Antigens of sporozoites of Theileria parva
Cell-mediated immunity as a tool for identification of protective antigens (Theileria parva)
Protective antigens of' African Trypanosomes
A. J. Musoke
Rapporteur's summary
Recent studies have indicated that sporozoites of several Theileria parva isolates possess a common antigenic determinant which induces neutralizing antibodies to this stage in cattle and rabbits. Therefore, efforts have concentrated on identifying the protein(s) bearing this determinant. Antigenic analysis of sporozoites using either recovery sera from cattle or immune sera produced by feeding infected ticks on rabbits revealed several specific proteins with approximate molecular masses of 105,000, 67,000, 43,000, 25,500 and 18,500 daltons. Using a monoclonal antibody directed against the epitope responsible for inducing neutralizing antibodies, only the 67,000-dalton protein was recognized. This protein has been isolated using an immunoabsorbent column on which the IgM monoclonal antibody was coupled.
Efforts are being made to produce polyvalent monospecific antisera to the 105,000-, 67,000- and 25,500-dalton proteins for screening the DNA libraries. This is done by eluting the proteins from gels before inoculating them into rabbits and mice.
Sera were collected from several farms in Kenya in order to investigate parasite antigens recognized by the immune system of cattle in endemic areas. The sera were first screened for antisporozoite antibodies by indirect immunofluorescence and neutralization of infectivity tests. Those sera which were found to stain and neutralize infectivity of sporozoites were used in Western Blots where the 105,000- and 67,000-dalton proteins were identified.
Points arising from the discussion
Polyvalent antisera were raised in rabbits using individual antigen bands cut out from SDS-PAGE gels. Antigen was eluted from the gel slices and various immunization schedules were performed by varying the amount of antigen in the different protocols. Complete and incomplete Freund's adjuvants were used.
Cattle were immunized to produce anti-sporozoite antibodies by first using the 'infection and treatment' protocol and then challenging with batches of infected ticks until sufficiently high anti-sporozoite antibody levels were achieved. Conditions for elusion of immunoabsorbent columns ranged from pH 2.5 to 11.5, monoclonal antibodies do not always work in Western blotting techniques and IgM class monoclonal antibodies are difficult to work with in this technique. It is not known whether the higher molecular mass antigens are glycosylated.
W.I. Morrison
Rapporteur's summary
Studies carried out over the last 6 years indicate that cell-mediated immune responses against macroschizont-infected cells are important in immunity to Theileria parva. Such responses are restricted by products of the major histocompatibility (MHC) gene loci and, in some instances, are specific for the infecting parasite strain. Attempts to identify the target antigen on the surface of the infected cell, using biochemical and monoclonal antibody techniques, have so far proved unsuccessful.
Recent studies have involved propagation and cloning of the cytotoxic effector cells in vitro. The potential uses of such cytotoxic cell populations include:
(1) clarification of the role of cell-mediated cytotoxicity in protective immunity, i.e.- Do the effector cells generated in vitro kill parasitized cells from infected animals?- Can an effect be demonstrated in vivo following adoptive transfer experiments (these will require genetically related cattle)?
(2) determination of whether the effectors show parasite strain specificity; if so, they could be used as strain typing reagents
(3) identification of relevant cell-surface antigens, i.e.
- screening for monoclonal antibodies which block the cytotoxic effector function- screening of target cells transfected with relevant parasite or host DNA.
Points arising from the discussion
Despite much effort, the antigen responsible for the induction of specific cytotoxicity for schizont-infected lymphocytes has not been identified. There was general agreement that, owing to the strain specificity of the cytotoxic response, parasite rather than host antigens were involved.
MHC-restricted cytotoxic T-cell clones which killed Theileria-infected cells and some uninfected blast cell populations did not kill the bovine leukaemic cell line BL-3.
S.Z. Shapiro
Rapporteur's summary
The classical approach to vaccines, i.e. immunization with' live attenuated or dead organisms, does not work with African trypanosomes because of the antigenic variation of the parasite. The neo-classical approach to vaccines, i.e. identification of 'the surface' antigens of the pathogenic organism 'for 'production of a subunit vaccine, shows little promise for the African trypanosome because its surface is covered by variable antigens.
One approach is the use of infection sera to detect antigens with vaccine potential. N'Dama and Zebu cattle are used because they differ in 'their resistance to African trypanosomiasis. Resistant and susceptible animals develop antibodies to common (nonvariant) trypanosome antigens and resistant animals develop antibodies to three antigens (110,000-, 150,000- and 300, 000-dalton molecules) not recognized by animals unable to self-cure.
Theoretically there are several antigens which might offer protection against African trypanosomiasis:
(1) receptors for receptor-mediated endocytosis; endocytosis occurs in the flagellar pocket; endocytotic vesicles have been purified and internalized host molecules are being studied(2) lysosomal enzymes; the lysozomes of trypanosomes are being purified to study their essential enzymes
(3) secreted or released pathogenic molecules including specific enzymes such as proteases or phospholipases and factors defined by pathogenic function such as mitogens or haemolysins
(4) procyclic surface antigens; where tsetse were fed on animals immunized with whole procyclics, infection rates in the flies were reduced; surface antigens of procyclic Trypanosoma brucei are being characterized
(5) metacyclic variable surface antigens; these are more limited in number than the bloodstream repertoire so it may be possible to vaccinate against individual serodemes
(6) vector antigens as targets: tsetse feeding enzymes and tsetse tissue antigens.
Points arising from the discussion
Data are scarce from well-controlled experiments which could determine the mechanisms involved in the control of parasitaemia by trypanotolerant livestock. Both phagocytic cells and antibodies from resistant wild animals have an enhanced effect compared with those from trypanosusceptible animals. It is not clear why antibodies to mammalian clatherin do not recognize the equivalent protein in trypanosomiasis.
Immunization of mammals with antigens from uncoated trypanosomes and/or tsetse antigens would only have an impact on the control of tsetse/trypanosomiasis risk where tsetse are feeding predominantly on domestic livestock. The result of antibody-enzyme interactions can be inhibitory, enhancing or have no effect at all on enzyme function.
The possibility of gene transfer as a means of improving genetic resistance to trypanosomiasis is being considered. The first stage is to identify the mechanisms responsible for trypanotolerance.
