- Email: [email protected]
Diversity and selection in Babesia bovis and their impact on vaccine use
21
Parasitology Today, vol, 8, no, i, 1992 Trans. R. Sac. Med, Hya~, 78, 641-644 17 Koppaka, V. et al. (1989),91oL Cell. Biochem. 91, 167-172 18 Deguercy, A. et al. (1986) Biochern. Int, 12, 21-31 19 Moll, G.N, et al. (1990)Biochim. Biophys. Acto 1024, 189-192 20 Yuthavong, Y. and Limpaiboon, T, (1987) Biochim. Biophys. Acta 929, 278-287 21 Wiser, M.F. (1988) Mol. Cell. Biochem, 84, 51-58 22 Tilley, L. et al. (1990) Biochim. Biophys, Acta 1025, 135-142 23 Murray, M.C. and Perkins, M.E. (1989) Mol. Biochem. Parasitol. 39, 229-236
24 AIIred, D.R, et ol. (1986)J. Cell Biol, 81, 1-16 25 Taylor, D.W. et aL (1987) Exp. PorositoL 64, 178-187 26 SimOes, A.P. et al. (1991) Biochim, Biophys. Acta 1063, 45-50 27 Moll, G.N. et aL (1990) Biochem. Cell Biol, 68, 579-585 28 Beaumelle, B.D. and Vial, H.J. (I 986) Biochim. Biophys. Acta 877, 262-270 29 Beaumelle, B.D. et al. (1988)J, Cell. Physiol. 135, 94-100 30 Sherman, I.W. and Maguire, P.A. (1990) Blood Cells 16, 556-558 31 Maguire, P.A. et al. (1991) Parasitology 102, 179-186
32 Moll, G.N. et al. (1988) FF__BS Lett. 232, 341-346 33 Grellier, P. et al, (1991)J. Cell Biol. 112, 267-277 34 Bannister, L.H. and Mitchell, G.H. (1986) J. ProtozooL 33, 271-275 35 Haldar, K. et aL (1989)J. Cell Biol. 108, 2183-2192
Ana Paula Sim6es, Ben RoelofsenandJos A. F. Op den Kamp are at the Centre for Biomembranes and Lipid Enzymology, University of Utrecht, PO Box 80054, 3508 TB, Utrecht, The Netherlands
Diversity and Selection in Babesia bovis and Their Impact on Vaccine Use B.P. Dalrymple In the past few years the prospect of a recombinant vaccine effective against the cattle haernoparasite Babesia bovis has almost become a realit/. However, in Australia, vaccination with live parasites has been practised since before the turn of the century and it has recently been proposed that selection of parasites resistant to irnrnunity induced by the B. bovis line Ka (since 1979 the only component of the live attenuated vaccine) may have occurred. Brian Dalrymple examines the evidence for and against this proposal and discusses examples of strain diversity and variation and their effect on the long-term viability of defined attenuated and recombinant vaccines. Babesia bovis is a tick-transmitted protozoan haemoparasite of cattle that is of worldwide economic importance. Since 1964 B. bovis has been very effectively controlled in Australia by the use of live parasites attenuated by rapid syringe passage through splenectomized calves'. Killed organisms 2'3, organisms cultured, in vitro 4, supernatant-derived soluble antigens4's and pJrified native6'7 and recombinant 8 (K.R. Gale et o1., abstract*) proteins of B. bovis have been tested as vaccines by a number of groups. Vaccination by these methods, with the exception of the culture supernatant-derived soluble antigens, generally protects animals against homologous and heterologous challenge. *Recent Developments in the Control of Anaplasmosis, Babesiosis and Cowdriosis (1991) Nairobi, Kenya ~} i992, ElsevierSciencePublishersLtd, (UK)
From 1979 until late 1990 the only source of parasites for the attenuated live vaccine distributed in Australia was the Ka line, an attenuated derivative of the Kv isolate ~. However, in the late 1980s, significant problems with the vaccine were observed, with increasingly large numbers of challenged vaccinated animals exhibiting pathology severe enough to be classified as clinical cases ~. It has been suggested that the intensive and extensive use of one line of parasite may have exerted pressure on the natural population to select for parasites that could evade the immune response induced by immunization with Ka ~. This may have important implications for the long-term use of antiB. bovis vaccines of unchanged composition, tn particular, recombinant vaccines, which will inevitably rely on a small number of components, may be vulnerable to such adaptations. The occurrence and extent of strain variation (predominantly due to phenotypic variation) and strain diversity (differences between genetically distinct populations) in B. bovis are likely to have implications for the success of vaccination.
ImmunologicalAnalyses Differences between isolates of B. boris were originally demonstrated by immunological methods. Agglutination tests are quite specific for the population of parasites that induce an immune response 9 and have revealed
that relapse populations of parasites in the same infected animal are distinct ~°. Likewise, passive transfer of serum from immune animals shows that different strains are antigenically distinct as homologous protection has been observed, but heterologous protection has not' '. However, in these experiments, relapse populations were controlled by transfer of serum obtained during earlier episodes of parasitaemia, suggesting that these populations were phenotypically, but not genetically, different II. Overall, each population of parasites appears to express a distinct subset of proteins that induces the major immune response in an infected animal. Indeed, proteins with very different levels of expression in the Kv, Ka, C and L isolates of B. bovis have been identified ~2.~3. These and others can be precipitated by homologous antisera from infected animals; heterologous sera will also precipitate some proteins. Despite these differences, the attenuated live vaccine strains and nonviable extracts of parasitized erythrocytes can successfully protect cattle against subsequent challenge with a heterologous line of parasites3'4. These observations also support the proposal that the immunogenic strain-specific proteins may not play a role in broad spectrum protective immunization. Alternatively, it has been proposed that the initial infection may prime the host for a secondary response against antigens in the heterologous challenge strain that are at least partially similar to those in the immunizing strain ~J.
22
Parasitology Today, vol. 8, no. I, 1992
KatypeA?a
~ii~/tir~llent ie~iiiiiill~
~
passaged in a varietyofways
~
attenuation
D
KatypeBa
D
KatypeCa
D
KatypeDa differentproportions
of the two lines, minor components or phenotypic variants
?
Ka1981
severalcloned linesderived,no ~._ geneticdifferences at theBabRlocus. but phen_otyplcally differentP
Kv1981
KaandKr 1983,1988 and 1990 Fig. I. Diagrammatic representation of populations of B. bovis after passage through different environments. Genetically distinct subpopulations are indicated by different hatching. See Refs 13(a) and 20, 21(b).
Genetic Analysis of Parasite Populations The genetic analysis of parasites is a powerful technique for determining strain diversity. In a search for genes expressed in attenuated lines of B. boris, but not in virulent lines, a number of genes were identified by Cowman et al. ~4 and their use as probes showed that strain diversity and the selection of genetically distinct subpopulations of parasites occurred during passaging (Fig. I and Refs 14-16). In particular, the BabR locus exhibited extensive strain diversity and possibly also strain variation ~s.~6. With other probes, significant diversity has been seen among American isolates and between American and Australian isolates ~7'~8. The ribosomal RNA (rRNA) genes are flanked by a number of polymorphic restriction sites and an rRNA-specific probe discriminates between all the isolates studied ~9. Three potentially informative restriction fragment length polymorphisms (RFLPs) that are closely associated with the rRNA units have been identified, one of which discriminates between an African isolate and all the Australian isolates examined 19. While the rRNA probe was able to distinguish between all the isolates originally analysed, the occurrence of
multiple gene copies made it difficult to correlate particular bands on Southern blots with particular subpopulations. Recently a probe (designated C51A), based on a variable-length, tandemly repeated sequence, has been used for the analysis of subpopulations of B. bovis (B.P. Dalrymple eta/., unpublished). This probe and associated sequences discriminate between almost all the genetically distinct subpopulations examined that were not resolved by the rRNA probe. Many samples of parasites appear to contain more than one genetically distinct subpopulation of parasites and changes in population structure can be followed during passaging and culturing. The application of C51A and other probes to the study of relapse populations will discriminate between the selection of phenotypic variants and genetically distinct subpopulations by the immune system.
Analysis of the Australian Attenuated Vaccine Lines The Kv line and the related Ka line (the Australian vaccine strain) exhibit significantly different two-dimensional (2-D) protein profiles ~2. Four phenotypically distinct derivatives of Ka have also been described 13. In an inde-
pendent genetic analysis of Kv and Ka, the Ka line appears to contain two major genetically distinct subpopulations, one of which is not detectable in the Kv isolate (Fig. I and Ref. 14). On subsequent passage through ticks, the majority of the Ka-specific subpopulation appears to have been lost (Fig. I and Ref. 14). Similarly, Carson et al. have shown that the composition of samples of parasites derived from Kv and Ka changes upon passaging ~6. The Ka line seems to contain one major avirulent, poorly tick-transmissible line and one major virulent, efficiently ticktransmitted line 14. Therefore the four types of Ka derivative identified by Kahl et al. ~3 may have included genetically distinct populations that were too minor to be detected by the probes, were mixtures of fewer subpopulations in different ratios, were phenotypic variants or were even a combination of these. Whatever the explanation, substantial differences in the antigenic profile of samples of Ka may have been present in the different samples of the vaccine distributed in Australia. Unlike the sample of Ka DNA purified in 1981 (Refs 14, 20), genetic analysis of samples of Ka DNA from 1983 (with one probe) and in 1988 and 1990 (many probes) identified only one allele of the target genes and these samples are essentially clonal (Fig. I; Ref. 20 and B.P. Dalrymple, unpublished). This suggests that the less virulent line becomes the dominant component of the vaccine in some, if not all, of the vaccines distributed. It is also possible that changes in expressed genes may also have occurred within this genetically clonal population. Indeed, cloned lines derived from Ka all appear to be genetically very similar, if not identical, but exhibit different 2-D protein profiles, virulence and vector transmissibility phenotypes 2°'21. Interestingly, the attenuated line of parasites, Ta, developed to replace Ka ~, contains two major subpopulations of genetically distinct parasites (B.P. Dalrymple, unpublished). The major subpopulation of the attenuated line is a minor subpopulation of the original isolate and vice versa, it is possible that the combination of two (or more) genetically distinct lines with different virulence phenotypes might constitute a more effective attenuated vaccine. Thus, not only the nature of the expressed antigens, but also the environment in which they are expressed, may play a role in effective immunization by the live attenuated vaccines ~'~3 The use of both phenotypic and genetic analysis of populations and of
23
ParasitoloD" Today, vol. 8, no. I, 1992
well-characterized cloned lines of parasites is required to determine the importance of variation in the vaccine and challenge strains of B. boris,
Development of a Recombinant Vaccine The nature of the protective epitopes is not known for either the attenuated or the nonlMng vaccine. In the absence of this in'Formation two major approaches to the development of a recombinant vaccine have been undertaken. In the first, parasite surface proteins have been targeted 18'22. In the second, protective fracl:ions of native proteins have been subfl-actionated and tested in vaccination-challenge experiments to identify purified proteins containing protective epitopes 6'7. The genes for a number of these proteins have been isolated and characterized 18'22. Four of the recombinant proteins have been tested for the induction of a protective immune response 8 and, for the 12D3, I IC5 and 21B4 proteins, significant protection is observed upon subsequent challenge with a heterologous virulent line of B. bovis, In a field trial with animals vaccinated with a combination of the 12D3 and I IC5 antigens, a mean maximum parasitaemia of 232 + 3451~1 was observed, with 10% of the animals requiring treatment. In the control groups a mean maximum parasitaemia of 2154 + 3065 I~1 and a 40% treatment rate was observed (see also K.R. Gale et aJ., abstract*), The probability of selection of resistant populations of B. boris by a recombinant vaccine depends on many factors, including the normal biological role of the components, the diversity of the proteins in the population, the ability of the proteins to remain active after mutation, the chromosomal location of the genes (for example, telomeric genes may be prone to deletion) and the linkage of the genes within the genome, The genes for the B. bovis proteins analysed so far are present in all 15 genetically different subpopulations examined and appear to be conserved in structure (B.P. Dalrymple, unpublished and R,E. Casu and K.R. Gale, pers. commun,), However, little other information is available and more data must be obtained before the long-term viability of a recombinant vaccine can be determined,
*Recent Developments in the Control of Anaplasmosis, Babesiosis and Cowdriosis (1991) Nairobi, Kenya
Conclusion There is clear evidence of strain diversity in B. bovis, but less evidence of strain variation, The recent problems with the attenuated vaccine could be attributable either to a change in the composition of the vaccine (both phenotypic and genetic) or to the selection of some resistant strains in the natural population, or to a combination of both factors, An apparent reduction in the complexity of some samples of the attenuated vaccine has been demonstrated and selection of parasite subpopulations clearly occurs during the passage of populations of parasites through ticks, splenectomized animals and during culture, in vitro. However, the selection of subpopulations of parasites by immune pressure that is clearly separable from innate strain variation has not yet been shown for B. bovis. In future the analysis of the population dynamics of defined lines and mixtures of such lines of parasites following inoculation of vaccinated animals may allow this to be demonstrated. Since the nature of the antigens involved in the induction of the protective immunity by the live attenuated vaccine are not known, good attenuated live vaccine strains can only be identified by trial and error, There is little prospect that this process can be improved, but the prediction that more than one component may constitute a more effective attenuated live vaccine is testable, The use of gene probes will also allow the composition of the distributed vaccine to be monitored. Strain variation, and in particular strain diversity, has been well documented for malaria parasites 23-2s. Many of the candidate vaccine antigens isolated from Plasmodium falciparum are highly polymorphic 23'24 and the proposal that this is a mechanism for the evasion of the immune response has been discussed in detail 26. The selection of mutations within, and the loss of a target antigen under, immune pressure has been shown for a P. knowlesi antigen 27 and, for the P. falciparum S-antigen, serotype-specific immunity applies selective pressure on the population 28. The information on strain variation and diversity currently available for B. boris may not be relevant to the recombinant vaccine, especially since the role of the native equivalents of these components is not known for the attenuated and killed vaccines. The success or failure of a recombinant vaccine in the face of strain variation,
diversity resistant on the included
and the selection of potentially strains will ultimately depend identity of the components in the distributed product.
Acknowledgements I would like to thank the staff of the Long Pocket Laboratories of the CSIRO and the Tick Fever ResearchCentre of the Queensland Department of Primary Industries for useful discussions,
References I De Vos,A.J.andJorgensen,W.K. in Tick Vector Biology: Medical and Veterinary Aspects (Fivaz, B.H., Petney, T.N. and Haruck, I.G., eds), Springer-Verlag(in press) 2 Mahoney, D.F. (1967) Exp. Parasitol. 20, 125-129 3 Mahoney, D.F. and Wright, I.G. (1976) Vet. Parasitol. 2, 273-282 4 Timms, P. et al. (1983)Aust. Vet. ]. 60, 75-77 5 Montenegro-James,S., Kakoma, I. and Ristic, M. (1989) in Veterinary Protozoan and Haemoparasite Vaccines (Wright, I.G., ed.), pp 61-97, CRC Press 6 Goodger, B.V. et al. (1985) Int. 1. Parasitol. 15, 175-179 7 Commins, MA., Goodger, B.V. and Wright, I.G. (1985) Int. J. ParasitoL 15, 491-495 8 Timms, P. et al. (1988) Res. Vet. Sci. 45, 267-269 9 Curnow, J.A. (I 968) Nature 217, 267-268 I 0 Curnow,].A. (1973)Aust. Vet. J. 49, 279-283 II Mahoney,D.F. eta/. (1979) Int. J. Parasitol. 9, 297-306 12 Kahl, L.P. et al. (1982) J. fmmunol. 129, 1700-1705 13 Kahl, L.P. et at. (1983) Exp. Parositol. 56, 222-235 14 Cowman, A.F. et oL (1984) ,91ol. Biochem. ParasitoL I I, 9 I- 103 15 Cowman,A.F. et al. (I 984) Cell 37, 653-660 16 Carson, C.A. et ol. (1990) Exp. ParasitoL 70, 404-410 17 Jasmer, D.P. et al. (1990)J. ParasitoL 76, 834-84 I 18 Suarez, C.E. et oL (1991) ,91ol. Biochem. Parasitol. 46, 45-52 19 Dalrymple, B.P. (1990) ,91ol. Biochem. ParasitoL 43, I 17-124 20 Gill, A.C. et al. (I 987) Exp. Parasitol. 63, 180-188 21 Timms, P., Stewart, N.P. and De Vos, A.J. (1990) Infect. Immun, 58, 217 I-2176 22 Reduker, D.W. et al. (1989) ,91ol. Biochem. Parasitol. 35, 239-248 23 Howard, R.J. (1987) Contrib. Microbial. Immunol. 8, 176-218 24 Kemp, D.J., Cowman, A.F. and Walliker, D. (1990) Adv. Parasitol. 29, 75-149 25 Mendis, K.N., David, P.H. and Carter, R. (1991) in Immunoparasitology Today (Ash, C. and Gallagher,R.B.,eds),pp A34--A37,Elsevier Trends Journals,Cambridge 26 Schofield, L. (I 991) Parasitolo~ Today 7, 99-105 27 Klotz, F.W. et al. (1987)J. EXp. Med. 165, 359-367 28 Forsyth, K.P.et al. (1988) Philos. Trans. R. Sac. London Ser.B: 321,485-493 Brian Dalrymple is at CSIRO, Division of Tropical Animal Production, Long Pocket Laboratories, Private Bag No. 3, PO, Indooroopilly, Queensland 4068, Australia.
Parasitology Today, vol, 8, no, i, 1992 Trans. R. Sac. Med, Hya~, 78, 641-644 17 Koppaka, V. et al. (1989),91oL Cell. Biochem. 91, 167-172 18 Deguercy, A. et al. (1986) Biochern. Int, 12, 21-31 19 Moll, G.N, et al. (1990)Biochim. Biophys. Acto 1024, 189-192 20 Yuthavong, Y. and Limpaiboon, T, (1987) Biochim. Biophys. Acta 929, 278-287 21 Wiser, M.F. (1988) Mol. Cell. Biochem, 84, 51-58 22 Tilley, L. et al. (1990) Biochim. Biophys, Acta 1025, 135-142 23 Murray, M.C. and Perkins, M.E. (1989) Mol. Biochem. Parasitol. 39, 229-236
24 AIIred, D.R, et ol. (1986)J. Cell Biol, 81, 1-16 25 Taylor, D.W. et aL (1987) Exp. PorositoL 64, 178-187 26 SimOes, A.P. et al. (1991) Biochim, Biophys. Acta 1063, 45-50 27 Moll, G.N. et aL (1990) Biochem. Cell Biol, 68, 579-585 28 Beaumelle, B.D. and Vial, H.J. (I 986) Biochim. Biophys. Acta 877, 262-270 29 Beaumelle, B.D. et al. (1988)J, Cell. Physiol. 135, 94-100 30 Sherman, I.W. and Maguire, P.A. (1990) Blood Cells 16, 556-558 31 Maguire, P.A. et al. (1991) Parasitology 102, 179-186
32 Moll, G.N. et al. (1988) FF__BS Lett. 232, 341-346 33 Grellier, P. et al, (1991)J. Cell Biol. 112, 267-277 34 Bannister, L.H. and Mitchell, G.H. (1986) J. ProtozooL 33, 271-275 35 Haldar, K. et aL (1989)J. Cell Biol. 108, 2183-2192
Ana Paula Sim6es, Ben RoelofsenandJos A. F. Op den Kamp are at the Centre for Biomembranes and Lipid Enzymology, University of Utrecht, PO Box 80054, 3508 TB, Utrecht, The Netherlands
Diversity and Selection in Babesia bovis and Their Impact on Vaccine Use B.P. Dalrymple In the past few years the prospect of a recombinant vaccine effective against the cattle haernoparasite Babesia bovis has almost become a realit/. However, in Australia, vaccination with live parasites has been practised since before the turn of the century and it has recently been proposed that selection of parasites resistant to irnrnunity induced by the B. bovis line Ka (since 1979 the only component of the live attenuated vaccine) may have occurred. Brian Dalrymple examines the evidence for and against this proposal and discusses examples of strain diversity and variation and their effect on the long-term viability of defined attenuated and recombinant vaccines. Babesia bovis is a tick-transmitted protozoan haemoparasite of cattle that is of worldwide economic importance. Since 1964 B. bovis has been very effectively controlled in Australia by the use of live parasites attenuated by rapid syringe passage through splenectomized calves'. Killed organisms 2'3, organisms cultured, in vitro 4, supernatant-derived soluble antigens4's and pJrified native6'7 and recombinant 8 (K.R. Gale et o1., abstract*) proteins of B. bovis have been tested as vaccines by a number of groups. Vaccination by these methods, with the exception of the culture supernatant-derived soluble antigens, generally protects animals against homologous and heterologous challenge. *Recent Developments in the Control of Anaplasmosis, Babesiosis and Cowdriosis (1991) Nairobi, Kenya ~} i992, ElsevierSciencePublishersLtd, (UK)
From 1979 until late 1990 the only source of parasites for the attenuated live vaccine distributed in Australia was the Ka line, an attenuated derivative of the Kv isolate ~. However, in the late 1980s, significant problems with the vaccine were observed, with increasingly large numbers of challenged vaccinated animals exhibiting pathology severe enough to be classified as clinical cases ~. It has been suggested that the intensive and extensive use of one line of parasite may have exerted pressure on the natural population to select for parasites that could evade the immune response induced by immunization with Ka ~. This may have important implications for the long-term use of antiB. bovis vaccines of unchanged composition, tn particular, recombinant vaccines, which will inevitably rely on a small number of components, may be vulnerable to such adaptations. The occurrence and extent of strain variation (predominantly due to phenotypic variation) and strain diversity (differences between genetically distinct populations) in B. bovis are likely to have implications for the success of vaccination.
ImmunologicalAnalyses Differences between isolates of B. boris were originally demonstrated by immunological methods. Agglutination tests are quite specific for the population of parasites that induce an immune response 9 and have revealed
that relapse populations of parasites in the same infected animal are distinct ~°. Likewise, passive transfer of serum from immune animals shows that different strains are antigenically distinct as homologous protection has been observed, but heterologous protection has not' '. However, in these experiments, relapse populations were controlled by transfer of serum obtained during earlier episodes of parasitaemia, suggesting that these populations were phenotypically, but not genetically, different II. Overall, each population of parasites appears to express a distinct subset of proteins that induces the major immune response in an infected animal. Indeed, proteins with very different levels of expression in the Kv, Ka, C and L isolates of B. bovis have been identified ~2.~3. These and others can be precipitated by homologous antisera from infected animals; heterologous sera will also precipitate some proteins. Despite these differences, the attenuated live vaccine strains and nonviable extracts of parasitized erythrocytes can successfully protect cattle against subsequent challenge with a heterologous line of parasites3'4. These observations also support the proposal that the immunogenic strain-specific proteins may not play a role in broad spectrum protective immunization. Alternatively, it has been proposed that the initial infection may prime the host for a secondary response against antigens in the heterologous challenge strain that are at least partially similar to those in the immunizing strain ~J.
22
Parasitology Today, vol. 8, no. I, 1992
KatypeA?a
~ii~/tir~llent ie~iiiiiill~
~
passaged in a varietyofways
~
attenuation
D
KatypeBa
D
KatypeCa
D
KatypeDa differentproportions
of the two lines, minor components or phenotypic variants
?
Ka1981
severalcloned linesderived,no ~._ geneticdifferences at theBabRlocus. but phen_otyplcally differentP
Kv1981
KaandKr 1983,1988 and 1990 Fig. I. Diagrammatic representation of populations of B. bovis after passage through different environments. Genetically distinct subpopulations are indicated by different hatching. See Refs 13(a) and 20, 21(b).
Genetic Analysis of Parasite Populations The genetic analysis of parasites is a powerful technique for determining strain diversity. In a search for genes expressed in attenuated lines of B. boris, but not in virulent lines, a number of genes were identified by Cowman et al. ~4 and their use as probes showed that strain diversity and the selection of genetically distinct subpopulations of parasites occurred during passaging (Fig. I and Refs 14-16). In particular, the BabR locus exhibited extensive strain diversity and possibly also strain variation ~s.~6. With other probes, significant diversity has been seen among American isolates and between American and Australian isolates ~7'~8. The ribosomal RNA (rRNA) genes are flanked by a number of polymorphic restriction sites and an rRNA-specific probe discriminates between all the isolates studied ~9. Three potentially informative restriction fragment length polymorphisms (RFLPs) that are closely associated with the rRNA units have been identified, one of which discriminates between an African isolate and all the Australian isolates examined 19. While the rRNA probe was able to distinguish between all the isolates originally analysed, the occurrence of
multiple gene copies made it difficult to correlate particular bands on Southern blots with particular subpopulations. Recently a probe (designated C51A), based on a variable-length, tandemly repeated sequence, has been used for the analysis of subpopulations of B. bovis (B.P. Dalrymple eta/., unpublished). This probe and associated sequences discriminate between almost all the genetically distinct subpopulations examined that were not resolved by the rRNA probe. Many samples of parasites appear to contain more than one genetically distinct subpopulation of parasites and changes in population structure can be followed during passaging and culturing. The application of C51A and other probes to the study of relapse populations will discriminate between the selection of phenotypic variants and genetically distinct subpopulations by the immune system.
Analysis of the Australian Attenuated Vaccine Lines The Kv line and the related Ka line (the Australian vaccine strain) exhibit significantly different two-dimensional (2-D) protein profiles ~2. Four phenotypically distinct derivatives of Ka have also been described 13. In an inde-
pendent genetic analysis of Kv and Ka, the Ka line appears to contain two major genetically distinct subpopulations, one of which is not detectable in the Kv isolate (Fig. I and Ref. 14). On subsequent passage through ticks, the majority of the Ka-specific subpopulation appears to have been lost (Fig. I and Ref. 14). Similarly, Carson et al. have shown that the composition of samples of parasites derived from Kv and Ka changes upon passaging ~6. The Ka line seems to contain one major avirulent, poorly tick-transmissible line and one major virulent, efficiently ticktransmitted line 14. Therefore the four types of Ka derivative identified by Kahl et al. ~3 may have included genetically distinct populations that were too minor to be detected by the probes, were mixtures of fewer subpopulations in different ratios, were phenotypic variants or were even a combination of these. Whatever the explanation, substantial differences in the antigenic profile of samples of Ka may have been present in the different samples of the vaccine distributed in Australia. Unlike the sample of Ka DNA purified in 1981 (Refs 14, 20), genetic analysis of samples of Ka DNA from 1983 (with one probe) and in 1988 and 1990 (many probes) identified only one allele of the target genes and these samples are essentially clonal (Fig. I; Ref. 20 and B.P. Dalrymple, unpublished). This suggests that the less virulent line becomes the dominant component of the vaccine in some, if not all, of the vaccines distributed. It is also possible that changes in expressed genes may also have occurred within this genetically clonal population. Indeed, cloned lines derived from Ka all appear to be genetically very similar, if not identical, but exhibit different 2-D protein profiles, virulence and vector transmissibility phenotypes 2°'21. Interestingly, the attenuated line of parasites, Ta, developed to replace Ka ~, contains two major subpopulations of genetically distinct parasites (B.P. Dalrymple, unpublished). The major subpopulation of the attenuated line is a minor subpopulation of the original isolate and vice versa, it is possible that the combination of two (or more) genetically distinct lines with different virulence phenotypes might constitute a more effective attenuated vaccine. Thus, not only the nature of the expressed antigens, but also the environment in which they are expressed, may play a role in effective immunization by the live attenuated vaccines ~'~3 The use of both phenotypic and genetic analysis of populations and of
23
ParasitoloD" Today, vol. 8, no. I, 1992
well-characterized cloned lines of parasites is required to determine the importance of variation in the vaccine and challenge strains of B. boris,
Development of a Recombinant Vaccine The nature of the protective epitopes is not known for either the attenuated or the nonlMng vaccine. In the absence of this in'Formation two major approaches to the development of a recombinant vaccine have been undertaken. In the first, parasite surface proteins have been targeted 18'22. In the second, protective fracl:ions of native proteins have been subfl-actionated and tested in vaccination-challenge experiments to identify purified proteins containing protective epitopes 6'7. The genes for a number of these proteins have been isolated and characterized 18'22. Four of the recombinant proteins have been tested for the induction of a protective immune response 8 and, for the 12D3, I IC5 and 21B4 proteins, significant protection is observed upon subsequent challenge with a heterologous virulent line of B. bovis, In a field trial with animals vaccinated with a combination of the 12D3 and I IC5 antigens, a mean maximum parasitaemia of 232 + 3451~1 was observed, with 10% of the animals requiring treatment. In the control groups a mean maximum parasitaemia of 2154 + 3065 I~1 and a 40% treatment rate was observed (see also K.R. Gale et aJ., abstract*), The probability of selection of resistant populations of B. boris by a recombinant vaccine depends on many factors, including the normal biological role of the components, the diversity of the proteins in the population, the ability of the proteins to remain active after mutation, the chromosomal location of the genes (for example, telomeric genes may be prone to deletion) and the linkage of the genes within the genome, The genes for the B. bovis proteins analysed so far are present in all 15 genetically different subpopulations examined and appear to be conserved in structure (B.P. Dalrymple, unpublished and R,E. Casu and K.R. Gale, pers. commun,), However, little other information is available and more data must be obtained before the long-term viability of a recombinant vaccine can be determined,
*Recent Developments in the Control of Anaplasmosis, Babesiosis and Cowdriosis (1991) Nairobi, Kenya
Conclusion There is clear evidence of strain diversity in B. bovis, but less evidence of strain variation, The recent problems with the attenuated vaccine could be attributable either to a change in the composition of the vaccine (both phenotypic and genetic) or to the selection of some resistant strains in the natural population, or to a combination of both factors, An apparent reduction in the complexity of some samples of the attenuated vaccine has been demonstrated and selection of parasite subpopulations clearly occurs during the passage of populations of parasites through ticks, splenectomized animals and during culture, in vitro. However, the selection of subpopulations of parasites by immune pressure that is clearly separable from innate strain variation has not yet been shown for B. bovis. In future the analysis of the population dynamics of defined lines and mixtures of such lines of parasites following inoculation of vaccinated animals may allow this to be demonstrated. Since the nature of the antigens involved in the induction of the protective immunity by the live attenuated vaccine are not known, good attenuated live vaccine strains can only be identified by trial and error, There is little prospect that this process can be improved, but the prediction that more than one component may constitute a more effective attenuated live vaccine is testable, The use of gene probes will also allow the composition of the distributed vaccine to be monitored. Strain variation, and in particular strain diversity, has been well documented for malaria parasites 23-2s. Many of the candidate vaccine antigens isolated from Plasmodium falciparum are highly polymorphic 23'24 and the proposal that this is a mechanism for the evasion of the immune response has been discussed in detail 26. The selection of mutations within, and the loss of a target antigen under, immune pressure has been shown for a P. knowlesi antigen 27 and, for the P. falciparum S-antigen, serotype-specific immunity applies selective pressure on the population 28. The information on strain variation and diversity currently available for B. boris may not be relevant to the recombinant vaccine, especially since the role of the native equivalents of these components is not known for the attenuated and killed vaccines. The success or failure of a recombinant vaccine in the face of strain variation,
diversity resistant on the included
and the selection of potentially strains will ultimately depend identity of the components in the distributed product.
Acknowledgements I would like to thank the staff of the Long Pocket Laboratories of the CSIRO and the Tick Fever ResearchCentre of the Queensland Department of Primary Industries for useful discussions,
References I De Vos,A.J.andJorgensen,W.K. in Tick Vector Biology: Medical and Veterinary Aspects (Fivaz, B.H., Petney, T.N. and Haruck, I.G., eds), Springer-Verlag(in press) 2 Mahoney, D.F. (1967) Exp. Parasitol. 20, 125-129 3 Mahoney, D.F. and Wright, I.G. (1976) Vet. Parasitol. 2, 273-282 4 Timms, P. et al. (1983)Aust. Vet. ]. 60, 75-77 5 Montenegro-James,S., Kakoma, I. and Ristic, M. (1989) in Veterinary Protozoan and Haemoparasite Vaccines (Wright, I.G., ed.), pp 61-97, CRC Press 6 Goodger, B.V. et al. (1985) Int. 1. Parasitol. 15, 175-179 7 Commins, MA., Goodger, B.V. and Wright, I.G. (1985) Int. J. ParasitoL 15, 491-495 8 Timms, P. et al. (1988) Res. Vet. Sci. 45, 267-269 9 Curnow, J.A. (I 968) Nature 217, 267-268 I 0 Curnow,].A. (1973)Aust. Vet. J. 49, 279-283 II Mahoney,D.F. eta/. (1979) Int. J. Parasitol. 9, 297-306 12 Kahl, L.P. et al. (1982) J. fmmunol. 129, 1700-1705 13 Kahl, L.P. et at. (1983) Exp. Parositol. 56, 222-235 14 Cowman, A.F. et oL (1984) ,91ol. Biochem. ParasitoL I I, 9 I- 103 15 Cowman,A.F. et al. (I 984) Cell 37, 653-660 16 Carson, C.A. et ol. (1990) Exp. ParasitoL 70, 404-410 17 Jasmer, D.P. et al. (1990)J. ParasitoL 76, 834-84 I 18 Suarez, C.E. et oL (1991) ,91ol. Biochem. Parasitol. 46, 45-52 19 Dalrymple, B.P. (1990) ,91ol. Biochem. ParasitoL 43, I 17-124 20 Gill, A.C. et al. (I 987) Exp. Parasitol. 63, 180-188 21 Timms, P., Stewart, N.P. and De Vos, A.J. (1990) Infect. Immun, 58, 217 I-2176 22 Reduker, D.W. et al. (1989) ,91ol. Biochem. Parasitol. 35, 239-248 23 Howard, R.J. (1987) Contrib. Microbial. Immunol. 8, 176-218 24 Kemp, D.J., Cowman, A.F. and Walliker, D. (1990) Adv. Parasitol. 29, 75-149 25 Mendis, K.N., David, P.H. and Carter, R. (1991) in Immunoparasitology Today (Ash, C. and Gallagher,R.B.,eds),pp A34--A37,Elsevier Trends Journals,Cambridge 26 Schofield, L. (I 991) Parasitolo~ Today 7, 99-105 27 Klotz, F.W. et al. (1987)J. EXp. Med. 165, 359-367 28 Forsyth, K.P.et al. (1988) Philos. Trans. R. Sac. London Ser.B: 321,485-493 Brian Dalrymple is at CSIRO, Division of Tropical Animal Production, Long Pocket Laboratories, Private Bag No. 3, PO, Indooroopilly, Queensland 4068, Australia.