4 + BoT8 + and BoT4- BoT8 +. It is possible that some of the BoT4- BoT8 + cells are derived from null lymphocytes. Nevertheless, the findings clearly indicate that most of the parasitised cells in cattle infected with T. parva are T lymphocytes and, if the conclusion that the infected BoT4 + BoT8 + cells are derived from BoT4 + BoT8lymphocytes is correct, then a majority of the infected cells must arise from the BoT4 + subpopulation of T lymphocytes.
3.4 Pathogenicity of different infected cell types In an attempt to investigate the pathogenicity and immunogenicity of different infected cell types, we have examined the responses of a group of cattle following inoculation with cloned autologous parasitised cells of defined phenotypes (W.I. Morrison, unpubl, data, 1987). Each animal was inoculated sub-
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cutaneously with 105 cells from a cloned cell line. Previous studies with uncloned cultured cells had shown that this dose of autologous cells produces severe infections, sometimes resulting in death (Buscher et al., 1984). The cell lines comprised four BoT4 + T cell lines, two BoT8 + T cell lines, four B cell lines and one null cell line. In general, the infections produced by these cloned lines were much milder than those previously observed with early passage uncloned lines (Buscher et al., 1984). Indeed, three of the four B cell lines and one BoT8 + T cell line did not give rise to detectable infection. In five animals, parasitosis was detected at levels below 1% in the regional lymph node only for 1-4 days. Only in the remaining two animals, both of which received BoT4 ÷ T cells, were the infections moderately severe, with the level of parasitosis in the regional lymph node exceeding 10%. Several weeks after remission of infection, all of the animals were challenged with a lethal dose of infective tick stabilate of the same stock of T. parva as that used to establish the cell lines. Two of the cattle which had received parasitised B cell lines were found to be susceptible to challenge, whereas the remaining nine animals were solidly immune. In this experiment, neither the severity of infection nor the development of immunity showed an absolute correlation with the phenotype of the parasitised cells which the animals received. Nevertheless, B cells tended to produce mild or inapparent infections and in some instances did not induce immunity, whereas the two cell lines which produced moderately severe infections were both BoT4 + T cells. These observations are consistent with the findings that a large proportion of the parasitised cells in infected cattle belong to the BoT4 + subpopulation of T cells whereas infected B cells are difficult to detect (Emery et al., 1988). The generally mild nature of the infections produced by most of the cloned cell lines may be due to loss of adaptation to growth in vivo as a result of cloning and passage in vitro. Alternatively, the greater virulence of uncloned cell lines may be due to complementary effects of different cell types on each other's growth in vivo. Interpretation of the results obtained with these cloned cell lines is also complicated by the fact that the starting parasite stock may contain a mixture of genetically different T. parva parasites which may differ in virulence and in cross-protective properties. Thus, the finding that two cattle inoculated with parasitised B cells did not develop patent infections and were susceptible to subsequent challenge with the parent stock, may be due either to failure of the parasitised cells to establish infection in the animals or to the parasites within these lines being antigenically different from those present in the other cloned lines. The application of recently developed DNA probes (Conrad et al., 1987a) which detect differences in parasite DNA between and within parasite stocks will be a useful means of investigating whether or not heterogeneity in the parasites was an important contributory factor to the results of this study.
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3.5 Possible influences of functional properties of infected cells on pathogenicity or immunogenicity The capacity of different infected cell types to produce lymphokines or to stimulate other cells in the animal to produce lymphokines may be important in influencing the growth of the parasitised cells in vivo. Brown and Logan (1986) have demonstrated that T cell growth factor (TCGF), in the form of conditioned medium from PBM stimulated with concanavalin A, can potentiate the growth of Theileria-infected cell lines. This effect was particularly apparent when the cells were seeded at low cell concentrations and indeed, TCGF can be used to clone Theileria-infected cell lines distributed at one cell per well in round-bottomed microtitre plates. Production of TCGF activity by parasitised cells has also been detected and this activity was localised to a 2025 kD fraction of culture supernatant (Brown and Logan, 1986), indicating that the active molecule may be interleukin 2 (IL-2) or interleukin 4. More recently, Dobbelaere et al. (1988) have shown that human recombinant IL-2 supports the growth of parasitised cells at low cell concentrations and that parasitised cells constitutively express IL-2 receptors. These findings suggest that constitutive production of interleukins by parasitised cells may be an important factor in maintaining growth of the cells. The cell line on which most of the studies of Brown and Logan (1986) were based is a TCGF-dependent T cell clone (Brown and Grab, 1985) which was infected in vitro with T. parva and which prior to infection did not produce detectable levels of TCGF activity; subsequently this clone was shown to have the BoT4- BoT8 + phenotype. Thus, infection with Theileria is capable of inducing production of lymphokines by cells which under normal circumstances are non-producers. Whether lymphokine production that is normally associated with T cells can also be induced upon infection of other cell types such as B cells or null cells has yet to be determined. The relative capacities of different cell types to produce growthstimulating lymphokines when infected with T. parva and their sensitivity to the lymphokines may preferentially favour the growth of particular cell types in vivo. A striking feature of infections with T. parva in cattle is the marked proliferative response of uninfected lymphocytes which occurs at the time of initial detection of parasites in the lymph nodes (Morrison et al., 1981). This response, which involves mainly T lymphocytes (Emery et al., 1988), is presumably stimulated by parasitised cells. Parasitised cells grown in vitro also stimulate potent proliferative responses in autologous PBM (Pearson et al., 1979; Goddeeris and Morrison, 1987). In immune cattle, a component of this autologous Theileria mixed leukocyte reaction (MLR) is clearly parasite antigenspecific (Goddeeris et al., 1986a; Goddeeris and Morrison, 1987). However, the magnitude and kinetics of the response of PBM from naive and immune cattle are similar, suggesting that there may be a non-specific or mitogenic
222 component to the response (Goddeeris and Morrison, 1987). It is tempting to speculate that induction of the equivalent response in vivo leads to production of lymphokines which in turn potentiate the growth of parasitised cells and thus contribute to the pathogenesis of the disease. Moreover, recent findings indicate that parasitised T cells stimulate an autologous Theileria MLR at lower cell concentrations than parasitised B cells (Goddeeris and Morrison, 1987), suggesting that they may be more efficient at inducing proliferative responses in vivo. The expression of other functional properties by parasitised cells may influ ence their pathogenicity in vivo. For instance, variation in the migratory capacities of different infected cell types, reflecting expression of cell surface homing receptors, might be important because cells with a limited capacity to recirculate or to exit from the regional lymph node would be expected to remain localised and thus have less widespread pathogenic effects. The expression of effector functions such as cytotoxicity by parasitised lymphocytes might also contribute to the pathogenesis of the disease. Recent experiments have shown that when alloreactive cytotoxic T cell clones are infected in vitro with T.parva they retain antigen-specific cytotoxic activity for 3-4 months after infection (Baldwin and Teale, 1987). In relation to this finding, it would be of interest to determine whether or not memory cytotoxic T cells when infected with the parasite will acquire cytotoxic activity. Such a phenomenon might account for the observation that PBM taken from cattle in the later stages of ECF exhibit polyspecific cytotoxic activity (Emery et al., 1981a), although it was not ascertained whether this activity resided in the parasitised or non-parasitised cell populations. The acquisition of cytotoxic activity by parasitised cells might be of relevance to the pathogenesis of the disease if cells such as NK cells, which recognise autologous cell surface determinants, were infected. There is evidence that parasitised cells may produce factors with immunosuppressive activity. The supernatants of parasitised T cell lines grown at relatively high cell concentrations were found to have marked inhibitory activity on the proliferation in allogeneic and autologous MLRs. Production of this suppressive activity was cell type-specific, in that similar supernatants from parasitised B cell lines had no suppressive activity (Goddeeris and Morrison, 1987). The production of such suppressive factors in vivo may have an inhibitory effect on the generation of parasite-specific T cell responses, although the findings in vitro suggest that this may only be of significance once high levels of parasitosis have been attained. The role of different parasitised cell types in induction of protective immunity and in establishment of the carrier state has not been studied. As will be discussed, T cell responses of immune cattle against Theileria-infected lymphoblasts involves both class I and class II MHC-restricted T cells. Studies of activated murine lymphocytes indicate that recycling of class I MHC molecules on the surface of activated lymphocytes differs between T and B cells; T
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lymphocytes spontaneously internalise and recycle class I molecules from the cell surface through endosomes, whereas B lymphocytes do not (Tse et al., 1986; Machy et al., 1987). The functional significance of this phenomenon is unknown. However, it would be of interest to determine whether or not parasitised bovine T and B lymphocytes showed similar differences and whether this or other differences between the cells influence their capacity to induce MHC-restricted T cell responses against the parasite. Such differences might also favour the survival of particular infected cell types in immune animals and thus permit establishment of the carrier state. 4.0 I M M U N I T Y TO T. PARVA
4.1 General features of immunity Cattle which recover spontaneously from infection with T. parva are immune to challenge with the same stock of the parasite. Such immunity has been shown to last for up to 3.5 years in the absence of further challenge (Burridge et al., 1972 ). Cattle can also be immunised against the parasite by infection and treatment. This involves inoculating animals with infective sporozoites and treating them either with tetracycline in the period immediately after infection or with one of the two available theilericidal drugs, Clexon (Wellcome, U.K.) or Teret (Hoechst, W. Germany), during the early stages of the clinical reaction (8-10 days after infection). The prophylactic effect of tetracyclines was initially observed over 30 years ago by Neitz (1953) who showed that treatment of cattle for several days after a lethal tick challenge with T. parva caused attenuation of infection resulting in a mild or inapparent clinical reaction with subsequent immunity to challenge. If treatment was delayed until after the onset of patent infection, the drug had no effect. Subsequently this method of immunisation was refined to involve a single inoculation with a slow release formulation of oxytetracycline at the time of infection (Radley et al., 1975c,d). The major effect of this treatment was shown to be exerted during the first 4-5 days of infection (Radley, 1978 ). Immunisation by treatment with the theilericidal drug, Clexon, is equally effective (Dolan et al., 1984 ) but has the disadvantages that animals must be mustered for inoculation on two occasions and the drug is more expensive than oxytetracycline. On the other hand, some isolates of T. parva are less readily controlled by oxytetracycline than others so that animals require two or more doses of the drug (Radley, 1978). Practical application of these methods of immunisation has two main limitations. First, they involve the use of live parasites, in the form of ground up ticks, which must be stored in liquid nitrogen until used. Second, the immunity engendered by one stock of the parasite does not provide protection against all other stocks (Cunningham et al., 1974; Radley et al., 1975a). However, the
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parasite strain heterogeneity is probably limited, as the use of mixtures of two or three stocks for immunisation by infection and treatment has provided protection against experimental challenge with a number of different stocks isolated in different geographical locations and against field challenge (Radley et al., 1975b; Morzaria et al., 1987). Reluctance to apply such "cocktail"vaccines on a wide scale in the field relates to the fact that vaccinated animals often become low level carriers of parasites (Dolan, 1986) and thus there is concern that vaccination might result in the introduction of the vaccine strains of the parasite into resident tick populations. Nevertheless, in several countries, field immunisation trials have been carried out with considerable success (Irvin, 1985), using parasite stocks isolated from the regions in which they are to be applied. On the basis of these trials, it is likely that vaccination programmes using these local isolates will be implemented in several locations. It should also be pointed out that the establishment of a carrier state not only occurs in animals immunised by infection and treatment but also in animals which recover spontaneously from natural infections (Young et al., 1986). In 1970, when schizont-infected lymphoblasts were first successfully cultivated, it was hoped that the cultured cells could be used for vaccination. However, protection in the majority of cattle could only be achieved by administering l0 s or more parasitised cells (Brown, 1981). As this represented at least 100 ml of cultured cell suspension, it was clearly not an economically viable proposition. This finding was disappointing considering the results achieved with the related Theileria parasite, T. annulata; 105 or fewer schizont-infected cells from cultures were sufficient to induce immunity against this parasite (Pipano, 1981 ). The difference between the two parasites appears to relate to the capacity with which schizonts can transfer from one cell to another. Thus with T. annulata, free schizonts will readily infect lymphocytes whereas this is apparently a very rare event with T. parva. There is good evidence to suggest that in cattle inoculated with T. parva schizont-infected cells, development of immunity is dependent on transfer of parasites into cells of the recipient animals (Brown, 1981; Emery et al., 1981b). Therefore, it is not the inoculated cells themselves that induce immunity but the transient infection established in the recipient animal's own cells. Indeed, the immunisation procedures which have been successful in inducing protective immunity have all involved establishment of infection in recipient cattle. Obviously in the longer term, major advantages would be gained from development of a vaccine which did not rely on the use of viable parasites. It must be assumed that the consistent failure of attempts to immunise cattle with dead parasites (Cunningham et al., 1973; Wagner et al., 1974; Emery et al., 1981b) were due to the use of insufficient quantities of the appropriate antigens and/or to the antigens being presented in a manner which was inappropriate for induction of protective immune responses. The formulation of rational strategies for developing alternative methods of immunisation is,
225 therefore, dependent on acquiring knowledge of the immune responses that are important in mediating immunity. 4.2 I m m u n e effector mechanisms
Because of the apparent lack of correlation of the presence of anti-schizont and anti-piroplasm antibodies with immunity to T. parva, it has been considered for some time that immunity is probably mediated by cellular rather than humoral immune responses. This belief was strengthened by the findings that immunity could not be transferred passively with serum from immune cattle (Muhammed et al., 1975) whereas adoptive transfer of thoracic duct lymphocytes from immune to naive chimaeric twin calves afforded protection against concurrent challenge with the parasite (Emery, 1981b). The immune animals used in the serum transfer experiments had been immunised by infection and treatment, and their sera were shown to contain high titres of antibody to piroplasms. The observation that immune cattle challenged with sporozoites frequently exhibit transient low levels of schizont parasitosis before infection is eliminated indicates that protective immune responses are directed against the schizont-infected cell. This is supported by the findings that cattle immunised by infection and treatment are resistant to challenge with large numbers of autologous schizont-infected cells (Emery et al., 1981b), and that cattle are immune to challenge with sporozoites following recovery from infections established by inoculation with schizont-infected cells, i.e., in the absence of exposure to sporozoites. While these conclusions are valid for cattle immunised by infection and treatment regimes, there is evidence that exposure of cattle to sufficient quantities of sporozoites results in production of anti-sporozoite antibodies which may play a role in protection. Thus, the serum of immune cattle repeatedly challenged with large numbers of infected ticks contains anti-sporozoite antibodies which in vitro neutralise the infectivity of sporozoites (Musoke et al., 1982). Moreover, monoclonal antibodies specific for 71. parva sporozoites have been produced and a number of these also have neutralising activity (Dobbelaere et al., 1984; Musoke et al., 1984). Efforts are currently underway to characterise the sporozoite antigens against which these antibodies are directed and to isolate the parasite genes which encode them, with the aim of testing the immunogenicity of the gene products. Herein, however, further discussion of immunity will focus on the immune responses directed against schizont-infected cells. 4.3 Class I MHC-restricted T cell responses to parasitised cells
The development of techniques to infect lymphocytes in vitro with sporozoites was an important advance which made it possible to study cell-mediated
226 immune responses of individual cattle to their own parasitised cells. Using such a system, Eugui and Emery (1981) showed that, in cattle undergoing immunisation or challenge with T. parva, cytotoxic cells specific for parasitised target cells are transiently detectable in P B M at the time of remission of infection. These cytotoxic cells killed autologous parasitised cells but did not kill cells from unrelated cattle, infected with the same parasite. This characteristic of genetic restriction suggested that the effector cells were probably T lymphocytes which recognise cell surface antigens in the context of major histocompatibility complex (MHC) molecules. The temporal relationship of this response with disappearance of parasitised cells indicated that it was important in control of the infection. Moreover, such a mechanism of immunity is compatible with the finding that establishment of infection in the animal is required for immunity, because of the need for T cells to recognise antigenic changes on the cell surface in conjunction with self MHC molecules. Recently with the availability of MHC typing antisera and MAb specific for class I and class II MHC molecules, it has been possible to investigate the role of MHC molecules in restricting these responses. Serological definition of polymorphic bovine MHC antigens, termed bovine lymphocyte antigens (BoLA), is confined to class I molecules. On the basis of three international comparison tests with antisera produced in a number of laboratories throughout the world, seventeen class I specificities have been defined (Anon, 1982). Population studies indicate that these specificities are all encoded by one locus, termed BoLA-A. While there is some evidence for the existence of one (or more ) additional class I locus, this has yet to be confirmed. Using antisera to the internationally-defined specificities and to several locally defined specificities in East African cattle, we have examined the restriction of the Theileria-specific cytotoxic cell response in relation to BoLA-A phenotype. A group of ten immune cattle of defined BoLA-A phenotype were challenged with T. parva sporozoites and their PBM assayed for cytotoxic activity on parasitised target cells derived from the autologous animals, from animals which shared one or other of the BoLA-A specificities with the effector cells and from animals of different BoLA-A phenotypes (Morrison et al., 1987b). In nine of the ten animals the cytotoxic cell response was genetically restricted and there was killing only of target cells which shared BoLA-A specificities with the effectors. That the majority of the effectors was indeed restricted by class I MHC products was confirmed by the finding that a MAb specific for a monomorphic determinant on class I MHC molecules significantly inhibited the cytotoxicity whereas two MAb specific for class II MHC products had no effect. Differences in the MHC haplotypes encoding the A locus-controlled wl0 and KN18 antigens, within the sets of target cells used in these experiments, have been demonstrated using alloreactive T cell clones specific for class II MHC products (Teale and Kemp, 1987; A.J. Teale, unpubl. data, 1986). This observation suggests that these target cells may also differ
227 with respect to putative non-A-locus class I molecules. The findings indicate, therefore, that the Theileria-specific cytotoxic cells are restricted predominantly by A-locus products or possibly by the products of class I genes showing linkage disequilibrium with BoLA-A. Perhaps of greater interest, however, was the finding that in eight of the nine animals there was a marked bias in the cytotoxic cell response to one or other of the BoLA-A products (all animals were heterozygous). Moreover, within the six BoLA-A specificities represented, responses restricted by w6, w8 or KN18 consistently predominated over responses restricted by w7, wl0 or w l l . The one animal which showed no bias in its response had the w6/w8 phenotype. The finding in one animal, with the w7/wl0 phenotype, of a strong cytotoxic cell response restricted by the wl0 haplotype suggested that the bias in restriction of the response is relative rather than absolute. Thus, there appears to be a hierarchy in dominance of the response within the class I MHC specificities that restrict the cytotoxic T cells. Such a hierarchy may be due either to differences within the T cell repertoires in the frequency or avidity o f T cells with specificity for Theileria antigen plus MHC or to differences in the avidity with which parasite-induced antigens can associate with different class I MHC molecules on the cell surface. The initial demonstration of genetically restricted T cell responses to Theileria-infected cells prompted a search for parasite-specific antigens on the surface of infected cells. Using several assay systems, including immunofluorescence, complement-dependent lysis and antibody-dependent cellmediated lysis, it was not possible to detect antibody reactive with the surface of parasitised cells in the serum of hyperimmunised cattle (Creemers, 1982; reviewed in Morrison et al., 1986a). Moreover, attempts to identify parasitespecific antigens on the cell surface with MAb have proved unsuccessful. In one instance, an apparently parasite-specific antigen was detected with a MAb (Newson et al., 1986); however, when the parasitised cells were cloned and used to infect an allogeneic animal, infected cells isolated from the recipient animal did not express the antigen. Similar difficulties have been encountered in direct identification of the antigens recognised by T cells on the surface of virus-infected cells. Indeed, cultured populations of antigen-specific T cells rather than antibodies have been used as reagents to identify the relevant viral antigens (Townsend et al., 1984).
4.4 Generation of Theileria-specific T cells in vitro In order to investigate the antigenic specificity of the T cell responses against Theileri~-infected cells and to exploit T cells for identification of relevant antigens, it was necessary to establish methods for propagation and cloning of Theileria-specific T cells. Pearson et al. (1979) were the first to show that genetically restricted cytotoxic cells could be generated in the autologous
228
Theileria MLR. However, the cytotoxic cells induced in their cultures killed uninfected lymphoblasts as well as autologous infected cells. Later, Emery and Kar (1983) found that non-MHC-restricted cytotoxic cells capable of killing infected and uninfected lymphoblasts were generated in autologous Theileria MLRs from naive as well as immune cattle. We now believe that the nonspecific nature of the cytotoxicity generated in these studies relates, in one instance, to the method used to immunise the cattle and in the other, to the cell population used to establish the cultures. The animal used in the experiments of Pearson et al. ( 1979 ) received large numbers of autologous parasitised cells from a cell line grown in vitro, a procedure which we have found to induce T cell responses to culture-associated antigens (Morrison et al., 1986a). The defibrination procedure used by Emery and Kar ( 1983 ) to obtain PBM results in considerable depletion of monocytes, which tends to give non-specific proliferative responses (Goddeeris et al., 1987 ). Utilising PBM harvested by standard density gradient centrifugation from blood collected in anticoagulant, we have been able to generate MHC-restricted cytotoxic cells specific for parasitised lymphoblasts, in autologous Theileria MLRs from immune cattle but not from naive cattle (Goddeeris et al., 1986a). Furthermore, restimulation of lymphoblasts from these cultures, at weekly intervals, with the appropriate parasitised cells in the presence of irradiated autologous PBM as filler cells, results in marked enrichment for specific cytotoxic cells. The culture are composed almost entirely of T lymphocytes including both BoT4 + and BoT8 + subpopulations. Experiments in which the cells have been separated into the two populations using the cell sorter, have demonstrated that virtually all of the cytotoxic cells are within the BoT8 + subpopulation of T cells (Goddeeris et al., 1986a). This is consistent with the observation that among bovine alloreactive T cell clones (Teale et al., 1986), class I-specific clones are BoT8 +, and with findings with the equivalent T cell markers in other mammalian species (Meuer et al., 1982). Cloned populations of T cells specific for parasitised lymphoblasts have been derived from cultures of Theileria-specific T cells after two to four stimulations in vitro. In some experiments where the intention was to obtain BoT8 + T cell clones, cultures were depleted of BoT4 + cells, immediately before cloning, by complement-mediated lysis using a MAb specific for BoT4. Clones were obtained following seeding of cells at 1.0 or 0.3 cells per well in the presence of parasitised stimulator cells and irradiated PBM as filler cells, in medium supplemented with TCGF (Goddeeris et al., 1986b). Two types of T cell clone were identified, namely BoT8 + cytotoxic cells (Goddeeris et al., 1986b; Morrison et al., 1987a) and BoT4 + non-cytotoxic cells (Baldwin et al., 1987). The BoT8 + clones were dependent on bo~h specific antigen, in the form of irradiated parasitised cells, and TCGF for maintenance of growth, whereas some of the BoT4 + clones proliferated in the presence of stimulator cells alone, although TCGF considerably enhanced their
229 proliferation. In agreement with observations on the response in vivo, experiments with MAb specific for class I or class II MHC molecules showed that the BoT8 + cytotoxic T cell clones were restricted by class I MHC determinants. By contrast the BoT4 + clones were found to be restricted by class II MHC determinants. This finding, together with the observation that some of the BoT4 + clones produced TCGF activity in response to stimulation with concanavalin A, suggested that they were helper T cells. Demonstration of Theileria-specific helper T cell responses had previously been difficult because of the similar levels of proliferation obtained in autologous Theileria MLRs from naive and immune cattle. 4.5 Parasite strain-specificity of the T cell responses If T cell responses are indeed important in mediating immunity to T. parva, differences in the capacity of parasite stocks to cross-protect would be expected to be reflected in parasite strain specificity of the T cell responses. To investigate this possibility we have examined the parasite strain specificity of cytotoxic T cells generated in vivo and T cell clones grown in vitro. This work has focussed on two stocks of the parasite, namely T. parva (Muguga) and T. parva (Marikebuni ). Cattle immunised with T. parva (Marikebuni ) are immune to challenge with either of the two stocks, whereas a proportion of animals immunised with T. parva (Muguga) are susceptible to challenge with the Marikebuni stock (Irvin et al., 1983 ). The strain specificity of the cytotoxic T cell response has been examined in detail in five cattle immunised with T. parva (Muguga) and in one animal immunised with T. parva (Marikebuni) (Morrison et al., 1987b; B. M. Goddeeris, unpubl, data, 1988). In order to preclude the argument that differences in cytotoxicity might be due to the two parasite stocks infecting different cell types, cloned T cells infected with the two parasites were prepared for use as target cells for each animal. In some instances, these cell lines were also cloned after infection, to ensure that they did not contain mixed parasite populations. Cytotoxic T cells from the animal immunised with T. parva (Marikebuni) killed target cells infected with either of the two parasite stocks. On the other hand, cytotoxic cells from two of the animals immunised with T. parva (Muguga) were completely specific for Muguga-infected target cells, whereas effector cells from the remaining three animals also showed killing of Marikebuni-infected targets. These findings suggest that the heterogeneity in crossimmunity induced in cattle with T. parva (Muguga) may be due to differences in the specificity of the T cell responses, although further experiments, in which both the cytotoxic T cell responses and the immune status are evaluated in a larger number of cattle, are required to confirm this. The interpretation of these results is also potentially complicated by the fact that the parasite populations are uncloned. Thus, for example, the capacity of
230 T. parva ( M a r i k e b u n i ) to provide protection against T. parva ( M u g u g a ) might be due to the presence within the Marikebuni stock of two or more strains, one of which is antigenically similar to Muguga. In such circumstances, the cytotoxic T cells generated in animals immunised with T. parva (Marikebuni) might be composed of either a population of predominantly cross-reactive T cells or two populations with specificity for each of the component parasites. To distinguish between these possibilities, it was necessary to examine the specificity of cloned populations of cytotoxic T cells. Six BoT8 + cytotoxic T cell clones derived from an animal immunised with 7". parva (Marikebuni) were all found to kill target cells infected with Marikebuni or Muguga, to similar levels (Morrison et al., 1987a). Cytotoxic T cell clones obtained from two cattle which showed strain specificity of the cytotoxic T cell response to T. parva (Muguga) in vivo, were found to be specific for Muguga-infected target cells (Goddeeris et al., 1986b; Morrison et al., 1986b). By contrast, preliminary findings with cytotoxic T cell clones from an animal immunised with T. parva (Muguga), which showed a cross-reactive T cell response in vivo, indicate that some of the clones are Muguga-specific whereas others are cross-reactive (Goddeeris, unpubl, data, 1988). Recent results also indicate that the Muguga-specific T cell clones give low levels of killing on some Marikebuni-infected cell lines (Goddeeris, unpubl, data, 1988). That there is indeed parasite heterogeneity within the Marikebuni stock has recently been demonstrated using the T. parva DNA probes described by Conrad et al. (1987a) and using schizont-specific MAb in Western blots (P.G. Toye and B.M. Goddeeris, unpubl, data, 1988 ). Nevertheless, the findings with T cell clones, albeit from a small number of animals, suggest that the T cells recognise two types of antigenic epitope, one of which is common to the Muguga and Marikebuni stocks and another which is absent from at least a component of the Marikebuni stock. The paradox remains as to why in some animals the immunity induced by T. parva (Muguga) does not protect against the Marikebuni stock, despite the two parasites possessing common antigenic epitopes. This may be explained by a relative dominance of strain-specific epitopes in induction of T cell responses to T. parva (Muguga), so that only in some animals is the response to common epitopes sufficient to provide cross-protection. If this is so, it is likely that the factor which determines whether the T cell response is strain-specific or cross-reactive is the MHC phenotype of the animal. Studies in mice of helper T cell responses to soluble protein antigens (Zanetti et al., 1987) and cytotoxic T cell responses to virus-infected cells (Vitiello and Sherman, 1983; Townsend and McMichael, 1985 ) have shown that mice of different MHC phenotypes respond to different epitopes on the immunogenic molecules. Moreover, in the case of cytotoxic T cell responses to influenza A virus, this phenomenon of determinant selection by the MHC resulted in differences between the mouse strains in the virus strain specificity of the T cell response (Vitiello and Sherman, 1983). Studies are underway in cattle to determine
231 whether the parasite strain specificity of the cytotoxic T cell response and of immunity induced by T. parva (Muguga) correlates with BoLA-A phenotype. Helper T cell clones specific for parasitised cells have been generated from two cattle immunised with T. parva (Muguga), which showed parasitic strain specificity of the cytotoxic T cell response in vivo. These helper clones were also found to be parasite strain-specific, in that Marikebuni-infected cells did not stimulate them to proliferate (Baldwin et al., 1987). In T cell responses to intracellular microorganisms, the helper T cell and cytotoxic T cell responses are not necessarily directed against epitopes on the same molecule. Thus, for example, in some strains of mice infected with influenza A virus, virus-specific helper T cells are mainly specific for epitopes on the haemagglutinin protein whereas the cytotoxic T cells are mainly specific for epitopes on the nucleoprotein (Townsend and McMichael, 1985). It therefore follows that, if both helper and cytotoxic T cell responses are required for protection of cattle against T. parva, consideration of the strain specificity of both responses will be necessary in any attempts to correlate the strain-specificity of T cell responses with cross-protection between strains. 5.0 IDENTIFICATIONOF PARASITE ANTIGENS RECOGNISEDBY T CELLS In the last few years, experimental studies of virus-specific T cell responses have provided valuable insight into how viral antigens are presented to T cells on the surface of infected cells. The results of these studies have important implications when considering potential strategies for identifying the parasite antigens recognised by T cells. The work on influenza A virus has been particularly informative. Although the haemagglutinin glycoprotein is readily de: tected on the surface of influenza virus-infected cells, the majority of the cytotoxic T cells elicited by the virus in man and in most strains of mice are specific for intracellular viral proteins, the most important of which appears to be the nucleoprotein (Townsend and Skehel, 1984; Townsend et al., 1984; Gotch et al., 1987). While haemagglutinin-specific cytotoxic T cells are generated in some strains of mice, experiments in which cells have been transfected with either the intact haemagglutinin gene or a truncated form of the gene from which the sequence encoding the membrane anchor region has been deleted, have demonstrated that recognition of target cells by the effectors does not require expression of the intact haemagglutinin molecule on the cell surface (Townsend et al., 1986a). T a m m i n e n et al. ( 1987 ) have recently produced MAb which detect small quantities of nucleoprotein on the surface of influenza-infected cells. However, these antibodies reacted with the surface of virus-infected cells from mice of different MHC phenotypes, and they had no inhibitory effect on specific cytotoxic T cells, suggesting that they recognised a form of the nucleoprotein which was not associated with MHC molecules on the cell surface. By transfecting overlapping fragments of the nucleoprotein
232 gene into mouse L cells, which can then be used as target cells, it has been possible to map some of the T cell epitopes on the molecule (Townsend et al., 1986b). Moreover, synthetic peptides of 13-16 amino acids, containing the epitopes, could be presented efficiently by uninfected cells to nucleoproteinspecific class I-restricted cytotoxic T cells (Townsend et al., 1986b; Bastin et al., 1987 ). This series of elegant experiments has provided strong evidence that class I-restricted T cells recognise short peptide sequences on the surface of infected cells, suggesting that, as with antigens presented by class II MHC, processing of the antigens is a necessary step for presentation by class I. This is consistent with recent information on the three-dimensional structure of class I MHC molecules, which indicates that the cleft in the molecule between the first and second domains, into which antigens are believed to bind, will only accommodate a peptide of less than 20 amino acids (Bjorkman et al., 1987 ). Such peptides will be difficult to detect with antibodies. Despite the apparent similarity in the requirements for processing and presentation of antigens recognised in the context of class I and class II MHC molecules, there appear to be differences in the intracellular pathways by which the antigens are processed (discussed by Bevan, 1987). Thus, presentation of viral antigens to class II-restricted T cells can occur following endocytosis of dead virus or soluble viral proteins by presenting cells and is dependent on lysosomal activity of the cell (L.A. Morrison et al., 1986). By contrast, presentation in the context of class I is independent of lysosomal activity and usually requires active synthesis of viral proteins within the presenting cells, which cannot be achieved with dead virus or purified viral proteins (L.A. Morrison et al., 1986). These findings have important implications in studies aimed at identifying antigens recognised by parasite-specific T cells, since antigen-presenting cells might be expected to present crude parasite antigens to class IIrestricted T cells but not to class I-restricted T cells. Accordingly, experiments have been initiated to attempt to identify the antigens recognised by BoT4 + Theileria-specific T cells. Two groups of helper T cell clones have been derived which recognize distinct antigenic fractions prepared from homogenized Theileria parva-infected cells (C. Brown, D.J. Grab and K.S. Logan, manuscripts in preparation). The first group proliferates in response to glutaraldehyde-fixed parasitised cells, purified schizont, and cell fractions enriched for schizont membranes, only in the presence of autologous antigen-presenting cells. The second group of clones proliferates in response to fixed infected cells in the absence of antigen-presenting cells, and to soluble protein antigen (s) prepared from the cell homogenate if autologous antigen-presenting cells are added. Thus, it is likely that the soluble antigen (s) is recognized by the BoT4 + T cell clones on the surface of the infected cell in association with class II molecules, whereas the schizont membrane antigen (s) which requires processing and presentation for the appropriate T cell clones to be stimulated, may not be associated with class II
233 molecules o n t h e surface o f i n f e c t e d cells. C h a r a c t e r i z a t i o n a n d p u r i f i c a t i o n of t h e s e a n t i g e n s is u n d e r w a y . Once t h e r e l e v a n t p a r a s i t e a n t i g e n s h a v e b e e n identified, specific a n t i b o d i e s can be p r o d u c e d for use in s c r e e n i n g p a r a s i t e D N A libraries. P a r a s i t e genes t h u s identified will n e e d to be e x p r e s s e d in m a m m a l i a n cells b e a r i n g t h e app r o p r i a t e b o v i n e M H C molecules, in o r d e r to c o n f i r m t h a t t h e gene p r o d u c t s are r e c o g n i s e d b y parasite-specific T cells. T h i s can be a c h i e v e d b y t r a n s f e c t i n g t h e gene into t h e g e n o m e or b y i n s e r t i n g t h e m into r e c o m b i n a n t virus vectors which can be used to i n f e c t b o v i n e cells. In this way, it will be possible to d e t e r m i n e w h e t h e r or n o t t h e gene p r o d u c t s are also recognised b y class Ir e s t r i c t e d c y t o t o x i c T cells. U l t i m a t e l y , if t h e t a r g e t p a r a s i t e a n t i g e n s can be identified, t h e y will require to be p r e s e n t e d to t h e h o s t ' s i m m u n e s y s t e m in such a m a n n e r t h a t t h e y efficiently induce t h e a p p r o p r i a t e M H C - r e s t r i c t e d T cell responses. H e r e again, r e c o m b i n a n t virus vectors could p l a y a n i m p o r t a n t role in a c h i e v i n g o p t i m a l a n t i g e n p r e s e n t a t i o n o f p a r a s i t e gene products.
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