Complement and parasitic trematodes

Parasitology Today, vol. 5, no. I, 1989 10 Castro, G.A. in Handbook of Physiology (Motility and Circulation of the Gastrointestinal System) (Wood, J.D., ed.), American Physiological Society (in press) 11 Gabella, G. (1975) Cell Tissue Res. 163,199-214 12 Kaiser L. et al. (1987) Am. J. Physiol. 253, H1325-H1329 13 Wolinsky, H. (1976) Cardiovasc. Med. 1, 41-54 14 Lin, T.M. and Olson, L.J. (1970)J. Parasitol. 56, 529-539 15 Farmer, S.G. and Laniyonu, A.A. (1984) Br. J. Pharmacol. 82,883-889 16 Vermillion, D.L. and Collins, S.M. (1980) Gastroenterology 90, 1680 17 Coulson, E.J. (1953)J. Allergy 24, 458--473 18 Olson, L.J. and Schultz, C.W. (1961)Ann. NYAcad. Sci. 113,441-455 19 Sharp, A.P. and Olson, L.J. (1962)J. Parasitol. 48, 362-367 20 Vermillion, D., Ernst, P.B., Scicchitano, R. and Collins, S.M. Am. J. Physiol. (in press) 21 Briggs, N.T. (1961)Ann. NYAcad. Sc/. 113,456-466 22 Alizadeh, H., Weems, W.A. and Castro, G.A. (1988) F A S E B J . 2, A325 23 Palmer, J.M. and Castro, G.A. (1986) Am. y. Physiol. 250, G266-G273 24 Luzzi, S. etal. (1987) Agents Actions 20, 181-184 25 Palmer, J.M., Tamura, K. and Wood, J.D. (1988) Fed. Am. Soc. Exp. Biol. 2, A325 26 Hopps, H.C. (1964) Principles of Pathology AppletonCentury-Crofts 27 Symons, L.E.A. and Fairbairu, D. (1962)Fed. Proc. 21,913-918 28 Ferguson, A. and Jarrett, E.E. (1975) Gut 16, 114-117 29 Manson-Smith, D.F., Bruce, R.G. and Parrot, D.M.V. (1979)Cell. Immunol. 47,285-292 30 Miller, H.R.P. and Nawa, Y. (1979) Exp. Parasitol. 47, 81-90 31 Castro, G.A. and Harari, Y. (1982) Mol. Biochem. Parasitol. 6,191-202

19 32 Harari, Y. and Castro; G.A. (1983) Mol. Biochem. Parasitol. 9, 73-81 33 Harari, Y. and Castro, G.A. (1988)J. Parasitol. 74, 244-248 34 Bland, P.W. and Warren, L.G. (1986)Immunology 58, 9-14 35 Mayrhofer, G., Pugh, C.W. and Barclay, A.N. (1983) Eur. J. Immunol. 13, 112-122 36 Crago, S.S. and Tomasi, T.B. (1988) in Immunology of the Gastrointestinal Tract and Liver (Heyworth, M.F. and Jones, A.L., eds), pp 105-124, Raven Press 37 Russell, D.A. and Castro, G.A. (1979)J. Infect. Dis. 139,304-312 38 Hessell, J., Ramaswamy, K. and Castro, G.A. (1982) J. Parasitol. 68,202-207 39 Castro, G.A., Hessell, J.J. and Walen, G. (1979) Parasite Immunol. 1,259-266 40 Russell, D.A. and Castro, G.A. (1985)Immunology 54, 573-579 41 Russell, D.A. (1986)Am.J. Physiol. 251, G253-G262 42 Harari, Y., Russell, D.A. and Castro, G.A. (1987)J. Immunol. 138,1250-1255 43 Castro, G.A., Harari, Y. and Russell, D. (1987)Am. J. Physiol. 253, G540-G548 44 Harari, Y. and Castro, G.A. Immunology (in press) 45 Miller, H.R.P. etal. (1986) inMastCellDifferentiation and Heterogeneity (Befus, A.D., Bienenstock, J. and Denburg, J., eds), pp 230-255, Raven Press 46 Baird, A.W., Cuthbert, A.W. and Pearce, F.L. (1985) Br. J. Pharmacol. 85,787-795 47 Castro, G.A. in Nerves and the Gastrointestinal Tract (Falk Symposium No. 50.) (Singer, M.V. and Goebell, eds), MTP Press (in press) 48 Castro, G.A. News Physiol. Sci. (in press) 49 Cooke, H.J. (1986) Gastroenterology90, 1057-1081 50 Jankovic, B.D. (1987) in Neuroimmune Interactions: Proceedings of the Second International Workshop on Neuroimmunomodulation (Jankovic, B.D., Markovic, B.M. and Spector, N.H., eds), pp 1-2, The New York Academy of Sciences

Acknowledgements I thank my colleagues, Hassan Alizadeh, William Weems and Sui Zhang for allowing me to refer to unpublished results, and Jim Pastore and Danny Morse for their preparation of final illustrations.

Complement and Parasitic Trematodes Z. Fishelson The complement ( C ) system acts as a barrier to protect our bodies against invading pathogens. It may react to cytophilic antibodies or directly to foreign molecules presented by the intruder. As well as their cytotoxic activity, C components can attract and attach leucocytes to the surface of the foreign body, and activate them to kill it. Zvi Fishelson describes various strategies used by a parasitic trematode to escape immune damage in the face of potent immune surveillance by C and other effector mechanisms. In the past few years, research on the interaction between parasites and the C system has gained momentum, for the following main reasons: (1) our understanding of the molecular organization and the dynamics of C has markedly increased; (2) new techniques for C research have been developed; (3) the extensive involvement of C in many pathological conditions is now more fully appreciated; and (4) many more immunologists/complementologists have become attracted to parasitological research and to the challenging phenomenon of immune evasion. ~) 1989. ElsevierSol(nee Publishers Ltd, (UK) 0165 6147189/$02.00

This review describes our current understanding of the complex interactions of a parasitic trematode with complement. It should be noted that most of the data available in this field have been obtained using Schistosoma mansoni, although some studies using other trematodes such as F asciola hepatica and Opisthorchis viverrini have been carried out. The reader is also referred to previous review articles on the interaction of bacteria and other parasites with complement 14. The complement system consists of 19 components, which occur in plasma, and an additional 11 C-related proteins which

Senior Scientist Department of Chemical Immunology The Weizmann Institute of Science PO Box 26, Rehovot 76100, Israel

20

Parasitology Today, vol. 5, no. I, 1989

are usually expressed on cell membranes (see Box 1). Figure 1 presents a general scheme of the C cascade. Cercaricidal activity of complement As early as 1936 Culbertson 12described the cercaricidal activity of normal serum from various animals on cercariae of several species of parasitic trematodes other than those infecting man. Thus, for example, cercariae of Schistosomatium douthitti are not killed by human, rat, duck or herring gull sera but are sensitive to sera from water snake and green frog. On the other hand, several species of Cercaria and Diplodiscus are killed by human, rat and herring gull serum as well as by water snake and green frog serum. Normal sera of guinea-pig, cat, human, dog, rat, mouse, pig, horse and sheep - but not of cattle - produced a strong-to-moderate lethal effect on cercariae of Schistosoma mansoni 13, and cercariae of S. mansoni activated the alternative pathway of C in normal human and guinea-pig sera 14. The latter observation is based on the findings

ClassiCal

C3(H~O,);Bb Alternative

y

that, (1) C4-deficient guinea-pig serum efficiently kills cercaria, and (2) cercaricidal activity is not affected by ethyleneglycoltetracetic acid (EGTA), which chelates Ca z+ ions much better than Mg 2+ ions, thus permitting the selective activation of the alternative pathway of C. Following C activation, C3 molecules are deposited on the cercaria 14. Mechanically excysted metacercariae of Opisthorchis viverrini are also killed by exposure to normal human serum 15 The cercariae of S. mansoni are covered by a high molecular weight glycocalyx composed of carbohydrates and proteins. Besides its antigenic properties, the glycocalyx is a strong activator of the alternative pathway of C (Refs 16, 17). Sodium periodate-treated glycocalyx shows a reduced capacity to activate C. Also, C3b molecules deposited during C activation on the glycocalyx are easily removed by hydroxylamine treatment (M. Marikovsky, R. Arnon and Z. Fishelson, unpublished). These results indicate that C3b binds covalently via an ester bond to carbohydrate residues in the glycocalyx.

Parasitology 7oday, vol. 5, no. I, 1989

Development of complement resistance Whether or not the free-swimming cercariae of Schistosoma spp or the encysted metacercariae of Fasciola, Opisthorchis or Paragonimus spp activate C in their definitive hosts is of little biological importance. It is the infective larvae and the adult flukes that face destruction by the action of potent effector mechanisms. Penetration of the cercariae of S. mansoni into the skin rapidly induces a sequence of morphological and physiological changes which permit the maturation of the schistosomulum in the hostile environment of the host is. Several substances which occur in the infected skin, such as lipids and plasma factors, may accelerate the rate of cercarial transformation into schistosomula. However, most of the transitional steps may progress spontaneously following separation of the tail and incubation of the cercarial bodies at 37°C in synthetic physiological medium 19-21. By applying force to the cercariae (e.g. by syringe passage or vortexing), efficient tail separation in vitro can be achieved giving rise to the mechanically transformed schistosomula (MTS). Alternatively, skintransformed schistosomula (STS) may also be obtained in vitro by infection of isolated rat skin. Interestingly, activation of the cytolytic alternative pathway of guinea-pig, rat, foetal calf, and human C by cercariae has been shown to induce rapid tail loss in vitro 22. Similarly to the cercariae, freshly transformed schistosomula of S. mansoni activate the alternative pathway of C (Refs 23, 24). Normal sera of chicken, guinea-pig, human, monkey, rat, hamster, rabbit and mouse (in decreasing order of efficiency) express schistosomulacidal activity which is mediated by the cytolytic alternative pathway of C (Ref. 24). Properdin and C3b, but not Clq, are found on the surface of STS following incubation with normal human serum 25. Ultrastructural analysis 18'26 has shown that the initial Cinduced damage occurs at the anterior end of the larva and then proceeds to the posterior end. In addition, the induced lesions progress from the outer membrane to the muscle layer until the tegument is completely lost. Serum taken from hyperimmune or infected mouse, rat, rabbit, monkey and man, contains C-activating antibodies which may trigger the classical pathway and produce killing of schistosomula ofS. mansoni (reviewed in Refs 18, 27). The fact that C l q binds to STS following

21

104

I

I

I

I

I

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I

1

I

I

1

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Fig. 2. Development in MTS of S. mansoni of complement-resistance and complementnon-activation. MTS were incubated in defined synthetic medium (DSM) alone. Results were taken from Ref. 16 with kind permission of Academic Press.

incubation with sera from patients heavily infected with S. mansoni 25 indicates activation of the classical pathway of C but does not rule out a simultaneous activation of the alternative pathway. It is conceivable that, in immune hosts that express acquired immunity, both classical and alternative pathways of C may be activated by the infecting parasite. MTS apparently activate C more efficiently than S T S 24'25,28 and are more sensitive to C killing in the presence 28 or absence 24 of antibodies. This is best explained by the occurrence of larger quantities of residual glycocalyx on MTS than on STS is. Schistosomula acquire, upon incubation at 37°C, resistance to C-mediated damage via the classical 29-31 or alternative 16 pathways. This conversion to C resistance occurs even in synthetic medium, but its rate is accelerated when the culture medium is supplemented with serum. During culture the schistosomula lose C-activating substances ~6, C3 acceptor sites and surface antigens 32'33, which could all be subcomponents of the shed glycocalyx. Both the shedding process and the conversion to C resistance may be divided into two phases, an initial rapid stage with a half-time of about 30-60 min at 37°C (Fig. 2) and a subsequent slower stage with a half-time of about 3-5 h 30-32. Figure 3 demonstrates that, during 60 min incubation at 37°C in synthetic medium, most of the glycocalyx of MTS of S. mansoni is lost. The conversion is blocked by puromycin 3° and at 4°C (Refs 32,34). Two mechanisms have been suggested to explain the removal of the glycocalyx from the tegument of the transforming schistosomula of S. mansoni: (1) microvilli which are formed and elimin-

22

Parasitology Today, vol. 5, no. I, 1989

Complement as a mediator of cell attack

Opsonization is used by the humoral immune system to label infectious agents for destruction and elimination by effector cells. The labelling molecules are usually immunoglobulins (mostly IgG and IgE subclasses) and/or C3b and its degradation products C3bi and C3dg. Specific killing of schistosomula of S. mansoni by 0 macrophages, eosinophils and platelets can be mediated by antibodies 27'42. However, these same effector cells carry on their surface several C3 receptors (CR1, CR3 and CR4) 9 as well as Fc receptors; and indeed binding of eosinophils 43, neutrophils 44 and macrophages 45 to schistosomula can be effected by C in the Cercoria O' 60' presence or absence of antibodies. Fig. 3. Immunofluorescence staining of cercariae and MTS of S. mansoni by rabbit Interestingly, human and rat eosinophils, anti-glycocalyx antibodies and goat anti-rabbit IgG conjugated with fluorescein isothiocyanate; light (A,C,E) and fluorescence (B,D,F) micrographs. After 60 min of culture in monocytes or neutrophils 44'46'47 kill defined synthetic medium at 37°C, most of the glycocalyx has disappeared from the M TS schistosomula faster and better if they (M. Marikovsky, R. Arnon and Z. Fishelson, unpublished). bind via C3 receptors than via Fc receptors. When cultured in vitro, MTS of S. ated after skin penetration may carry with mansoni become progressively refractory them the associated glycocalyxlS; and (2) to C-dependent killing by eosinoserine proteases secreted by transforming phils 33'37. Similarly, lung schistosomula schistosomula from acetabular glands or are completely resistant to damage mediexposed within the glycocalyx (M. Mari- ated by human and rat eosinophils and kovsky, R. Arnon and Z. Fishelson, murine macrophages 37'3s'45. What are the C components to which unpublished) may proteolytically detach the glycocalyx34. In this context, when a the schistosomacidal leucocytes bind, and protease of molecular mass 28 kDa, what receptors are engaged? Preliminary released from schistosomula, is added in a results (M. Marikovsky, R. Arnon and Z. highly purified form to transforming Fishelson, unpublished) suggest that the schistosomula, it markedly accelerates C3b deposited on schistosomula by alterboth shedding of glycocalyx and the native pathway activation of human C is development of C-non-activation and C- almost completely converted within 10 resistance in the schistosomula (Fig. 4). min at 37°C to C3bi, probably by the Rapid shedding of glycocalyx has also action o f the plasma factors H and I. been reported for newly excysted juv- Therefore, CR3 and CR4 may be the eniles of Fasciola hepatica 35 which are not receptors involved in the adherence and killed by bovine, human or rat serum and cytolytic cellular processes. Recently, fail to deposit rat C3 even in immune Capron et al. 48 have shown that IgEserum 36. dependent adherence of hypodense Lung stage and adult S. mansoni are human eosinophils to schistosomula of S. relatively refractory to C lysis 37'38. mansoni and schistosomacidal activity are Whether or not this is due to poor C blocked by monoclonal antibodies antiactivation and lack of C3b deposition is Fc~ receptor or anti-CR3 s-chain but not still controversial. However, it is clear that by anti-CR1, anti-LFA1 or anti-CR3 both adults and eggs orS. mansoni contain 13-chain. Whether the eosinophil CR3 in extractable, soluble antigenic substances the latter experimental system binds to that can efficiently activate both classical deposited C3bi or to surface carbohyand alternative pathways of human C in drates on the schistosomula (or both) the absence of antibodies 39'4°. Tegu- awaits clarification. mental damage has been demonstrated by scanning electron microscopy in adults of Complement evasion mechanisms Rapid shedding of the glycocalyx, as S. mansoni exposed to guinea-pig C (Ref. 41). Unfortunately, these beautiful micro- described above, is the first of a series of graphs were not accompanied by assess- C-evasion mechanisms used by transment of the extent of schistosome forming cercarial bodies ofS. mansoni and juveniles ofF. hepatica. This conversion is mortality.

Parasitology Today, vol. 5, no. I, 1989

largely autonomous; however, some serum factors may accelerate it. Phosphate-buffered saline extracts of F. hepatica adults inhibit both the classical and alternative pathways of bovine and human complement49. Montgomery et al. 49 have suggested that this adult fluke extract contains three inhibitory molecules, two of which (one protein and one non-protein) inhibit the classical pathway, whereas the third molecule is a heat-labile inhibitor of the alternative pathway. However, it is not clear yet whether these molecules regulate or consume C. Anticomplementary antigens have been similarly isolated from adult S. mansoni 39. In both parasitic models, there is no evidence to suggest that these molecules express any activity within the intact parasite. Lung stage and cultured MTS of S. mansoni are refractory to C damage even after binding of C-fixing antibodies 31'5°. This strongly indicates regulatory mechanisms active at the surface of the parasite. Mild treatment of the complementresistant cultured MTS with trypsin or pronase makes them highly sensitive to the cytolytic alternative pathway of human and guinea-pig C (Fig. 5). Rabbit antibodies raised against the proteins removed by trypsin bind to nontrypsinized, resistant schistosomula and ac.tivate their killing by C4-deficient guinea-pig serum (M. Marikovsky, R. Arnon, Z. Fishelson, unpublished). These results suggest the spontaneous expression of a C-regulatory protein on the surface of cultured MTS. Receptors for C3b and for C l q have been detected on the surface of MTS and S T S 3. C3 receptors such as CR1 and CR2 have indeed been shown to exert regulatory activity in C activation 9. Similarly, since C l q can act as a regulator of alternative pathway of C activation 51, C l q receptors may serve the same purpose. C3 receptors have also been detected in adult S. mansoni 52. However, there are as yet no published data on C regulatory activity of these C receptors on parasite surfaces. Secreted or membranal proteases may also contribute to parasite resistance to C. Schistosomular enzymes degrade the C3, C3b and C9 C components (M. Marikovsky, R. Arnon and Z. Fishelson, unpublished) and may thus inhibit C activation. Furthermore, the proteases may also protect the schistosomes by cleaving IgG and producing IgG peptides which activate the classical pathway of C and consume C1, C4 and C2 (Ref. 3).

?_3

REMOVAL OF GLYCOCALYX re} i

g

Fig. 4. Schistosomular protease (28 kDa) accelerates release of glycocalyx (upper panel), and development in M TS of S. mansoni of complement-resistance (middle panel) and complement-nonactivation (bottom panel). (A) Control MTS incubated with medium alone, (B) M TSincubated with crude enzyme preparation; (C) M TSincubated with purified 28 kDa protease. Incubation: 10-30 min at 37°C. The percentage difference from the control is indicated above the bar.

+102%

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What are the serum factors that confer C resistance on cultured parasites or those derived in vivo? Phospholipids such as phosphatidyl choline, sphingomyelin and phosphatidyl ethanolamine reduce sensitivity of MTS and STS to antibodydependent C killing 53. Serum-induced phospholipid methylation in MTS may also somehow increase the refractoriness of schistosomula to C damage 54. Incidentally, in adult S. mansoni, complement C3 triggers incorporation of phosphatidyl choline into the outer apical bilayer com-

T

r----q--'f~--T--

Fig. 5. Conversion of complement-resistant 7 h MTS of S. mansoni to complement-sensitive by trypsin and pronase. M TS were treated for 20 min at 37°C with the proteases shown (M. Marikovsky, R. Arnon and Z. Fishelson, unpublished).

Protease (/zgIml) Trypsin (100) Acetyloted trypsJn (100)

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40

60

Mortality (%)

[ 80

I I00

Parasitology Today, vol. 5, no. I, 1989

24

Acknowledgements ZF is the recipient of a Health SciencesGrant from the Rockefeller Foundation, and holds the Barecha Foundation Career Development Chair. The author thanks Dr Moshe Marikovsky for his contributions to the field, as represented in Figs2-5, aswell as in our published and as yet unpublished manuscripts. The author also thanks Professor Ruth Arnon for her valuable collaboration.

plex sS. Thus the activation of C components may stimulate rapid turnover of the outer membrane bilayer which then in turn will clear the deposited C proteins faster. This may also explain the very rapid membrane renewal and shedding of bound C3b observed in vivo but not during in vitro culture of schistosomes 56. Finally, acquisition of host decay accelerating factor (DAF) by schistosomula of S. mansoni has recently been suggested as an evasion mechanism from C killing (Ref. 57 and F. J. Ramalho-Pinto, unpublished).

Concluding remarks Most species of trematodes parasitic in man have either settled in relatively immunoprotected sites such as the bile ducts (eg. F. hepatica, Opisthorchis sinensis) or have isolated themselves from the environment by encapsulation (eg. Paragonimus westermani). It is the Schistosoma spp that have adapted to life within the host's most immunopotent tissue, the blood. In vivo indications of C activation in infected patients (reviewed in Refs 3, 4), may suggest that the C system is attempting to combat the parasite. However, it is probably the soluble released antigens and immune complexes and not the parasites that systematically activate and consume C. Experiments performed in mice to assess the role of C in parasite immunity have provided contrasting data. Depletion of C3 in vivo by injection of cobra venom factor has interfered with primary and acquired immunity, and increased infection rates 3'23'58. These results could not be confirmed by others 59-61. Primary infection is similar in C5-deficient and normal mice 3'62, which suggests that, in murine schistosomiasis, C may play a role, not in killing the parasite, but rather in potentiating cellular effector mechanisms, by opsonizing the infective or migrating larvae or by releasing biologically active C fragments, or both. C involvement in innate and acquired immunity to S. mansoni has also been reported in the rat system 61. In contrast to the C-resistance of lung worms in vitro, the principal C-mediated attrition in vivo in rats appears to be at the lung stage. This further emphasizes the gap that exists between data obtained in an isolated experimental system and in situ. Also debatable is the question of which animal model is closest to man in respect of, for example, host interaction with S. mansoni. Clearly, multiple effector mechanisms

exist, and their relative importance in immune protection may vary between animal species 63. Perhaps the question should be, which animal species produces a C response comparable to that of man? In that respect, since schistosomacidal activity of the mouse and rat complement is inferior z4, and that of the guinea-pig C is superior 64, relative to that of the human, it is possible that the C system of monkeys will provide more relevant data on the role of human C in parasite immunity. Evidently, parasites exercise multiple evasion strategies to resist complement damage. Basic research in the C field has recently provided more sophisticated techniques which should be used to unveil those escape mechanisms and to develop counteracting reagents. Thus, immune protection may eventually be achieved by combining vaccination with sensitization in vivo of the parasites to complement damage. References

1 Taylor, P.W. (1983)Microbiol. Rev. 47, 46-83 2 Joiner, K.A., Brown, E.J. and Frank, M.M. (1984) Annu. Rev. Immunol. 2,461--491 3 Santoro, F. (1982) Clin. Immunol. Allergy 2,639-654 4 Leid, R.W. (1988)Adv. Parasitol. 27,131-168 5 MiJller-Eberhard, H.J. (1983) Springer Semin. Immunopathol. 6, 117-398 6 Mialler-Eberhard, H.J. (1984) Springer Semin. Immunopathol. 7, 93-270 7 Campbell, R.D., Carroll, M.C. and Porter, R.R. (1986) Adv. Immunol. 38,203-244 8 Cooper, N.R. (1985)Adv. Immunol. 37,151-216 9 Ross, G.D. and Medof, M.E. (1985) Adv. Immunol. 37, 217-267 10 F ishelson , Z. (198 5) I mmunol. Lett. 11,261-276 11 Miiller-Eberhard, H.J. (1988)Annu. Rev. Biochem. 57, 321-347 12 Culberston, J.T. (1936)J. Parasitol. 22,111-125 13 Standen, O.D. (1952)J. Helminthol. 26, 25-42 14 Machado, A.J. etal. (1975)Exp. Parasitol. 38, 20-29 15 Sirisinha, S. etal. (1986) Int. J. Parasitol. 16,341-346 16 Marikovsky, M. etal. (1986) Exp. Parasitol. 61, 86-94 17 Samuelson, J.C. and Caulfield, J.P. (1986) Infect. Immun. 51,181-186 18 McLaren, D.J. (1980)Schistosoma mansoni: The Parasite Surface in Relation to Host Immunity John Wiley & Sons 19 Brink, L.H., McLaren, D.J. and Smithers, S.R. (1977) Parasitology 74, 73-86 20 Sitrewalt, M.A., Cousin, C.E. and Dorsey, C.H. (1983) Exp. Parasitol. 56,358-368 21 Samuelson, J.C., Caulfield, J.P. and David, J.R. (1980) Exp. Parasitol. 50,369-383 22 Greenblatt, H.C., Eveland, L.K. and Morse, S.L. (1979) Exp. Parasitol. 48, 100-108 23 Tavares, C.A.P. et al. (1978) Exp. Parasitol. 46, 145-151 24 Santoro, F. etal. (1979)J. Immunol. 123,1551-1557 25 Ruppell, A. etal. (1983) Parasitology 87, 75-86 26 Ouaissi, M.A., Santoro, F. and Capron, A. (1980) Exp. Parasitol. 50, 74-82 27 Capron, A. etal. (1982) Immunol. Rev. 61,41-66 28 McLaren, D.J. and Incani, R.N. (1982) Exp. Parasitol. 53,285-298 29 Dean, D.A. (1977)J. Parasitol. 63,418-426 30 Tavares, C.A.P. etal. (1980)Parasitology 80, 95-104 31 Levi-Schaffer, F., Schryer, M.D. and Smolarsky, M. (1982)J. Immunol. 129, 2744-2751

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32 Samuelson, J.C., Sher, A. and Caulfield, J.P. (1980) J. Immunol. 124, 2055-2057 33 Dessein, A. et al. (1981) Parasitology 82,357-374 34 Marikovsky, M., Amon, R. and Fishelson, Z. (1988) J. lmmunol. 141,273-278 35 Duffus, W.P.H. and Franks, D. (1980) Clin. Exp. Immunol. 41,430-440 36 Davies, C. and Goose, J. (1981)Parasite Immunol. 3, 81-96 37 Bickle, Q.D. and Ford, M.J. (1982)J. Immunol. 128, 2101-2106 38 Payares, G. etal. (1985) Parasite lmmunol. 7, 45-61 39 Santoro, F. etal. (1980)Immunol. Lett. 2, 43-46 40 Van Egmond, J.G., Deelder, A.M. and Daha, M.R. (1981)Exp. P arasitol. 51,188-194 41 McCormick, S.L. and Damian, R.T. (1987)J. Parasitol. 73,130-143 42 Butterworth, A.E. et al. (1982) Immunol. Rev. 61, 5-39 43 Ramalho-Pinto, F.J., McLaren, D.J. and Smithers, S.R. (1978)J. Exp. Med. 147,147-156 44 Anwar, A.R.E., Smithers, S.R. and Kay, A.B. (1979) J. Immunol. 122,628-637 45 Sher, A. etal. (1982)J. Immunol. 128, 1876-1879 46 McLaren, D.J. and Ramalho-Pinto, F.J. (1979) J. Immunol. 123,1431-1438 47 McKean, J.R., Anwar, A.R.E. and Kay, A.B. (1981) Exp. Parasitol. 51,307-317 48 Capron, M. etal. (1987)J. Immunol. 139, 2059-2065

il¸

49 Montgomery, T.D., Leid, R.W. and Wescott, R.B. (1986) Vet. Parasitol. 19, 55-65 50 Moser, G., Wassom, D.L. and Sher, A. (1980) J. Exp.Med. 152,41-53 51 Fishelson, Z. and Mtiller-Eberhard, H. J. (1987)Mol. Immunol. 24,987-993 52 Tarleton, R.L. and Kemp, W.M. (1981)J. Immunol. 126, 379-384 53 Billecocq, A. (1987) Mol. Biochem. Parasitol. 25, 133-142 54 Parra, J.F.C. et al. (1986) Mol. Biochem. Parasitol. 21, 151-159 55 Young, B.W. and Podesta, R.B. (1986)J. Parasitol. 72,802-803 56 Ruppel, A. and McLaren, D.J. (1986) Exp. Parasitol. 62,223-236 57 Pearce, E.J., Hall, B.F. and Sher, A. (1988)FASEB y. 2, A905 58 Kassis, A.I., Warren, K.S. and Mahmoud, A.F. (1979)J. Immunol. 123,1659-1662 59 Doenhoff, M. and Long, E. (1979) Parasitology 78, 171-183 60 Sher, A. etal. (1982)J. Immunol. 128, 1880-1884 61 Vignali, D.A.A. etal. (1988)Immunology 63, 55-61 62 Ruppel, A., Rother, U. and Diesfeld, H.J. (1982) Parasitology 85, 315-323 63 Capron, M. and Capron, A. (1986) Parasitology Today 2, 69-75 64 Marikovsky, M. and Fishelson, Z. (1985) Complement 2,51

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Immunoselection Techniques for Cloning DNA-encoding Parasite-specific Antigens Michael A, Miles and J. Louise Clarke New techniques sometimes generate a 'band-waggon' effect, with research workers keen to jump on and apply the technique in their own favourite field without always pausing to consider why. Michael Miles and Louise Clarke describe a new technique which they are applying to a well-defined and valuable end- improvement in the differential diagnosis of parasitic infections, A recent editorial in the journal Nature ~ suggests that the fashionable haste to collect molecular biological data may leave insufficient time for 'reflection'. In parasitology, gene cloning is indeed fashionable but it can also contribute to real and specific rewards such as the selection of probes for identification and taxonomy, the production of specific diagnostic reagents, vaccine development, and the characterization of new chemotherapeutic targets. But in the haste to employ recombinant DNA technology, it is possible to lose sight of applied objectives and the best strategies to reach them. Recent improvements in immunoselection techniques suggest short-cuts towards one objective - the isolation of recombinant clones corresponding to parasitespecific (diagnostic) antigens. Here we describe how well-characterized antisera, from patients or from immunized animals, can be used to select such recombinants rapidly. The aim of this approach is to stimulate the develop~) 1989 ElsevierSoepcePubh~hers Ltd (UK) 016S 6147/89/$0200

ment of recombinant reagents or synthetic peptides that may help to solve the many problems of differential serological diagnosis.

DiagnosticAntigens Specific diagnostic antigens of many parasites have not yet been purified, and corresponding monospecific antisera or monoclonal antibodies are not available: the isolation and characterization of diagnostic antigens from such parasites by expression cloning can be extremely time-consuming. A single high titre serum or serum pool may be used to isolate large numbers of recombinants from an expression library*, but the subsequent characterization of these * A n expression library is constructed by transferring fragments of parasite D N A into a recipient host (eg. E. coil) in such a way that parasite antigens are expressed in the new host and can be detected by antibodies.

recombinants is usually prolonged and tedious. They are commonly rescreened to homogeneity; DNA is then prepared from each clone, and relationships between clones established by cross-hybridization studies. In addition, lysogens are prepared (which can be difficult for large DNA inserts), and the serological potential of each recombinant evaluated using strip western blots of lysogen lysates against large panels of antisera. Only then can fusion proteins, assessed to be potentially useful for diagnosis, be further analysed by subcloning of the DNA inserts, sequence determination, and epitope mapping with the ultimate aim of producing purified diagnostic reagents 2s. These endeavours may take years of intensive effort with the tenuous hope that one of the recombinants will 'turn up trumps'. The initial screening of expression libraries is 'serological' and directly analogous to the final objective - the diagnostic assay. There is no reason why pairs of well-characterized serum samples or serum pools, which differ in the specificity to be distinguished by the ultimate assay, cannot be used directly to select recombinants atthe first screening stage. Similarly, single recombinant/nonrecombinant mixtures can be used to