The use of experimental artefacts in African trypanosome research

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Parasrtology Today, vol. 6, no.

I4 Sims, T.A.. Hay, J. and Talbot, I.C. (I 988) 1. Pothoi. I56,255-26 I I5 Sims, T.A.. Hay, J, and Talbot. I.C. (I 989) 8r.j. trp. Patho/.70.3 17-325

I6 Luft. B.J.et of.(I 984)/. Am. Med. Assoc.252, 913-917 I 7 Helweg-Larsen. S.et al. ( l986)Acto Neural. Stand. 74,467-474

Reply We thank Dr Hay for his thoughtful comments on our paper’. We are pleased to have the opportunity to reply. We do appreciate that there is a relationship between the prevalence of Toxoplasma In a particular environment and the rate of rise of anti-Toxoplasma antibody levels. However, the risk of an AIDS patient developingcerebral toxoplasmosis is related to the overall prevalence oftoxoplasmosis in their environment; thus we stressed the geographical relationship with seroprevalence as belngthe most important factor. We are grateful to Dr Hay for drawing our attention to the rare reports of extra-cranial toxoplasmosis. However, a careful reading of the references cited shows that Luft et 01.~ reported extra-cranial toxoplasmosis in lmmunocompromlsed but not AIDS patients. The ‘skin toxoplasmosis’3 that Dr Hay alludes

to was in fact a rash associated with disseminated toxoplasmosis infection. No Toxoplasmaorganisms were seen in the skin biopsy and the authors make It quite clear thatthey were reporting an epiphenomenon in the skin rather than skin infection with toxoplasmosis. On both anatomical and embryological grounds toxoplasmic retinochoroiditis may be considered as a cranial manifestation of Toxoplasma infection. An estimated 20 000 cases of CNS toxoplasmosis have occurred worldwlde to date in contrast to two cases each of pneumonitis and orchitis and one case of Toxoplasma peritonitis. This emphasizes the pre-eminence of cerebral toxoplasmosis. The experlmental model oftoxoplasmic retinochoroiditis to which Dr Hay refers is a model of acute congenital Toxoplasma infection4. We referred in our paper to a model of chronic Toxoplasmainfection5, which we feel is more germane to the Toxoplasma infection seen in AIDS patients who usually have serologlcal evidence of past infection. The observation that Toxoplasmacysts acquire a coating of neurofilament protein is of interes@ However, this observation still fails to provide a causal mechanism for the reactivation of Toxoplasma in the immunocompromised host. Finally, we note Dr Hay’s last comment concerning the histological appearance of Toxoplasma encephalitis in AIDS and non-

I, I990

AIDS patients’. It ISperhaps not surprising thatthe bralns of the AIDS patients lack a mononuclear cell Infiltrate, as this represents the pathological basis for HIV disease. Thus these differences are again descriptive without shedding any further light on the mechanism of this tragic complication of AIDS. D.N.J. Lockwood Department of ClinIcal Sciences London School of Hygiene and Tropical Medicine Keppel Street London WC I E7HT. UK J.N. Weber Department of Medlclne Royal Postgraduate Medical School Du Cane Road London W I ZOHS. UK References I Lockwood,

D.N.J. and Weber, J.N.( 1989) PorosrtologyToday5.3 I G3 I6

2 Luft, B.J.,Conley, F.K. and Remqton.

J.S. ioncet I, 78 I-784 3 Hirschmann, J.V. and Chu, A.C. (I 988)Arch. Dermotol. I 24, 1446 I 447 4 Dutton. G.N. and Hay, J.( 1983) Trans. Ophtholmol. Sot. 103.503-508 5 Vollmer, T.L. etol. ( 1987)). Immunol. 138,

(I 983)

3737-374 I 6 S~ms,T.A.,Hay.J.andTalbot,I.C.(l989)Br.j. Exp. Pathol.70,3 17-325 7 Luft, B.J.et al. (I 984)). Am. Med. Assoc.252, 913-917

The Use of Experimental Artefacts in African Trypanosome Research C.M.R. Turner When trypanosomes are removed from the field and maintained in laboratory conditfons, phenotypic changes commonly occur such that the lines used by many investigators in routine work show several differences from the populations that affect humans and cattle in Africa’. Whether these differences are important or irrelevant ofcourse depends on the purpose ofeach particular experiment, but an awareness ofwhat the differences are con be a useful aid in the interpretation of results. Furthermore, trypanosomes can be manipulated in the laboratory to possessparticular characteristics that ard in the testing ofhypotheses that are difficult to test using ‘wild-type’ trypanosomes. In this article, Mike Turner describes how some defined trypanosome lines have been created, how they differ from one another andseveral oftheir uses. The course of a trypanosome infection in its mammalian host is controlled by five interacting processes. Four of these are intrinsic to the parasite, replication, differentiation from dividing to nondividing forms, antigenic variation and dispersion from the bloodstream into extravascular sites. The fifth process involves the trypanosome-specific immune responses of the host. The interactions between these processes provide a complex mechanism for controlling an InfectIon, and our understand-

ing of the individual components of this mechanism is considerably facilitated by the study of trypanosome populations that differ in one or more process. Fortunately, such model populations are readily available because the phenotypic characteristics of trypanosome populations change during laboratory maintenance of the parasites’. By selecting for or against particular changes, therefore, populations can be manipulated to create lines (see Box I ) that differ in any, or all, of the four parasite-dependent

processes. Changes in any of these will affect the fifth process, namely, how trypanosomes interact with the immune system. By manipulating trypanosomes to create lines that can be used as experimental models, several aspects of trypanosome biology become amenable to investigation. The discussion here is restricted to Jrypanosoma brucei brucef and 1. b. rhodesiense. Although many of the general points are likely to apply to T. b. gambiense, T. congolense and T. vivax, comparatively little work has been done on these species and the interpretation of data is accordingly less clear cut. Growth of a trypanosome population is caused by multiplication of the socalled ‘slender’ forms. The slender forms may also differentiate into non-dividing ‘stumpy’ forms that are infective to the tsetse fly vector and this differentiation provides a mechanism by which the size of the population may be limlted. Of @1990.

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considerably greater importance in controlling population size, however, are the immune-mediated killing mechanisms of the host. Protective immune responses appear to be exclusively directed against the coat of variable surface glycoprotein (VSG) that enwraps each organism. Trypanosomes evade the immune responses by spontaneously switching from the expression of one molecular species of VSG [or variable antigen type (VAT)] to another, in the process of antigenic variation. It is assumed that the switching of antigen types is linked to the cell cycle, and that only dividing (ie. slender forms) are capable of undergoing antigenic variation, whereas nondividing stumpy forms are not. A further difference between the slender and stumpy forms lies in their distribution within the host. Slender forms disseminate from the bloodstream into the lymphatics and extravascular tissues where they cause much of the pathology associated with infection, whereas stumpy forms are restricted to the blood vasculature3. How these processes combine to determine the course of an infection vanes, depending on the laboratory history of a trypanosome line. (It also depends on the parasite genotype and host species, but these are not discussed here.) An outline of the ways in which trypanosome populations from a single primary isolate can be manipulated to create lines of differing characteristics is shown in Fig. I. As the figure shows, these lines can be considered to be of five general types: the characteristics of each type of line are summarized in Table I. Types of Trypanosome

Line

of this type Type I. Trypanosomes most closely resemble those in the field; they differ only in that they have been cloned and are (usually) in a rodent host. The cyclical transmission of this type through tsetse flies causes no major changes in characteristics. This type generally produces chronic infections that will last for several weeks or months in many strains of mouse (see, for example, Refs 4, 5), and stumpy-form parasites are readily detected at the peak and duringthedecliningphaseofeach parasitaemic wave’. Many different VATS are present in a type I population during each wave of infection. In three of the most extensive investigations, the numbers of VATS detected were I9 positive out of 20 for which populations were screened7, 2 I out of 24 (Ref. 8) and 8 out

Box I. Some Nomenclature: a Few Terms that have S ecificStandardized Meaningsin Trypanosome ResearchP Whxy isolate. The successfullntroductlon of trypanosomes from a naturally infected host into culture or an experimental animal. @Stock.A population derived from a primary isolate. Nine. A laboratory derivative of a stock maintained In different condltrons (eg. cultures, host species)or in differentgeographtcal locations. @Variable antigen repertoire. All the VATS expressed by a clone. The repertoire of a cloned stock is designated according to the laboratory In which a series of VATS was characterized, eg. GUTAR(Glasgow UniversityTrypanozoon Antigen Repertoire)7. .VAT. The antigenic identity ofa single trypanosome. Each VAT ISdesignated according to the repertoire of which it IS a member, eg. GUTat (Glasgow University Trypanozoon antigen type) 7. I 3 is the thirteenth type of the GUTAR 7 repertoire.

of I2 (Ref. 9). It is likely that many other VATS were also present. Type 2. The rapid passaging of type I trypanosomes by syringe inoculation (typically, 30-50 passages at two- to three-day intervals) produces populations with very different characteristics. Populations of this type typically kill a mouse within one week of inoculation, and the rate at which stumpy forms are produced is reduced so that the population is observed to consist only of slender forms. That the lack of stumpy forms is a quantitative rather than a qualitative change is shown by the observation that a monomorphic line of trypanosomes can produce stumpy forms when inoculated into cattle”. More than 99% of the trypanosomes in a type 2 population consist of a single VAT (see, for example, Ref. I I) and this homogeneity is in large part maintained by a low rate of switching between VATS’ 2. Trypanosome lines of types I and 2 have been generally available for several decades and have been used in many laboratories. Lines of the other three types, however, have become available only relatively recently and there are only a few examples of each. Type 3. If type I trypanosomes are not passaged rapidly by syringe inoculation to produce type 2 lines, but are passaged more slowly with continual recloning, a type with a novel combination of characteristics is produced. Such lines share with type I lines the feature that large numbers of stumpy forms are produced and that a mouse host can resolve the first wave of an infection. However, they share with type 2 lines the characteristic of VAT homogeneity at 95-98%. An example of a type 3 line is the one that expresses the VAT GUTat 7. I3 (Ref. 13). To produce this line the parent stock (a type I line) was transmitted through tsetse flies, a metacyclic form was cloned and then recloned seven times during the next 27 syringe pas-

sages until the line reliably produced antigenically homogeneous populations. Types 4 and 5. Type 2 trypanosomes can be cycllcally transmitted through tsetse flies only with considerable difficulty. Very few flies develop mature infections, and those that do, produce few metacyclic trypanosomes. Infections derived from these metacyclics are of type 4 and retain two of the characteristics of type 2 trypanosomes in that they are virulent and monomorphic. In terms of their antigenic heterogeneity, however, they are similar to type I (C.M.R. Turner and J.D. Barry, unpublished). Furthermore, it is possible, with care, to make clones from these lines that are homogeneous with respect to VAT expression and are of line type 5. They retain their virulence but are highly unstable with respect to VAT expression because of their high rate of antigenic switching14. Some Uses of the Different Types Type I trypanosomes have an overriding advantage compared with trypanosomes of other types, that of their resemblance to field isolates. The only two differences are that type I trypanosomes are cloned, and they are in a rodent host. Their resemblance to wildtype trypanosomes makes them particularly useful in many applied research investigations including pathology and chemotherapy, and where the chronicity of infection is particularly important, for example in studies on immunosuppression. The principle restriction with using type I lines is that there are many aspects of trypanosome biology that cannot be studied in isolation from other, potentially confounding, processes. It is by the use of other types that this restriction can be overcome for some processes. The most important feature of type 2 lines is their virulence, which has

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Table

I. A summary of some of the characteristics of the trypanosome lines shown in Fig. I

Trypanosome line

(I) Heterogeneous pleomorphic (2) Homogeneous monomorphic (3) Homogeneous pleomorphic (4) Heterogeneous monomorphic (5) Homogeneous monomorphic

Virulent?’

Stumpy forms produced?

Homogeneous StableVAT VAT expression?b expression?b

No

Yes

No

(No)

Yes

No

Yes

Yes

No

Yes

Yes

Yes

No

No

(No)

Yes

No

Yes

No

aA line hasbeen arbitrarily definedasvirulent if it killsa mouseat the first peak of a parasitaemicwave. b If more than 95% of a populationexpressthe sameVAT it isconsideredhomogeneous,and VATS are considered to be stably expressed if the rate of switching is less than IO-’ switches per cell per generation.(Resultsin bracketshavebeen inferred, not directly observed.)

rendered them particularly amenable to biochemical and molecular biological analyses. This is because high virulence facilitates the task of growing the large number of trypanosomes that;are routinely required for such studies. Also, the potential confounding influence of conducting experiments on a mixture of two stages in the life cycle is avoided. Antigenic variation in trypanosomes has been most extensively studied using type 2 lines. The antigenic homogeneity of populations of this type has considerably assisted basic technical tasks such as the eneration of VAT-specific antisera’? , the purification ofVSGs” and the cloning of VSG genes’6”7. These tasks would have been much more difficult if type I trypanosomes had had to be used as starting materials. Type 3 lines resemble type I (and wild-type) lines in terms of virulence and pleomorphism, but are antigenically homogeneous. This has proved to be useful in two particular areas of research. First, in the elucidation of VAT-specific immune responses to pleomorphic populations, well illustrated by the studies of Black and colleagues (reviewed in Ref. 18). Second, in experiments requiring the cyclical transmission of antigenitally homogeneous populations (see, for example, Ref. 13). Type 5 lines are of particular value in studies of antigenic variation. To date, this has been best illustrated by analyses of the rate of antigenic switching. Clones derived from fly-transmitted type I infections have been observed to be antigenically heterogeneous to an extent that seemed incompatible with published values of the rate of antigenic switchingI (J.D. Barry and C.M.R. Turner, unpublished). However, it was only with the development of this type of line that the measurement of VAT-

specific switching rates in fly-transmitted infections became possible. The results showed that the overall rate was approximately I 0d2- I 0p3 switches per cell per generationI as opposed to approximately I O+- I Oe7 in syringepassaged lines of parasites12. This type has also enabled study of the activation of VSG enes by metacyclic trypano% somes ’9.2 Problems with Interpretation? It is inevitable, perhaps, that the use of several types of line has not only overcome several difficulties but also caused Primary

a few new ones. Two examples, both relating to trypanosome growth, illustrate this point. Example I. It is standard procedure to describe an antigen’cally homogeneous line by the VAT that it expresses2. Lines expressing different VATS have been observed to vary in their growth rates2’.22, and this variation has sometimes been attributed to the VATS. Although this may indeed be true, such a conclusion seems premature. A potential confounding problem is that the lines may also differ in their laboratory histories, which will also cause variations ‘n growth rates. Furthermore, lines of differing laboratory history, but identical VAT have been observed to grow at different rates23,24. Example 2. When trypanosomes are rapidly syringe-passaged to produce type 2 lines, the rate of replication increases and the rate of differentiation decreases. This observation has led to the tacit assumption that the two processes are linked. However, there is no formal evidence for this linkage, and the results shown in Table 2 suggest that the assumption may be false. The pleomorphic line GUTat 7. I3 and the monomorphic line GUTat 7. I are both derived from the same stock. The former line not only differentiates more rapidly than the latter but also multiplies at a higher rate. Experimental artefacts are often regarded as sources of potential problems that need to be minimized in, or isolate

Clone

(1) Heterogeneous

pleomorphic

pleomorphic

line

Rapid passaging by syringe inoculation

Passaging with continual recloning

(3) Homogeneous

c

line

Clone

f (2) Homogeneous

monomorphic

line

Transmission through tsetse flies

(4) Heterogeneous

monomorphic

line

fig. 1. An outline of a method ofderivation from 0 common source of tryponosome lines with five different sets ofcharacteristics. (The differences between the fines are summarized in Table I .)

f

Clone +

(5) Homogeneous

monomorphic

line

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Table 2. A comparison of population growth rates (expressed as population doubling time, PDT) of two lines of trypanosomes, one that produces stumpy forms (GUTat 7. I 3) and one that does not (GUTat 7. I) Trypanosome line (2) Homogeneous pleomorphic (3) Homogeneous monomorphic

PDT(h)=

5.3,

4.6,

4.4

6. I,

5.9,

6.3

I7 Hoeijmakers, J.A.J. et a/. (1980) Gene 8, 391-417 I8 Black. S.J. et a/. (I 985) Curr. Top. Microbrol. Immunol. I 17,93-I I8 I9 Barry. J.D. ( 1989) in New Strategies ,n Parasito/ogy (McAdam. K.P.W.J., ed.), pp 101-l 13, Churchill Livingstone 20 Matthews, K. et al. in Parasitology: Molecular Biology, Drug ond Vaccine Design (Agabian, N. and Cerami. A., eds), Alan R. Liss(In press) 21 Myler, P.], et a/. (1985) Infect. Immun. 47, 684-690

Acknowledgements The

author

is a Royal

Society

University

The contribution of Dave Barry to this work is gratefully acknowledged. I am also grateful for the financial support of the trustees of the Beit Memorial Research Fellowships, the Medical Research Council and the UNDP/World BanbWHO Special Programme (TDR). Research

Fellow.

References

I

2 3 4 5 6 7 8 9 IO

II I2 13 I4 I5 I6

Herbert, W.J. and Parratt, D. (I 979) in E%o/ogy of the Kinetoplastida Vol. 2 (Lumsden. W.H.R. and Evans, D.A., eds), pp 48 l-52 I, Academic Press Anon. (I 978)Bull. WHO 56,467480 Ssenyonga, G.S.Z. and Adam, K.M.G. (1975) Porositology 70,255-26 I Hudson, K.M. and Terry, R]. (1979) Parasite Immunol. I, 3 17-326 Campbell, G.H. et al. (I 979) Am. /. lrop. Med. Hyg. 28,974-983 Balber, A.E. (I 972)Exp. Parasml. 3 I, 307-3 I9 Le Ray, D. et al. (I 977) Ann. Sot. 6e/ge Med. Trap. 57,369-38 I Hajduk, S.L. and Vickerman, K (I 98 I) Paras& ology 83,602-62 I Barry, J.D. and Emery, D.L. (I 984) Parosrtology 88,67-84 Black, S.J.,Jack, R.M. and Morrison. W.I. (I 983) Acta Trap. 40, I I - I8 Van Melrvenne, N., Janssens. P.G. and Magnus. E. ( I975)Ann. Sot. BelgeMed. Trap. 55, I-23 Lamont. G.S., Tucker, R.S. and Cross, G.A.M. ( 1986) Parasito/ogy92,355-367 Turner, C.M.R., Barry, J.D. ant Vickerman, K. (I 986)Parasito/ogy92.67-73 Turner, C.M.R. and Barry, J.D. (I 989) Parasitology 99,67-75 Cross, G.A.M. (I 975)Parasfto/ogy7 I, 393-4 I7 Williams, R.O.. Young, J.R. and Majiwa. P.A.O. ( I979)Nature 282,847-850

Med. Hyg. 80,824-830

Mike Turner is at the Deportment ofZoology, University ofGlasgow, Glasgow G I2 8QQ, UK.

A Way Round the ‘Real Difficulties’of Malaria Sporozoite Vaccine Development?

a Each value represents the result of a separate experiment. The method of determining PDTs was as given in Ref. 25.

eliminated from, a scientific investigation. In this article I have tried to show that this is not necessarily the case. In research on African trypanosomes, a number of at-tefacts that can arise from various methods of maintaining trypanosomes in the laboratory have been deliberately exploited with considerable success, most notably to date in the study of antigenic variation. The use of these artefacts to create experimental models has played an important part in advancing our understanding of several aspects oftrypanosome biology.

22 Seed, J.R. (I 97811. Protozool. 25,526529 23 Barry, I.D., Le Ray, D. and Herbert. W.J. ( 1979) J. Comp. Pathol. 89,465-470 24 Inverso, J.A.. De Gee, A.W.L. and Mansfield, J.M. ( 197811. Immunol. l40,289-293 25 Turner, C.M.R. et a/. ( 1986) Trans. R. Sot. Trap.

F. Sinigagliaand J.R.L. Pink Vaccines against the sporozoite stages ofmalaria, based on circumsporozoite (CS) protein epitopes, have had poor results in clinical trials. It has recent/y been suggested that attention should be switched to looking for new sporozoite, or hepatic-stage proteins from which to develop vaccines. Here Francesco Sinigaglia and Richard Pink argue the case for reconsidering CSproteins as vaccine candidates. In a recent issue of Immunology Today, Good et al.’ outlined the rational basis for and the difficulties associated with the development of malaria vaccines, especially those designed to induce protective immunity against the circumsporozoite (CS) protein, the major surface protein of sporozoites. Mice can be protected by anti-CS protein antibodies, which can prevent sporozoites from entering hepatocy-tes203,or by CS-protein-specific cytotoxic T-lymphocytes4 which may eliminate infected hepatocytes, or at least prevent sporozoites from developing within them. These findings form the rational basis for attempts to develop sporozoite vaccines. Unfortunately, the first clinical trials with vaccines containing components of the CS protein gave unsatisfactory results5-7. Populations living in malaria-endemic regions appear to develop poor sporozoite immunity*. Only 40% of adults living in endemic areas were found to have T-cells that proliferated in response to at least one of a large number of overlapping peptides covering the entire CS protein sequence of 400 residues’. Most peptides to which responses were obtained corresponded to one of the three polymorphic regions of the CS protein. Good et al. attributed these findings to immune selection against parasites expressing highly immunogenic CS proteins, and discussed several alternatives @1990.

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to the use of polymorphic T-cell epitopes for the construction of CS protein vaccines, such as the incorporation of epitopes from non-malaria proteins (eg. tetanus toxoid). However, since none of the strategies appeared satisfactory, they suggested that the search must be continued for other sporozoite or hepaticstage proteins that may stimulate protective immunity. Although we agree with this suggestion, we would here like to reconsider the possibility of using CS protein epitopes to construct a sporozoite vaccine. Non-polymorphic CS protein epitopes do not seem to be efficient inducers of protective immunity in endemic areas, but we will argue that such determinants could still be used for vaccination. Non-polymorphic CS Protein Epitopes The repetitive sequence in the central region of the CS protein is a B-cell epitope that would be a good candidate for a vaccine. Such repetitive sequences are found in all CS proteins, and antibodies directed against the repetitive sequence of the P. berghei CS protein can protect mice against sporozoite infection2. However, as a T-cell epitope, the repetitive (NANP) sequence of P. falcgarum is unsatisfactory, since most mouse”.’ ’or humanI MHC class II proteins are