- Email: [email protected]
On the function of repetitive domains in protein antigens of Plasmodium and other eukaryotic parasites
Parositobgy Today, vol. 7, no. 5, 199 /
with follow-up treatments. It is estimated that 155 million Africans harbour Ascaris and the infection is often associated with severe morbidity and a variety of complications L°. The large-scale regular distribution of ivermectin ~br onchocerciasis control will thus provide a unique opportunity, at no extra cost, to remove many of the intestinal worms from millions of Africans and keep them clear for a decade or more. Thus, the project has great potential in aiding the introduction and acceptance of PHC and other health programmes. The control of onchocerciasis in Nigeria would greatly reduce the global prevalence of this infection. However, the attainment of this goal will require the optimal application of existing resources with absolute dedication,
99
strong political will and generous international support. The adverse public health and socioeconomic consequences of onchocerciasis make it a disease high on the W H O list for research and control. Now that impressive commitment and progress towards its ultimate control have already been made as a result of the larviciding successes in the OCP area, the free donation of ivermectin, and the continuing efforts to develop an ideal macrofilaricide and/or an effective vaccine, the launching of a globally coordinated initiative to control onchocerciasis in all endemic areas may shortly become feasible. References
I WHO Technical Report SeriesNo. 752 (I 987)
Third Report of the WHO Expert Committee on
OnchocercJasis 2 Crosskey, R.W. (I 981 ) Tropenmed. Parasitol,
32,2-16 3 Aziz, M.A. (1986) Parasitology Today 2, 233-235 4 Cupp, E.W. et al. (I 986)Science23 I, 740-742 5 Duke, B.O.L. (1990) Parasitology Today 6, 82-84 6 Trpis, M. et al. (1990) Am.J. Trap.Med. Hyg. 42, J4-156
7 Pond,B. (1990) Lancet 335, 1539 8 Naquira, C. et al. (1989) Am.J. Trap. Med. Hyg. 40, 304-309 9 Freedman, D.O. et al. (1989)J. Infect. Dis. 159, 1151-1153 10 Crompton, D.W.T. and Tulley, JJ. (1987) Para-
sitologyToday 3, 123-127
Luke Edungbola is at the Faculty of Health Sciences, University o[ llorin, PMB 1515, Ilorin, Kwara State, Nigeria.
On The Function of Repetitive Domains in Protein Antigens of Plasmodium and Other Eukaryotic Parasites L. Schofield Highly reiterated repetitive domains occur within the protein antigens of many parasitic taxa, including Plasmodium, Trypanosoma, Leishmania and Toxoplasma. In malaria it has been proposed that repeat regions may function as ligands for host proteins, or serve to suppress the development of immunity through a strategy of serological' crossreactivity. In this article Louis Schofield presents a novel hypothesis, based on empirical evidence, that repetitive domains in antigens do not elicit protective immune responses and instead have evolved as a mechanism of immune evasion by their ability to induce thymus-independent B-cell activation. It is also proposed that this unusual response is associated with several forms of immunosuppression. The hypothesis has the added attraction of helping to explain several distinctive features of the molecular biology, evolution and immunology of repetitive regions in protein antigens of parasites. Tandemly repeated regions within proteins are not unique to eukaryotic parasites but the frequency and magnitude of the phenomenon in these taxa suggest they represent a special case. In most other organisms repetitiveness ,within proteins serves a clear
Louis Schofield is at the National Institute for Medical Research, The Ridgeway, Mill Hill, London N W 7 I AA, UK. ~) 199I, E~sevierSciencePublishersLtd,(UK)0169J~707/91/$02.00
physico-chemical or structural function, as seen in the case of collagen or glue proteins 1. However, in parasites, and particularly in Plasmodium, repeats are widespread among proteins from different developmental stages, with various types of cellular organizations and apparently divergent functions. Such proteins include soluble antigens released during schizogony (S-antigens), proteins associated with the infected red blood cell (RESA), rhoptry organelle proteins and surface antigens of sporozoites and merozoites. Even a phylogenetically conserved heatshock protein shows unique repeat elements2. The repetitive unit, of a variety of different sizes, can be reiterated from once to more than 50 times. The repeat structure is quite distinct among different proteins and, furthermore, allelic variants of a protein, or phylogenetically related homologues, often show completely unique repeats within otherwise conserved protein sequences. In addition, more than one type of repeat can occur within some proteins. For reasons of space I will not examine in depth the variety and structural features of repeats but refer the reader to the primary literature 3-9. The overall picture is one of great tolerance by the parasite for diverse repetitive structures of different shapes and sizes inserted promiscuously into a wide range of proteins with a variety of functions. Despite the great diversity in size, number and distribution of repetitive elements, many distinctive
I O0
features are held in common. First is the property of repetitiveness itself. Second, there appears to be a noticeable bias in the component amino acids 10, with residues such as N, A, D, V, P, E, G, Q and S highly represented but C, M, F, W, Y, I and L rarely present. The third distinctive feature of these regions is their immunodominance. For example, antibody to sporozoites appears to be directed almost entirely towards the repetitive domain of the circumsporozoite (CS) protein. When sera from individuals naturally or experimentally exposed to sporozoites is absorbed with this repeat peptide, reactivity to whole parasites is lost, indicating that this is the single immunodominant epitope recognized by naturally induced antibody 11. Conversely, antibody to adjacent nonrepeat epitopes on the CS and other repeat-containing proteins is much less prevalent. Similar types of repeat immunodominance are exhibited by S-antigens and RESA 12. A fourth and final property of many of these regions is their unusual genetics and evolutionary history. Based on an analysis of the evolutionarily conserved CS protein family, Enea and colleagues 13-18 have convincingly shown that the evolution and maintenance of the repeat structure must be through a mechanism operating at the DNA level, similar to that described for satellite DNA. Simple phenotypic selection acting on point mutations could not simultaneously generate highfidelity repeat structures within a protein and yet cause a massive shift in sequence in allelic variants. In contrast, the postulated genetic mechanism could easily cause a DNA sequence to be reiterated and spread, and at the same time result in the divergence of the repeated unit a c r o s s t a x a 14. Strong support for this view comes from a study of the unrelated Santigen family 19, in which a simple frameshift in the decoding of a conserved repetitive DNA sequence results in a phenotypically distinct allelic variant with structurally and serologically distinct tandem repeats. Thus it is the structure of the DNA, rather than the protein, that is conserved in both gene families. One mechanism by which this may occur is through biased gene conversion 17. As well as proposing a function for repeats, therefore, a satisfactory theory of their biology must provide answers for the following questions: (1) why are these regions repetitive? (2) why are repeats found in so many different types of protein? (3) why is amino acid choice biased in repeats? (4) why are most, but not all, of these regions immunodominant? (5) how is it possible to have completely different repeat structures within phylogenetic homologues and allelic variants of the same protein (that have the same function)? and (6) can the postulated function of the repeats be reconciled with their highly unusual genetics and evolution? Clearly, the repeats constitute a related family of structures and we should avoid constructing different theories to account for different repeats and their various properties if they can be dealt with satisfactorily under one unifying explanation. In other
Parasitology Today, vol. 7, no. 5, 199 /
words, epistemological criteria demand that a theory of repeats should be not only simple and falsifiable but also comprehensive.
Existing theories To date, two theories of the function of repeats have been forwarded. In the first, each repeat unit is proposed to act as a ligand for host structures such as red blood cell receptors and hepatocytes 2°. In this view, the binding units are repeated because this allows a multimeric high-avidity interaction between parasite and receptor. The high fidelity of the repeats is thought to reflect the constraints imposed by purifying selection to ensure binding. While providing a plausible answer to the first two questions, this postulate has several major weaknesses: it does not explain why choice of amino acids should be constrained for so many different types of host-parasite interaction (question 3); it does not explain, and is at odds with, the immunodominance of the repeats (question 4); it is impossible to reconcile a repetitive unit constrained in structure by a requirement for interactive ligand properties, with the fact that radically different repeats may occupy the same region of allelic variants of a protein that has the same function (question 5); similarly, the molecular evolution of these domains cannot be explained by reference to purifying selection and despite considerable research effort over many years no convincing evidence has surfaced to suggest that the repeated regions have any ligand properties whatsoever. In the second theory, the repeats are proposed to enable the parasite to evade immunity by presenting to the host an extensive network of crossreactive epitopes that abort the affinity maturation of the response to 'protective' epitopes 21. This proposition has several attractive features: it can provide an explanation of the immunodominance of repeat epitopes; amino acid choice can be said to be biased to favour crossreactions and there is clear evidence that a number of repeat domains are serologically crossreactive with each other (but no evidence as yet that the presence in the host of several domains leads to a reduction in antibody affinity). However, I suggest that the proposal is incomplete for five reasons. First, it does not in any way explain why these regions are repetitive. Why would epitopes designed to be crossreactive with 'protective' domains require this tandem structure? Second, swamping the B-cell repertoire with crossreactive analogues is a 'global' or trans-acting strategy, which fails to explain why repeats are maintained in any specific example of a protein. The postulate would simply require that a large mass of crossreactive epitopes be released freely into the circulation to compete for B cells. Third, and more important, there is a tautologous element to the proposal. The crossreactivity actually described by Anders is among different repeat structures 2~. If 'protective' epitopes lie within the repeats, then more questions are raised than answered, as it fails to explain why
Parasitology Today, vol. 7, no. 5. 1991
I 01
Table I. Antigen-specific T cells are not required for secondary responses to the CS protein of sporozoites a
T cells
B cells
Challenge
T i t r e versus C S
T i t r e versus T T
T (naive)
B (naive)
SPZ "IT
64 ND b
128 ND
T (naive)
B (SPZ + l'T)
SPZ TT
4096 ND
32 512
T (SPZ + 3-1-)
B (SPZ + l-F)
SPZ 1-F
8192 32
64 65536
~Donor A/J mine were primed separately to Tetanus toxoid (TT) or P. berghei sporozoites (SPZ). Spleen cells from each group were then pooled in equal numbers before purification of T cells (by removal of adherent and slg+ cells) and B cells (by removal of adherent and Thy I + cells). Following T-cell transfer, light irradiation and B-cell tran:,Yer,the syngeneic recipients were boosted separately with either SPZ or 3-1"and the serological titre to TT and NANP3 determined after eight days, by solid phase radioimmunoassay(adapted from Ref. 29). b Not determined.
repeats could evolve with this property. In other words, the repeats are proposed to have evolved to crossreact with each other to avoid immunity directed to the repeats. We may avoid this dilemma only by adopting two auxiliary hypotheses, namely that undefined protective epitopes are indeed outside the repeats, and their crossreactivity with repeats exists but has yet to be shown. However, these necessary assumptions threaten the empirical basis of the postulate, for now the inter-repeat crossreactivity can only be of second2a'y relevance. Fourth, a considerable degree of repeat serological crossreactivity is observed across malarial taxa, which parasitize different hosts 22'23. Thus 'co-ordinate' immune evasion cannot account for the phenomenon. Finally, and most importantly, the theory is in conflict with what we know about the molecular evolution of repetitive elements. This is because it requires that crossreactive repeats and 'protective' epitopes must necessarily evolve in reference to each other, constrained by the need to be structurally related (to crossreact), with phenotypic selection responsible for various appropriate amino acid substitutions. However, this is incompatible with the molecular evolution of repetitive domains, which proceeds by quantum leaps 15, with no mechanism for concerted evolution and little evidence for adaptive amino acid substitutions. A novel hypothesis In contrast to the foregoing propositions, I would like to offer a novel theory of repeat biology. It is well known that the repetitiveness of a wide variety of agents causes T-cell-independent activation of B cells by crosslinking hapten-specific surface immunoglobulin (Ig)24. Examples are repetitive structures such as polyacrylamide, bacterial carbohydrates, (Fab)2 anti-sIg and repetitive proteins such as collagen and flagellin2s-27. Many infectious agents, in particular the gram-negative bacteria, favour the strategy of presenting repetitive T-cellindependent antigens on their surface. Following this line of thought we recently sought to determine whether repetitive antigens of malaria might also induce such a response. This proposal was strongly at odds with the conventional wisdom that held the antibody response to malaria repeated proteins to be
under Ir gene control28 and therefore the opposite of what we proposed. However, the conventional view is based entirely on the immunological behaviour of various vaccine constructs and not of the parasite itself. Therefore, to test the postulate, we undertook an investigation into the regulation of the antibody response to the repetitive domain of the CS protein on intact sporozoites29, and observed that the parasites do indeed induce a thymus-independent B-cell response to the repeats (Table 1). In other words, the sporozoite CS protein induces activation of B cells leading to production of antibody to the repeats without a requirement for MHC-restricted cognate interaction with CS-specific helper T cells. This phenomenon is antigen-specific because the sporozoites do not induce antibody secretion from bystander B cells specific for Tetanus toxoid (Table 1). Mice hyperimmunized with sporozoites have high levels of anti-repeat antibodies and, according to conventional wisdom, their T cells must be able to recognize the CS protein. When such T cells are made available to the B cells in a manner allowing the expression of MHC-restricted helper activity, there is little apparent booster effect (Table 1). In contrast to the behaviour of sporozoites, the response to the antigen Tetanus toxoid exhibited classical T-celldependence under identical experimental conditions. In further experiments, T cells derived from priming with CS-based vaccine constructs, and known to be reactive with CS sequences, were shown under these conditions to have little ability to influence the T-cell-independent response to parasites (Table 2). It is important to appreciate that these experimental conditions are designed to allow the expression of helper activity and therefore provide a critical falsifying test of the T-cell epitope-Ir genecontrol theory of the anti-CS response. A subsequent study has similarly provided evidence that immunization with sporozoites does not prime T cells to CS epitopes3°. Thus, based upon this empirical demonstration of a T-cell-independent response to the CS protein repeats, we propose the following: (I) It is already well established that tandemly repeated structures crosslink surface immunoglobulin
Parasitology Today, vol. 7, no. 5, 1991
102
Table 2. Failure of CS-specific T cells t o boost the antibody response t o sporozoites a T cells
B cells
Challenge
Titre versus NANP4o
T (FCA)
NANP40
SPZ CS.T3 + NANP3
32768 512
T (CS.T3)
N A N P40
SPZ CS.T3 + NANP3
32768 131072
aBalb/c mice were immunized twice with 50 ug of the peptide DIEKKIAKMEKASSVFNVVNS, corresponding to amino acids 378-398 of the CS protein, a conserved peptide T-cell site (CS.T3) widely recognized by murine and human MHC 39, in Freunds' adjuvant (FCA). Control mice received adjuvant alone. After light irradiation they received purified B cells from donors primed to the NANP40 sequence conjugated to bovine serum albumen (BSA) and were boosted with P. falciparum sporozoites (SPZ) or a peptide construct of CS.T3 conjugated to the B epitope NANP3. Sera were diluted twofold in phosphate buffer saline-BSA and titres were determined by radioimmunoassay using NANP4o-BSA assolid phaseantigen.
on B cells, providing the cells with a thymusindependent activation signal that is not given by single or nonrepetitive epitopes 25. Thus we may propose that repeat domains are repetitive (question 1) simply to provide this activation signal to B cells, as the first step of a strategy to force them down an inappropriate (T-cell-independent) differentiation pathway. (2) A thymus-independent response is generally considered to be inferior to a T-cell-dependent response, lacking in affinity maturation and adequate T-cell and B-cell memory formation. Thus the response induced by parasites will be low grade and non-neutralizing. (3) While inducing low-grade non-neutralizing antibody, the repeats can be immunodominant, and will suppress the formation of antibody to important adjacent areas on the same molecule, ie. epitopic suppression (question 4). In other words, this form of immunosuppression is cis-acting and therefore requires a repeat domain to exist within each protein participating in the strategy (question 2). (4) The thymus-independent response is likely to be associated with lesions in T-cell priming or T-cell-B-cell cooperation, which may be an additional form ofimmunosuppression, and is consistent with our own and other observations of low anti-CS priminffz9'3°. (5) The repeats will be constrained to amino acids that allow the domains to be efficient B-cell epitopes, favouring hydrophilicity and an appropriate conformation (question 3). (6) The precise structure of repetitiveness within the domain is relatively unimportant, provided it is an effective B-cell epitope and can contribute to the induction of T-cell-independent B-cell activation. Thus, in addition to allowing a wide diversity of repetitive structures, the responsible genetic mechanism can with impunity insert very different repeats into the same location within allelic variants of a protein (questions 5 and 6). The frequency of such
equally fit alleles within the population is therefore not the result of immune selection but of random drift 18. The same genetic mechanism may also create unconstrained and selectively neutral repeats in a variety of different proteins.
Objections to the hypothesis How can these regions be a mechanism of immune evasion when they are the target of protective immunity to the parasite? The view that the repeat domains of malaria proteins are the target of protective immunity, derived from the need to rationalize vaccine development, has led to a major paradox: why should the parasite display on its surface highly immunogenic antigens that induce protective immunity? The answer to this dilemma comes from understanding that, despite the desire to render repetitive regions into vaccines, a considerable body of evidence has for many years shown that antibody to these regions induced by natural or experimental exposure to parasites is in fact nonprotective. Sera with very high levels of antibody to the CS repeats from mice immune to sporozoites cannot transfer any protection to recipients 31. Even in hyperimmune animals, sporozoites escape the antibody and gain access to the liver where they may be destroyed by cellular mechanisms 32'33. In general, anti-repeat antibody shows inhibitory activity only in artificial experimental systems where parameters can be modified to promote the desired observation, for example by the use of unrealistically high levels of monoclonal antibodies, culture systems modified, in vitro, prolonged incubation periods, or using selected hosts with genetically low levels of susceptibility to infection. Most importantly, field studies show that the highest levels of naturally acquired antibody to the CS repeat is not associated with reduced risk of infection 34, which is also the case for antibody to other repetitive antigens such as RESA 35. I f repetitive antigens of parasites are T-cellindependent, how are they able to induce IgG? It is a common fallacy to assume that T-cell-independent antigens induce only IgM and T-cell-dependent antigens induce IgG, and that this can distinguish between the two. This is not always the case, as shown by the fact that much naturally occurring Ig to bacterial T-cell-independent antigens is of the IgG isotype. This is because infectious agents induce high levels of trans-acting factors that cause classswitch in B cells activated by T-cell-independent antigens. In other words, MHC-unrestricted (factordriven, noncognate) help may be responsible for the emergence of IgG to T-cell-independent malarial antigens. In fact there is evidence that this occurs in response to sporozoites 29, and may well account for IgG formation to the CS protein. The natural human antibody response to the CS protein following exposure to sporozoites also has the characteristics of an anti-carbohydrate response 36. How can the response to sporozoites be thymusindependent when much recent work has demonstrated
Parasitology Today, vol. 7, no. 5, 199 /
T-cell sites on the CS protein? The answer to this is that the work in question was carried out with synthetic peptides and recombinant protein analogues of repetitive proteins and not with the parasite itself 37-39. It should be appreciated that this is a highly artificial approach representing a type of minimalist condition; when presented with synthetic peptides or simple soluble proteins, the immune system is constrained to only one of two possible outcomes, ie. responsiveness leading to a full IgG response (requiring/viHC-restricted cognate interactions between the recognizing B cell and an antigen-specific helper T cell) or nonresponsiveness (when these conditions are not met). This accounts for the phenomenon of MHC restriction. In contrast, the immune response to an infectious agent is of a higher level of complexity and involves MHCunrestricted effects. It is therefore no surprise that previous studies have shown several synthetic peptides based on the CS protein to be T-cell epitopes, since being a peptide T-cell site is merely the trivial consequence of being non-self and able to associate with one or other of the experimentally available MHC. The only important question, which is whether any of these regions function as helper T-cell sites in the context of anti-parasite responses, has unfortunately never been demonstrated or even clearly addressed by any of these studies. It is of course possible to show that sporozoites can boost antibodies to the repeats in mice primed with peptide constructs 4°'41 but these studies fail to address whether this is due to the action of helper T-cell sites or the T-cell-independent activation of the repeatspecific B cells by native sporozoites. Thus the conclusions concerning the MHC restriction of the antibody response to sporozoites are extrapolations from the simplified immunological behaviour of synthetic replicas, but the response to the parasite is unlike that to a nominal peptide antigen (for review, see Ref. 29). I f repetitiveness pnrvides an activation signal to B cells, why are repetitive synthetic peptides and recombinant proteins not T-cell-independent antigens? The activation and differentiation of B cells leading to antibody secretion can occur through several pathways, each involving different signals. Typically, three signals are required to initiate a T-cell-dependent developmental pathway: (1) binding of (monomeric) antigen to surface Ig; (2) recognition of B-cell Ia (MHC) molecules by histocompatible, antigen-specific T-cell receptors (TcR) followed by (3) T-cell growth and differentiation factors 42. Clearly this sequence corresponds to events in T-cell-B-cell cooperation. In contrast, crosslinking of surface Ig by repetitive epitopes leads to the induction of phosphatydylinositol turnover 43, Ca 2+ flux44, B-cell cycle entry (Go--G1 progression)45 and the acquisition of responsiveness to soluble lymphokines (factor-driven help)46. However, in the absence of further signals of this type, crosslinking, while providing an initial state of T-I activation
103
distinct from that induced by normal ag-Ig binding, is not sufficient to drive the B cell fully to antibody secretion. Thus repetitive recombinant proteins will crosslink slg but this is insufficient for full expression of antibody formation which, in the absence of further appropriate signals, may proceed through MHC-restricted events. By way of a corollary we must invoke an additional or secondary signal of parasite or host origin to fully account for our observation of T-cell-independent antibody formarion to the repetitive antigens of parasites. Possible candidates for this signal could be host factors induced by infection, eg. interleukins 4 and 5, or a covalently bound lipid such as the sn-l,2diacylglycerol moiety present within the glycosylphosphatidylinositol complex of several parasite surface antigens47'4s. Many of the other repetitive T-I antigens on the surface of infectious agents provide more than one such signal, eg. lipopolysaccharide and the E. coli surface lipoprotein. At high concentration, potent T-cell-independent antigens such as lipoproteins or glycolipid mitogens gain access to B cells in an antigen-independent manner through the cell membrane, activating cellular growth and differentiation pathways internally and causing polyclonal Ig secretion. However, at lower concentrations they gain access to B cells by interaction with antigen-specific slg. I f immunodominance and crosslinking of surface lg is important, how do we explain the existence of nonimmunodominant repeats, minor repeats, variant repeats and unconserved or degenerate repeats? Based on the ideas of Enea x~'~4, repetitive DNA is introduced promiscuously into coding regions, where we propose it can acquire fitness by inducing a T-I response. Naturally, selection will reject maladaptive mutations. However, the organism will also tolerate selectively neutral intrusions of this type, eg. minor repeats and those reiterated only once or twice, and these will tend also to be less immunodominant, eg. the small repeats in PMMSA. Indeed, most of the repeats in P. falciparum antigens may be 'neutral', as shown by their allelic diversity, eg. both PMMSA and MSA-2 (Ref. 49), demonstrating on one hand that their presence is not lethally disruptive of protein function and on the other that their absence is not prejudicial to parasite survival. The presence in Plasmodium of unique repeats in the otherwise phylogenerically conserved heat-shock protein 2 is further evidence that the genetic mechanism inserts repeats wholesale, without reference to protein function, to be acted upon by selection or allowed to drift. Even large immunodominant repeat structures may also in time become selectively less constrained and then drift, tolerating a number of amino acid replacements over evolutionary time because these substitutions do not disrupt the capacity of the molecule to crosslink slg, eg. degenerate repeats in FIRA. In these cases (drifting or unconstrained repeats) we would expect a certain allelic heterogeneity among isolates. However, we would
104
not expect to see major high-fidelity repeats that are not immunodominant. Similarly, a protein may tolerate the emergence of variant but related dimorphic repeats such as in RESA and several CS proteins, the divergence of these regions being constrained by the need to provide crosslinking of surface Ig when focused on the B cell (although here lower-affinity interactions are tolerated). Thus the serological crossreactivity in cis noted by Anders 21 would be a consequence of these constraints. How do we explain the serological crossreactivity of repeat antigens? In Anders' theory, crossreactivity is seen within repeats in a given protein and among repeats from different proteins of one or more developmental stage. The repeats have evolved to collaborate in trans in aborting antibody affinity maturation to putative protective epitopes that may be within or external to the repeats themselves 21. In our view, repeats are 'designer' B-cell epitopes that have evolved both to interact with B cells at much higher efficiency than adjacent nonrepeat epitopes (thereby inducing epitopic suppression in cis) and to crosslink the surface Ig of the recognizing B cells. As noted above, crossreactions between two or more repeat domains within a protein (which are usually closely related) simply reflect the constraints on divergent repeat sequences to co-operate in cis to promote crosslinking on the B-cell surface. Crossreactivity among domains of different proteins may arise as the result of several factors. First, repeat sequences may have ancestral relationships among and within species and would therefore be structurally related. Thus the CS proteins ofP. falciparum and P. berghei share NANP sequences and are serologically crossreactive. However, these parasites exist in different hosts. Crossreactivity between RESA and other antigens is cited as being central to the postulate of crossreactive immune evasion21. However, two studies show that this antigen also crossreacts with P. chabaudi, P. cynomolgi and P. vivax 22"23. It is clearly impossible in these major examples, from taxa separated far in evolutionary time, to account for serological relatedness by invokhag joint immune evasion or co-ordinated sequence evolution. Therefore, the crossreactivity among P. falciparum proteins such as Pfl 1.1, RESA, Ag 332 and FIRA, may simply reflect an ancestral relationship among their EEVV, EENV, EE-V and EPV repeat sequences, respectively, and may not indicate a shared interactive function. Second, there is considerable evidence that the germ-line repertoire of IgV genes is biased towards the recognition of the repetitive carbohydrate antigens of bacteria, and repetitive antigens of parasites may exploit this to react with a large number of somatically unmutated B-cell lineages, ie. the most fit sequences are those with a certain bias in amino acid choice. This, together with the known degeneracy of the antibody repertoire, could lead to extensive crossreactivity. The question therefore is whether crossreactions cause failure of affinity maturation, and whether the
Parasitology Today, vol. 7, no. 5, 1991
repetitive domains have evolved to promote this end, or if crossreactivity is a side consequence of other strategies and constraints. As in any area of biology, major features such as the repeat regions will generate a number of phenotypic consequences (which may act as selective constraints on the evolution of the system). This does not mean that there are distinct functions associated with each phenomenon, and we should avoid needlessly multiplying separate hypotheses to account for all observations. Nonetheless, until put to further experimental test, we would accept the proposal of Anders that crossreactivity among the repeats themselves (but not with 'protective' epitopes) results in the expansion of the repeatreactive repertoire 21. Conclusions Optimal regulation of the humoral immune response requires processing of antigen, via catabolic proteases, that allows antigen presentation in the context of MHC class II to the helper T-cell-effector arm by B cells. Infectious bacteria evade this proteolytic processing by exposing polymeric carbohydrates on their surface. However, constraints on the synthetic enzymes involved limit the serotypic diversity available to any one strain. Unable to process these antigens, the host adaptive response has been to respond to polymerism per se (via crosslinking of Ig on recognizing B cells). However, the resulting (T-cell-independent) response is considerably less efficient than the MHC-restricted variety. We propose that eukaryotic parasites, especially malaria, have evolved a mechanism to exploit this phenomenon to the full. As proposed by Enea, Arnot and colleagues 14'15'19, an unusual genetic recombinational process inserts repetitive DNA into genes encoding proteins (not involving transposable elements). This mechanism acts without reference to existing protein function, with lethal, neutral or adaptive outcome. While no doubt generating some lethal mutations, the mechanism overall has been selected because, in contrast to the slow pace of selection acting on point mutations, it is a most efficient method for obtaining repetitiveness in a variety of antigens. The repeats thus generated fall into one of two classes. A certain proportion (perhaps the majority) are structurally unconstrained, nonimmunodominant and selectively neutral (without function), but many also acquire fitness by acting as 'designer' B-cell epitopes. As potent epitopes they serve to focus important molecules onto antigenspecific B cells, and in the process suppress antibody to adjacent regions (epitopic suppression). Crosslinking of the surface Ig by the tandem array of repeated epitopes is not sufficient for a full T-cellindependent response, but is simply the first step in a sequence of signals that sends the B cells on an abortive or inappropriate differentiation pathway. This may lead to failures in affinity maturation, memory formation and T-cell activation or cooperation. The immune response to the repetitive
Parasitology Today, vol. 7, no. 5, 199 I
105
antigens of malaria is therefore closer to the antibacterial rather than Ir gene controlled type. References
1 2 3 4 5 6 7 8 9
Garfmkel,M.D. et al. (1983)J. Mol. Biol. 168,765-789 Yang,Y.F. etal. (1987)Mol. Biochem. Parasitol. 26, 61-67 Dame,J.B. etal. (1984)S~.'nce225, 593-599 Cowman,A.F. et al. (1985) Cell 40,775-783 Favaloro, M.J. et al. (1986)NucleicAcidsRes.14, 8265-8277 Stahl, H.D. et al. (1987)Mol.Biol. Med. 4, 199-211 Coppel, R. et al. (1986) Mol. Biochem. Parasitol. 20,265-277 Weber, J.L. etal. (1988)J. Biol. Chem. 263, 11421-11425 Wellems, T.E. and Howard, R.J. (1986)Proe. NatlAcad. Sci. USA 83, 6065-6069 10 Nussenzweig,V. (1986) Am.J. Trap. Med. Hyg. 35,678-688 11 Zavala, F. etal. (1985)Science228, 1436-1438 12 Perlmann, P. et al. (1986) in Vaccines 86 (Lerner, R.A., Chanock, R.M. and Brown, F., eds), pp 86-91, Cold Spring Harbor 13 Enea, V. etal. (1986)J. Mol. Biol. 188,721-726 14 Galinski,M. etal. (1987)Cell48, 311-319 15 Di Giovanni,L. et al. (1990) Exp. Parasitol. 70, 373-381 16 Colomer-Gould,V. and Enea, V. (1990) Mol. Bioehem. Parasitol. 43, 51-58 17 Arnot, D. etal. (1988) Proc. NatIAcad. Sci. USA 85, 8102-8106 18 Arnot, D. Acta Leiden. (in press) 19 Saint, R.B.etal. (1987)Mo.1.CelIBiol. 7,2968-2973 20 Nussenzweig, V. and Nussenzweig, R. (1989) Adv. Immunol. 45, 283-334
21 Anders, R.F. (1986) Parasit. Immunol. 8, 529-539 22 Holmquist, G. etal. (1988) Infect. Immun. 56, 1545-1550 23 Wandiworanun, C. et al. (1989) Am. J. Trap. Med. Hyg. 40, 579-584 24 Mosier, D. and Subbarao, B. (1982)Immunol. Today 3, 217-219 25 Dintzis, I.M. etal. (1976)Proc. NatlAcad. Sci. USA 73, 3671-3675 26 Feldmann, M. (1972)J. Exp. Med. 135,735-753 27 Moiler, G. (1980)Immunol.Rev. 52, 1-231 28 Good, M.F. et al. (1988) Immunol. Today 9,351-355 29 Schofield,L. and Uadia, P. (1990)J. Immunol. 144,2781-2788 30 Link, H. et al. (1990)Am.J. Trap. Med. Hyg. 43,452-463 31 Nussenzweig,R.S. et al. (1972) Exp. P arasitol. 31,88-97 32 Schofield,L. etal. (1987) Nature240, 663-666 33 Weiss,W.R. et al. (1988) Proc. NatI Acad. Sci. USA 85,573-575 34 Burkot, T.R. etal. (1989)J. Clin. Microbial. 27,1346-1351 35 Petersen, E. et al. (1990)Trans. R. Sac. Trap. M ed. Hyg. 84, 339-345 36 Webster, K. etal. (1988)J. Clin. Immunol. 8,342-348 37 Good, M.F. etal. (1987)Science 235, 1039-1062 38 Good, M.F. et al. (1988)J. lmmunol. 140,1645-1650 39 Sirfigaglia, F. et al. (1988 ) Nature 336, 778-7 80 40 Hoffman, S. et al. (1987)Exp. P arasitol. 64, 64-70 41 Nardin, E. etal. (1988)Eur.J. Immunol. 17, 1763-1767 42 Cambier, J.C. (1986) B-lymphocyte Differentiation, CRCPress 43 Coggeshall, K.M. and Cambier, J.C. (1984) J. Immunol. 133, 3382-3386 44 Tsien, R.Y. etal. (1982)J. Cell. Biol. 94, 325-334 45 Defranco, A.L. etal. (1982)J. Exp. Med. 155,1523-1536 46 Howard, M. and Paul, W.E. (1983)Annu.Rev. Immunol. 1,307-334 47 Haldar, K. et al. (1983)J. Biol. Chem. 260, 4969-4974 48 Smythe, J.A. et al. (1988) Proc. NatI Acad. Sci. USA 85, 5195-5199 49 Smythe, J.A. et al. (1990 ) Mol. Biochem. P arasitol. 39,227-234
The Evolutionary Origin of Glycosomes P,A.M. Michelsand F.R.Opperdoes The glycolytic pathway of the Kinetoplastida is organized in a unique manner: the majority of its enzymes are contained in organelles called glycosomes. In this article Paul Michels and Fred Opperdoes argue that the glycosomes are equivalent to the microbodies and peroxisomes identified in other eukaryotic cells. They explore the possible evolutionary origin of the glycosome by comparing many cf its structural and functional properties with those of other members of the microbody family and with some .features of other organeIles, the mitochondria and chloroplasts, which have been studied in much more detail. In all Kinetoplastida examined, the major part of the glycolytic pathway has been located in an organelle called the glycosome. This is the case in representatives from the two major evolutionary divisions of this order, the Trypanosomatina (Trypanosoma, Leishmania, Crithidia, Phytomonas) and the Bodonina (Trypanoplasma) 1-3. Such a compartmentalization of the glycolytic pathway is unique: in all other eukaryotes studied, glycolytic activity is only found in the cytosol. The glycosome has many lea-
Paul Michels and Fred Opperdoes are at the International Institute of Cellular and Molecular Pathology, Research Unit for Tropical Diseases,Avenue Hippocrate 74, B- 1200 Brussels, Belgium. ~) 199I, ElsevierSciencePublishersLtd, (t3K)01694707191/$02.00
tures that have led to its classification in the microbody family, to which peroxisomes and glyoxysomes also belong. The glycosome's morphology is reminiscent of that of most other microbodies, being a small organelle with an electron-dense matrix, surrounded by a single phospholipid bilayer membrane (Fig. 1 and Refs 4,5). Moreover, glycosomes contain some enzymes that are usually associated with microbodies and peroxisomes, such as those involved in [3-oxidation and ether-lipid biosynthesis 3'6. The typical peroxisomal enzyme, catalase, is not detectable in most representatives of the Kinetoplastida. However, when it is present, such as in C. luciliae, Phytomonas sp. and Trypanoplasma borelli, it is located in the glycosome. Another feature that the glycosome has in common with other microbodies is its route of biosynthesis. Its proteins are encoded on nuclear chromosomes, synthesized on free ribosomes in the cytosol and imported posttranslationally into the organelle without any detectable form of processing 7-9. The function of microbodies in the cell It is difficult to define a specific function common to all types of microbodies: the hallmark of peroxisomes is peroxide metabolismS; yeast microbodies mainly harbour key enzymes required for metabolism of the carbon and/or nitrogen source present in
with follow-up treatments. It is estimated that 155 million Africans harbour Ascaris and the infection is often associated with severe morbidity and a variety of complications L°. The large-scale regular distribution of ivermectin ~br onchocerciasis control will thus provide a unique opportunity, at no extra cost, to remove many of the intestinal worms from millions of Africans and keep them clear for a decade or more. Thus, the project has great potential in aiding the introduction and acceptance of PHC and other health programmes. The control of onchocerciasis in Nigeria would greatly reduce the global prevalence of this infection. However, the attainment of this goal will require the optimal application of existing resources with absolute dedication,
99
strong political will and generous international support. The adverse public health and socioeconomic consequences of onchocerciasis make it a disease high on the W H O list for research and control. Now that impressive commitment and progress towards its ultimate control have already been made as a result of the larviciding successes in the OCP area, the free donation of ivermectin, and the continuing efforts to develop an ideal macrofilaricide and/or an effective vaccine, the launching of a globally coordinated initiative to control onchocerciasis in all endemic areas may shortly become feasible. References
I WHO Technical Report SeriesNo. 752 (I 987)
Third Report of the WHO Expert Committee on
OnchocercJasis 2 Crosskey, R.W. (I 981 ) Tropenmed. Parasitol,
32,2-16 3 Aziz, M.A. (1986) Parasitology Today 2, 233-235 4 Cupp, E.W. et al. (I 986)Science23 I, 740-742 5 Duke, B.O.L. (1990) Parasitology Today 6, 82-84 6 Trpis, M. et al. (1990) Am.J. Trap.Med. Hyg. 42, J4-156
7 Pond,B. (1990) Lancet 335, 1539 8 Naquira, C. et al. (1989) Am.J. Trap. Med. Hyg. 40, 304-309 9 Freedman, D.O. et al. (1989)J. Infect. Dis. 159, 1151-1153 10 Crompton, D.W.T. and Tulley, JJ. (1987) Para-
sitologyToday 3, 123-127
Luke Edungbola is at the Faculty of Health Sciences, University o[ llorin, PMB 1515, Ilorin, Kwara State, Nigeria.
On The Function of Repetitive Domains in Protein Antigens of Plasmodium and Other Eukaryotic Parasites L. Schofield Highly reiterated repetitive domains occur within the protein antigens of many parasitic taxa, including Plasmodium, Trypanosoma, Leishmania and Toxoplasma. In malaria it has been proposed that repeat regions may function as ligands for host proteins, or serve to suppress the development of immunity through a strategy of serological' crossreactivity. In this article Louis Schofield presents a novel hypothesis, based on empirical evidence, that repetitive domains in antigens do not elicit protective immune responses and instead have evolved as a mechanism of immune evasion by their ability to induce thymus-independent B-cell activation. It is also proposed that this unusual response is associated with several forms of immunosuppression. The hypothesis has the added attraction of helping to explain several distinctive features of the molecular biology, evolution and immunology of repetitive regions in protein antigens of parasites. Tandemly repeated regions within proteins are not unique to eukaryotic parasites but the frequency and magnitude of the phenomenon in these taxa suggest they represent a special case. In most other organisms repetitiveness ,within proteins serves a clear
Louis Schofield is at the National Institute for Medical Research, The Ridgeway, Mill Hill, London N W 7 I AA, UK. ~) 199I, E~sevierSciencePublishersLtd,(UK)0169J~707/91/$02.00
physico-chemical or structural function, as seen in the case of collagen or glue proteins 1. However, in parasites, and particularly in Plasmodium, repeats are widespread among proteins from different developmental stages, with various types of cellular organizations and apparently divergent functions. Such proteins include soluble antigens released during schizogony (S-antigens), proteins associated with the infected red blood cell (RESA), rhoptry organelle proteins and surface antigens of sporozoites and merozoites. Even a phylogenetically conserved heatshock protein shows unique repeat elements2. The repetitive unit, of a variety of different sizes, can be reiterated from once to more than 50 times. The repeat structure is quite distinct among different proteins and, furthermore, allelic variants of a protein, or phylogenetically related homologues, often show completely unique repeats within otherwise conserved protein sequences. In addition, more than one type of repeat can occur within some proteins. For reasons of space I will not examine in depth the variety and structural features of repeats but refer the reader to the primary literature 3-9. The overall picture is one of great tolerance by the parasite for diverse repetitive structures of different shapes and sizes inserted promiscuously into a wide range of proteins with a variety of functions. Despite the great diversity in size, number and distribution of repetitive elements, many distinctive
I O0
features are held in common. First is the property of repetitiveness itself. Second, there appears to be a noticeable bias in the component amino acids 10, with residues such as N, A, D, V, P, E, G, Q and S highly represented but C, M, F, W, Y, I and L rarely present. The third distinctive feature of these regions is their immunodominance. For example, antibody to sporozoites appears to be directed almost entirely towards the repetitive domain of the circumsporozoite (CS) protein. When sera from individuals naturally or experimentally exposed to sporozoites is absorbed with this repeat peptide, reactivity to whole parasites is lost, indicating that this is the single immunodominant epitope recognized by naturally induced antibody 11. Conversely, antibody to adjacent nonrepeat epitopes on the CS and other repeat-containing proteins is much less prevalent. Similar types of repeat immunodominance are exhibited by S-antigens and RESA 12. A fourth and final property of many of these regions is their unusual genetics and evolutionary history. Based on an analysis of the evolutionarily conserved CS protein family, Enea and colleagues 13-18 have convincingly shown that the evolution and maintenance of the repeat structure must be through a mechanism operating at the DNA level, similar to that described for satellite DNA. Simple phenotypic selection acting on point mutations could not simultaneously generate highfidelity repeat structures within a protein and yet cause a massive shift in sequence in allelic variants. In contrast, the postulated genetic mechanism could easily cause a DNA sequence to be reiterated and spread, and at the same time result in the divergence of the repeated unit a c r o s s t a x a 14. Strong support for this view comes from a study of the unrelated Santigen family 19, in which a simple frameshift in the decoding of a conserved repetitive DNA sequence results in a phenotypically distinct allelic variant with structurally and serologically distinct tandem repeats. Thus it is the structure of the DNA, rather than the protein, that is conserved in both gene families. One mechanism by which this may occur is through biased gene conversion 17. As well as proposing a function for repeats, therefore, a satisfactory theory of their biology must provide answers for the following questions: (1) why are these regions repetitive? (2) why are repeats found in so many different types of protein? (3) why is amino acid choice biased in repeats? (4) why are most, but not all, of these regions immunodominant? (5) how is it possible to have completely different repeat structures within phylogenetic homologues and allelic variants of the same protein (that have the same function)? and (6) can the postulated function of the repeats be reconciled with their highly unusual genetics and evolution? Clearly, the repeats constitute a related family of structures and we should avoid constructing different theories to account for different repeats and their various properties if they can be dealt with satisfactorily under one unifying explanation. In other
Parasitology Today, vol. 7, no. 5, 199 /
words, epistemological criteria demand that a theory of repeats should be not only simple and falsifiable but also comprehensive.
Existing theories To date, two theories of the function of repeats have been forwarded. In the first, each repeat unit is proposed to act as a ligand for host structures such as red blood cell receptors and hepatocytes 2°. In this view, the binding units are repeated because this allows a multimeric high-avidity interaction between parasite and receptor. The high fidelity of the repeats is thought to reflect the constraints imposed by purifying selection to ensure binding. While providing a plausible answer to the first two questions, this postulate has several major weaknesses: it does not explain why choice of amino acids should be constrained for so many different types of host-parasite interaction (question 3); it does not explain, and is at odds with, the immunodominance of the repeats (question 4); it is impossible to reconcile a repetitive unit constrained in structure by a requirement for interactive ligand properties, with the fact that radically different repeats may occupy the same region of allelic variants of a protein that has the same function (question 5); similarly, the molecular evolution of these domains cannot be explained by reference to purifying selection and despite considerable research effort over many years no convincing evidence has surfaced to suggest that the repeated regions have any ligand properties whatsoever. In the second theory, the repeats are proposed to enable the parasite to evade immunity by presenting to the host an extensive network of crossreactive epitopes that abort the affinity maturation of the response to 'protective' epitopes 21. This proposition has several attractive features: it can provide an explanation of the immunodominance of repeat epitopes; amino acid choice can be said to be biased to favour crossreactions and there is clear evidence that a number of repeat domains are serologically crossreactive with each other (but no evidence as yet that the presence in the host of several domains leads to a reduction in antibody affinity). However, I suggest that the proposal is incomplete for five reasons. First, it does not in any way explain why these regions are repetitive. Why would epitopes designed to be crossreactive with 'protective' domains require this tandem structure? Second, swamping the B-cell repertoire with crossreactive analogues is a 'global' or trans-acting strategy, which fails to explain why repeats are maintained in any specific example of a protein. The postulate would simply require that a large mass of crossreactive epitopes be released freely into the circulation to compete for B cells. Third, and more important, there is a tautologous element to the proposal. The crossreactivity actually described by Anders is among different repeat structures 2~. If 'protective' epitopes lie within the repeats, then more questions are raised than answered, as it fails to explain why
Parasitology Today, vol. 7, no. 5. 1991
I 01
Table I. Antigen-specific T cells are not required for secondary responses to the CS protein of sporozoites a
T cells
B cells
Challenge
T i t r e versus C S
T i t r e versus T T
T (naive)
B (naive)
SPZ "IT
64 ND b
128 ND
T (naive)
B (SPZ + l'T)
SPZ TT
4096 ND
32 512
T (SPZ + 3-1-)
B (SPZ + l-F)
SPZ 1-F
8192 32
64 65536
~Donor A/J mine were primed separately to Tetanus toxoid (TT) or P. berghei sporozoites (SPZ). Spleen cells from each group were then pooled in equal numbers before purification of T cells (by removal of adherent and slg+ cells) and B cells (by removal of adherent and Thy I + cells). Following T-cell transfer, light irradiation and B-cell tran:,Yer,the syngeneic recipients were boosted separately with either SPZ or 3-1"and the serological titre to TT and NANP3 determined after eight days, by solid phase radioimmunoassay(adapted from Ref. 29). b Not determined.
repeats could evolve with this property. In other words, the repeats are proposed to have evolved to crossreact with each other to avoid immunity directed to the repeats. We may avoid this dilemma only by adopting two auxiliary hypotheses, namely that undefined protective epitopes are indeed outside the repeats, and their crossreactivity with repeats exists but has yet to be shown. However, these necessary assumptions threaten the empirical basis of the postulate, for now the inter-repeat crossreactivity can only be of second2a'y relevance. Fourth, a considerable degree of repeat serological crossreactivity is observed across malarial taxa, which parasitize different hosts 22'23. Thus 'co-ordinate' immune evasion cannot account for the phenomenon. Finally, and most importantly, the theory is in conflict with what we know about the molecular evolution of repetitive elements. This is because it requires that crossreactive repeats and 'protective' epitopes must necessarily evolve in reference to each other, constrained by the need to be structurally related (to crossreact), with phenotypic selection responsible for various appropriate amino acid substitutions. However, this is incompatible with the molecular evolution of repetitive domains, which proceeds by quantum leaps 15, with no mechanism for concerted evolution and little evidence for adaptive amino acid substitutions. A novel hypothesis In contrast to the foregoing propositions, I would like to offer a novel theory of repeat biology. It is well known that the repetitiveness of a wide variety of agents causes T-cell-independent activation of B cells by crosslinking hapten-specific surface immunoglobulin (Ig)24. Examples are repetitive structures such as polyacrylamide, bacterial carbohydrates, (Fab)2 anti-sIg and repetitive proteins such as collagen and flagellin2s-27. Many infectious agents, in particular the gram-negative bacteria, favour the strategy of presenting repetitive T-cellindependent antigens on their surface. Following this line of thought we recently sought to determine whether repetitive antigens of malaria might also induce such a response. This proposal was strongly at odds with the conventional wisdom that held the antibody response to malaria repeated proteins to be
under Ir gene control28 and therefore the opposite of what we proposed. However, the conventional view is based entirely on the immunological behaviour of various vaccine constructs and not of the parasite itself. Therefore, to test the postulate, we undertook an investigation into the regulation of the antibody response to the repetitive domain of the CS protein on intact sporozoites29, and observed that the parasites do indeed induce a thymus-independent B-cell response to the repeats (Table 1). In other words, the sporozoite CS protein induces activation of B cells leading to production of antibody to the repeats without a requirement for MHC-restricted cognate interaction with CS-specific helper T cells. This phenomenon is antigen-specific because the sporozoites do not induce antibody secretion from bystander B cells specific for Tetanus toxoid (Table 1). Mice hyperimmunized with sporozoites have high levels of anti-repeat antibodies and, according to conventional wisdom, their T cells must be able to recognize the CS protein. When such T cells are made available to the B cells in a manner allowing the expression of MHC-restricted helper activity, there is little apparent booster effect (Table 1). In contrast to the behaviour of sporozoites, the response to the antigen Tetanus toxoid exhibited classical T-celldependence under identical experimental conditions. In further experiments, T cells derived from priming with CS-based vaccine constructs, and known to be reactive with CS sequences, were shown under these conditions to have little ability to influence the T-cell-independent response to parasites (Table 2). It is important to appreciate that these experimental conditions are designed to allow the expression of helper activity and therefore provide a critical falsifying test of the T-cell epitope-Ir genecontrol theory of the anti-CS response. A subsequent study has similarly provided evidence that immunization with sporozoites does not prime T cells to CS epitopes3°. Thus, based upon this empirical demonstration of a T-cell-independent response to the CS protein repeats, we propose the following: (I) It is already well established that tandemly repeated structures crosslink surface immunoglobulin
Parasitology Today, vol. 7, no. 5, 1991
102
Table 2. Failure of CS-specific T cells t o boost the antibody response t o sporozoites a T cells
B cells
Challenge
Titre versus NANP4o
T (FCA)
NANP40
SPZ CS.T3 + NANP3
32768 512
T (CS.T3)
N A N P40
SPZ CS.T3 + NANP3
32768 131072
aBalb/c mice were immunized twice with 50 ug of the peptide DIEKKIAKMEKASSVFNVVNS, corresponding to amino acids 378-398 of the CS protein, a conserved peptide T-cell site (CS.T3) widely recognized by murine and human MHC 39, in Freunds' adjuvant (FCA). Control mice received adjuvant alone. After light irradiation they received purified B cells from donors primed to the NANP40 sequence conjugated to bovine serum albumen (BSA) and were boosted with P. falciparum sporozoites (SPZ) or a peptide construct of CS.T3 conjugated to the B epitope NANP3. Sera were diluted twofold in phosphate buffer saline-BSA and titres were determined by radioimmunoassay using NANP4o-BSA assolid phaseantigen.
on B cells, providing the cells with a thymusindependent activation signal that is not given by single or nonrepetitive epitopes 25. Thus we may propose that repeat domains are repetitive (question 1) simply to provide this activation signal to B cells, as the first step of a strategy to force them down an inappropriate (T-cell-independent) differentiation pathway. (2) A thymus-independent response is generally considered to be inferior to a T-cell-dependent response, lacking in affinity maturation and adequate T-cell and B-cell memory formation. Thus the response induced by parasites will be low grade and non-neutralizing. (3) While inducing low-grade non-neutralizing antibody, the repeats can be immunodominant, and will suppress the formation of antibody to important adjacent areas on the same molecule, ie. epitopic suppression (question 4). In other words, this form of immunosuppression is cis-acting and therefore requires a repeat domain to exist within each protein participating in the strategy (question 2). (4) The thymus-independent response is likely to be associated with lesions in T-cell priming or T-cell-B-cell cooperation, which may be an additional form ofimmunosuppression, and is consistent with our own and other observations of low anti-CS priminffz9'3°. (5) The repeats will be constrained to amino acids that allow the domains to be efficient B-cell epitopes, favouring hydrophilicity and an appropriate conformation (question 3). (6) The precise structure of repetitiveness within the domain is relatively unimportant, provided it is an effective B-cell epitope and can contribute to the induction of T-cell-independent B-cell activation. Thus, in addition to allowing a wide diversity of repetitive structures, the responsible genetic mechanism can with impunity insert very different repeats into the same location within allelic variants of a protein (questions 5 and 6). The frequency of such
equally fit alleles within the population is therefore not the result of immune selection but of random drift 18. The same genetic mechanism may also create unconstrained and selectively neutral repeats in a variety of different proteins.
Objections to the hypothesis How can these regions be a mechanism of immune evasion when they are the target of protective immunity to the parasite? The view that the repeat domains of malaria proteins are the target of protective immunity, derived from the need to rationalize vaccine development, has led to a major paradox: why should the parasite display on its surface highly immunogenic antigens that induce protective immunity? The answer to this dilemma comes from understanding that, despite the desire to render repetitive regions into vaccines, a considerable body of evidence has for many years shown that antibody to these regions induced by natural or experimental exposure to parasites is in fact nonprotective. Sera with very high levels of antibody to the CS repeats from mice immune to sporozoites cannot transfer any protection to recipients 31. Even in hyperimmune animals, sporozoites escape the antibody and gain access to the liver where they may be destroyed by cellular mechanisms 32'33. In general, anti-repeat antibody shows inhibitory activity only in artificial experimental systems where parameters can be modified to promote the desired observation, for example by the use of unrealistically high levels of monoclonal antibodies, culture systems modified, in vitro, prolonged incubation periods, or using selected hosts with genetically low levels of susceptibility to infection. Most importantly, field studies show that the highest levels of naturally acquired antibody to the CS repeat is not associated with reduced risk of infection 34, which is also the case for antibody to other repetitive antigens such as RESA 35. I f repetitive antigens of parasites are T-cellindependent, how are they able to induce IgG? It is a common fallacy to assume that T-cell-independent antigens induce only IgM and T-cell-dependent antigens induce IgG, and that this can distinguish between the two. This is not always the case, as shown by the fact that much naturally occurring Ig to bacterial T-cell-independent antigens is of the IgG isotype. This is because infectious agents induce high levels of trans-acting factors that cause classswitch in B cells activated by T-cell-independent antigens. In other words, MHC-unrestricted (factordriven, noncognate) help may be responsible for the emergence of IgG to T-cell-independent malarial antigens. In fact there is evidence that this occurs in response to sporozoites 29, and may well account for IgG formation to the CS protein. The natural human antibody response to the CS protein following exposure to sporozoites also has the characteristics of an anti-carbohydrate response 36. How can the response to sporozoites be thymusindependent when much recent work has demonstrated
Parasitology Today, vol. 7, no. 5, 199 /
T-cell sites on the CS protein? The answer to this is that the work in question was carried out with synthetic peptides and recombinant protein analogues of repetitive proteins and not with the parasite itself 37-39. It should be appreciated that this is a highly artificial approach representing a type of minimalist condition; when presented with synthetic peptides or simple soluble proteins, the immune system is constrained to only one of two possible outcomes, ie. responsiveness leading to a full IgG response (requiring/viHC-restricted cognate interactions between the recognizing B cell and an antigen-specific helper T cell) or nonresponsiveness (when these conditions are not met). This accounts for the phenomenon of MHC restriction. In contrast, the immune response to an infectious agent is of a higher level of complexity and involves MHCunrestricted effects. It is therefore no surprise that previous studies have shown several synthetic peptides based on the CS protein to be T-cell epitopes, since being a peptide T-cell site is merely the trivial consequence of being non-self and able to associate with one or other of the experimentally available MHC. The only important question, which is whether any of these regions function as helper T-cell sites in the context of anti-parasite responses, has unfortunately never been demonstrated or even clearly addressed by any of these studies. It is of course possible to show that sporozoites can boost antibodies to the repeats in mice primed with peptide constructs 4°'41 but these studies fail to address whether this is due to the action of helper T-cell sites or the T-cell-independent activation of the repeatspecific B cells by native sporozoites. Thus the conclusions concerning the MHC restriction of the antibody response to sporozoites are extrapolations from the simplified immunological behaviour of synthetic replicas, but the response to the parasite is unlike that to a nominal peptide antigen (for review, see Ref. 29). I f repetitiveness pnrvides an activation signal to B cells, why are repetitive synthetic peptides and recombinant proteins not T-cell-independent antigens? The activation and differentiation of B cells leading to antibody secretion can occur through several pathways, each involving different signals. Typically, three signals are required to initiate a T-cell-dependent developmental pathway: (1) binding of (monomeric) antigen to surface Ig; (2) recognition of B-cell Ia (MHC) molecules by histocompatible, antigen-specific T-cell receptors (TcR) followed by (3) T-cell growth and differentiation factors 42. Clearly this sequence corresponds to events in T-cell-B-cell cooperation. In contrast, crosslinking of surface Ig by repetitive epitopes leads to the induction of phosphatydylinositol turnover 43, Ca 2+ flux44, B-cell cycle entry (Go--G1 progression)45 and the acquisition of responsiveness to soluble lymphokines (factor-driven help)46. However, in the absence of further signals of this type, crosslinking, while providing an initial state of T-I activation
103
distinct from that induced by normal ag-Ig binding, is not sufficient to drive the B cell fully to antibody secretion. Thus repetitive recombinant proteins will crosslink slg but this is insufficient for full expression of antibody formation which, in the absence of further appropriate signals, may proceed through MHC-restricted events. By way of a corollary we must invoke an additional or secondary signal of parasite or host origin to fully account for our observation of T-cell-independent antibody formarion to the repetitive antigens of parasites. Possible candidates for this signal could be host factors induced by infection, eg. interleukins 4 and 5, or a covalently bound lipid such as the sn-l,2diacylglycerol moiety present within the glycosylphosphatidylinositol complex of several parasite surface antigens47'4s. Many of the other repetitive T-I antigens on the surface of infectious agents provide more than one such signal, eg. lipopolysaccharide and the E. coli surface lipoprotein. At high concentration, potent T-cell-independent antigens such as lipoproteins or glycolipid mitogens gain access to B cells in an antigen-independent manner through the cell membrane, activating cellular growth and differentiation pathways internally and causing polyclonal Ig secretion. However, at lower concentrations they gain access to B cells by interaction with antigen-specific slg. I f immunodominance and crosslinking of surface lg is important, how do we explain the existence of nonimmunodominant repeats, minor repeats, variant repeats and unconserved or degenerate repeats? Based on the ideas of Enea x~'~4, repetitive DNA is introduced promiscuously into coding regions, where we propose it can acquire fitness by inducing a T-I response. Naturally, selection will reject maladaptive mutations. However, the organism will also tolerate selectively neutral intrusions of this type, eg. minor repeats and those reiterated only once or twice, and these will tend also to be less immunodominant, eg. the small repeats in PMMSA. Indeed, most of the repeats in P. falciparum antigens may be 'neutral', as shown by their allelic diversity, eg. both PMMSA and MSA-2 (Ref. 49), demonstrating on one hand that their presence is not lethally disruptive of protein function and on the other that their absence is not prejudicial to parasite survival. The presence in Plasmodium of unique repeats in the otherwise phylogenerically conserved heat-shock protein 2 is further evidence that the genetic mechanism inserts repeats wholesale, without reference to protein function, to be acted upon by selection or allowed to drift. Even large immunodominant repeat structures may also in time become selectively less constrained and then drift, tolerating a number of amino acid replacements over evolutionary time because these substitutions do not disrupt the capacity of the molecule to crosslink slg, eg. degenerate repeats in FIRA. In these cases (drifting or unconstrained repeats) we would expect a certain allelic heterogeneity among isolates. However, we would
104
not expect to see major high-fidelity repeats that are not immunodominant. Similarly, a protein may tolerate the emergence of variant but related dimorphic repeats such as in RESA and several CS proteins, the divergence of these regions being constrained by the need to provide crosslinking of surface Ig when focused on the B cell (although here lower-affinity interactions are tolerated). Thus the serological crossreactivity in cis noted by Anders 21 would be a consequence of these constraints. How do we explain the serological crossreactivity of repeat antigens? In Anders' theory, crossreactivity is seen within repeats in a given protein and among repeats from different proteins of one or more developmental stage. The repeats have evolved to collaborate in trans in aborting antibody affinity maturation to putative protective epitopes that may be within or external to the repeats themselves 21. In our view, repeats are 'designer' B-cell epitopes that have evolved both to interact with B cells at much higher efficiency than adjacent nonrepeat epitopes (thereby inducing epitopic suppression in cis) and to crosslink the surface Ig of the recognizing B cells. As noted above, crossreactions between two or more repeat domains within a protein (which are usually closely related) simply reflect the constraints on divergent repeat sequences to co-operate in cis to promote crosslinking on the B-cell surface. Crossreactivity among domains of different proteins may arise as the result of several factors. First, repeat sequences may have ancestral relationships among and within species and would therefore be structurally related. Thus the CS proteins ofP. falciparum and P. berghei share NANP sequences and are serologically crossreactive. However, these parasites exist in different hosts. Crossreactivity between RESA and other antigens is cited as being central to the postulate of crossreactive immune evasion21. However, two studies show that this antigen also crossreacts with P. chabaudi, P. cynomolgi and P. vivax 22"23. It is clearly impossible in these major examples, from taxa separated far in evolutionary time, to account for serological relatedness by invokhag joint immune evasion or co-ordinated sequence evolution. Therefore, the crossreactivity among P. falciparum proteins such as Pfl 1.1, RESA, Ag 332 and FIRA, may simply reflect an ancestral relationship among their EEVV, EENV, EE-V and EPV repeat sequences, respectively, and may not indicate a shared interactive function. Second, there is considerable evidence that the germ-line repertoire of IgV genes is biased towards the recognition of the repetitive carbohydrate antigens of bacteria, and repetitive antigens of parasites may exploit this to react with a large number of somatically unmutated B-cell lineages, ie. the most fit sequences are those with a certain bias in amino acid choice. This, together with the known degeneracy of the antibody repertoire, could lead to extensive crossreactivity. The question therefore is whether crossreactions cause failure of affinity maturation, and whether the
Parasitology Today, vol. 7, no. 5, 1991
repetitive domains have evolved to promote this end, or if crossreactivity is a side consequence of other strategies and constraints. As in any area of biology, major features such as the repeat regions will generate a number of phenotypic consequences (which may act as selective constraints on the evolution of the system). This does not mean that there are distinct functions associated with each phenomenon, and we should avoid needlessly multiplying separate hypotheses to account for all observations. Nonetheless, until put to further experimental test, we would accept the proposal of Anders that crossreactivity among the repeats themselves (but not with 'protective' epitopes) results in the expansion of the repeatreactive repertoire 21. Conclusions Optimal regulation of the humoral immune response requires processing of antigen, via catabolic proteases, that allows antigen presentation in the context of MHC class II to the helper T-cell-effector arm by B cells. Infectious bacteria evade this proteolytic processing by exposing polymeric carbohydrates on their surface. However, constraints on the synthetic enzymes involved limit the serotypic diversity available to any one strain. Unable to process these antigens, the host adaptive response has been to respond to polymerism per se (via crosslinking of Ig on recognizing B cells). However, the resulting (T-cell-independent) response is considerably less efficient than the MHC-restricted variety. We propose that eukaryotic parasites, especially malaria, have evolved a mechanism to exploit this phenomenon to the full. As proposed by Enea, Arnot and colleagues 14'15'19, an unusual genetic recombinational process inserts repetitive DNA into genes encoding proteins (not involving transposable elements). This mechanism acts without reference to existing protein function, with lethal, neutral or adaptive outcome. While no doubt generating some lethal mutations, the mechanism overall has been selected because, in contrast to the slow pace of selection acting on point mutations, it is a most efficient method for obtaining repetitiveness in a variety of antigens. The repeats thus generated fall into one of two classes. A certain proportion (perhaps the majority) are structurally unconstrained, nonimmunodominant and selectively neutral (without function), but many also acquire fitness by acting as 'designer' B-cell epitopes. As potent epitopes they serve to focus important molecules onto antigenspecific B cells, and in the process suppress antibody to adjacent regions (epitopic suppression). Crosslinking of the surface Ig by the tandem array of repeated epitopes is not sufficient for a full T-cellindependent response, but is simply the first step in a sequence of signals that sends the B cells on an abortive or inappropriate differentiation pathway. This may lead to failures in affinity maturation, memory formation and T-cell activation or cooperation. The immune response to the repetitive
Parasitology Today, vol. 7, no. 5, 199 I
105
antigens of malaria is therefore closer to the antibacterial rather than Ir gene controlled type. References
1 2 3 4 5 6 7 8 9
Garfmkel,M.D. et al. (1983)J. Mol. Biol. 168,765-789 Yang,Y.F. etal. (1987)Mol. Biochem. Parasitol. 26, 61-67 Dame,J.B. etal. (1984)S~.'nce225, 593-599 Cowman,A.F. et al. (1985) Cell 40,775-783 Favaloro, M.J. et al. (1986)NucleicAcidsRes.14, 8265-8277 Stahl, H.D. et al. (1987)Mol.Biol. Med. 4, 199-211 Coppel, R. et al. (1986) Mol. Biochem. Parasitol. 20,265-277 Weber, J.L. etal. (1988)J. Biol. Chem. 263, 11421-11425 Wellems, T.E. and Howard, R.J. (1986)Proe. NatlAcad. Sci. USA 83, 6065-6069 10 Nussenzweig,V. (1986) Am.J. Trap. Med. Hyg. 35,678-688 11 Zavala, F. etal. (1985)Science228, 1436-1438 12 Perlmann, P. et al. (1986) in Vaccines 86 (Lerner, R.A., Chanock, R.M. and Brown, F., eds), pp 86-91, Cold Spring Harbor 13 Enea, V. etal. (1986)J. Mol. Biol. 188,721-726 14 Galinski,M. etal. (1987)Cell48, 311-319 15 Di Giovanni,L. et al. (1990) Exp. Parasitol. 70, 373-381 16 Colomer-Gould,V. and Enea, V. (1990) Mol. Bioehem. Parasitol. 43, 51-58 17 Arnot, D. etal. (1988) Proc. NatIAcad. Sci. USA 85, 8102-8106 18 Arnot, D. Acta Leiden. (in press) 19 Saint, R.B.etal. (1987)Mo.1.CelIBiol. 7,2968-2973 20 Nussenzweig, V. and Nussenzweig, R. (1989) Adv. Immunol. 45, 283-334
21 Anders, R.F. (1986) Parasit. Immunol. 8, 529-539 22 Holmquist, G. etal. (1988) Infect. Immun. 56, 1545-1550 23 Wandiworanun, C. et al. (1989) Am. J. Trap. Med. Hyg. 40, 579-584 24 Mosier, D. and Subbarao, B. (1982)Immunol. Today 3, 217-219 25 Dintzis, I.M. etal. (1976)Proc. NatlAcad. Sci. USA 73, 3671-3675 26 Feldmann, M. (1972)J. Exp. Med. 135,735-753 27 Moiler, G. (1980)Immunol.Rev. 52, 1-231 28 Good, M.F. et al. (1988) Immunol. Today 9,351-355 29 Schofield,L. and Uadia, P. (1990)J. Immunol. 144,2781-2788 30 Link, H. et al. (1990)Am.J. Trap. Med. Hyg. 43,452-463 31 Nussenzweig,R.S. et al. (1972) Exp. P arasitol. 31,88-97 32 Schofield,L. etal. (1987) Nature240, 663-666 33 Weiss,W.R. et al. (1988) Proc. NatI Acad. Sci. USA 85,573-575 34 Burkot, T.R. etal. (1989)J. Clin. Microbial. 27,1346-1351 35 Petersen, E. et al. (1990)Trans. R. Sac. Trap. M ed. Hyg. 84, 339-345 36 Webster, K. etal. (1988)J. Clin. Immunol. 8,342-348 37 Good, M.F. etal. (1987)Science 235, 1039-1062 38 Good, M.F. et al. (1988)J. lmmunol. 140,1645-1650 39 Sirfigaglia, F. et al. (1988 ) Nature 336, 778-7 80 40 Hoffman, S. et al. (1987)Exp. P arasitol. 64, 64-70 41 Nardin, E. etal. (1988)Eur.J. Immunol. 17, 1763-1767 42 Cambier, J.C. (1986) B-lymphocyte Differentiation, CRCPress 43 Coggeshall, K.M. and Cambier, J.C. (1984) J. Immunol. 133, 3382-3386 44 Tsien, R.Y. etal. (1982)J. Cell. Biol. 94, 325-334 45 Defranco, A.L. etal. (1982)J. Exp. Med. 155,1523-1536 46 Howard, M. and Paul, W.E. (1983)Annu.Rev. Immunol. 1,307-334 47 Haldar, K. et al. (1983)J. Biol. Chem. 260, 4969-4974 48 Smythe, J.A. et al. (1988) Proc. NatI Acad. Sci. USA 85, 5195-5199 49 Smythe, J.A. et al. (1990 ) Mol. Biochem. P arasitol. 39,227-234
The Evolutionary Origin of Glycosomes P,A.M. Michelsand F.R.Opperdoes The glycolytic pathway of the Kinetoplastida is organized in a unique manner: the majority of its enzymes are contained in organelles called glycosomes. In this article Paul Michels and Fred Opperdoes argue that the glycosomes are equivalent to the microbodies and peroxisomes identified in other eukaryotic cells. They explore the possible evolutionary origin of the glycosome by comparing many cf its structural and functional properties with those of other members of the microbody family and with some .features of other organeIles, the mitochondria and chloroplasts, which have been studied in much more detail. In all Kinetoplastida examined, the major part of the glycolytic pathway has been located in an organelle called the glycosome. This is the case in representatives from the two major evolutionary divisions of this order, the Trypanosomatina (Trypanosoma, Leishmania, Crithidia, Phytomonas) and the Bodonina (Trypanoplasma) 1-3. Such a compartmentalization of the glycolytic pathway is unique: in all other eukaryotes studied, glycolytic activity is only found in the cytosol. The glycosome has many lea-
Paul Michels and Fred Opperdoes are at the International Institute of Cellular and Molecular Pathology, Research Unit for Tropical Diseases,Avenue Hippocrate 74, B- 1200 Brussels, Belgium. ~) 199I, ElsevierSciencePublishersLtd, (t3K)01694707191/$02.00
tures that have led to its classification in the microbody family, to which peroxisomes and glyoxysomes also belong. The glycosome's morphology is reminiscent of that of most other microbodies, being a small organelle with an electron-dense matrix, surrounded by a single phospholipid bilayer membrane (Fig. 1 and Refs 4,5). Moreover, glycosomes contain some enzymes that are usually associated with microbodies and peroxisomes, such as those involved in [3-oxidation and ether-lipid biosynthesis 3'6. The typical peroxisomal enzyme, catalase, is not detectable in most representatives of the Kinetoplastida. However, when it is present, such as in C. luciliae, Phytomonas sp. and Trypanoplasma borelli, it is located in the glycosome. Another feature that the glycosome has in common with other microbodies is its route of biosynthesis. Its proteins are encoded on nuclear chromosomes, synthesized on free ribosomes in the cytosol and imported posttranslationally into the organelle without any detectable form of processing 7-9. The function of microbodies in the cell It is difficult to define a specific function common to all types of microbodies: the hallmark of peroxisomes is peroxide metabolismS; yeast microbodies mainly harbour key enzymes required for metabolism of the carbon and/or nitrogen source present in