- Email: [email protected]
Genetic control of the immune repertoire in nematode infections
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Parasitology Today, vol. 5, no. I O, 1989 62 Burnham, W.R., Reeve, R.R. and Finch, R. (1980) Gut21, 1097-1099 63 Sargeaunt, P.G., Williams, J.E. and Grene, J.D. (1978) Trans. R. Soc. Trop. Med. Hyg. 27, 519-521 64 Goldmeier,D. etal. (1986) Lancet 1,641-644 65 Nguyen-Dinh, P. et al. (1987) Third International Conference on AIDS (Abstr.)
66 Collins, W.E. et al. (1971) Trans. R. Soc. Trop. Med. Hyg. 65,43-58 67 Arnot, D. (1989) Parasitology Today 5, 138-142 68 Hiatt, R.A. et al. (1979)J. Infect. Dis. 139,659 69 Shapiro, S.Z. and Pearson, T.W. (1986)Parasite Antigem: Towards New Strategies for Vaccines (Pearson, T.W., ed.), p. 225, Marcel Dekker
Genetic Control of the Immune Repertoire in Nematode Infections M.W. Kennedy I
Wellcome Laboratories for Experimental Parasitology University of Glasgow Bearsden GlasgowG61 I QH, UK
Mammals vary considerably, both within and between species, in the way in which their innate and adaptive immune systems respond to infections. An understanding of the processes involved in such variability will not only contribute to explaining heterogeneity in susceptibility and pathology, but will also be relevant to vaccination. This will be particularly important for the new generation of vaccines that are likely to be composed of one or a few cloned or synthesized antigens. For helminth infections, this could have particular relevance to hypersensitivity responses. The adaptive immune response is fundamentally constrained by the genetic constitution of an individual, and the need to avoid reactivity to self. This will have important implications for the dynamic relationship between host defences and parasite evasion mechanisms at both physiological and evolutionary levels. In this review, Malcolm Kennedy examines the genetic control of the specificity of the immune response to nematode infections, and in particular, the role of the major histocompatibility complex. The adaptive immune system has the capacity to respond to an almost inconceivable array of antigens, including those that had never been encountered until the advent of synthetic chemistry. Despite this, there are strict limitations which mean that every individual probably has its own unique immunological repertoire. There are two principal constraints. First is the requirement for tolerance to an individual's own tissues, to harmless or beneficial organisms such as those occurring on the skin or in the alimentary tract, and to potentially antigenic materials inhaled or present in food. Second, an immune response to most antigens depends on the successful presentation of processed antigen to lymphocytes, and their subsequent activation. The cell surface proteins encoded by the major histocompatibility gene complex (MHC), originally discovered as a barrier to tissue transplantation, are at the heart of this process. The MHC comprises a set of closely linked genes in all species so far investigated. Its structure and function are best understood in humans and mice, in which the gene complexes are known as the HLA and H-2, respectively (see Box 1). The products of the class I region are involved
in direct cell-mediated killing of, for example, virus-infected cells. The class II region is primarily associated with antigen presentation and cooperation between T- and B-lymphocytes in the induction of an antibody response. It is thought that a response depends not only on whether an individual's MHC molecules have an affinity for given processed fragments of an antigen, but also on the manner in which the fragments are presented to T-cells 1-4. Both of these functions are regulated by class II-associated immune response (Ir) genes 5.
The advantage of polymorphism Since there are materials to which certain individuals cannot, or must not, respond, this leaves loop-holes in the immune repertoire that pathogens can exploit to their advantage. This could be achieved by mimicking host molecules, by the selection of parasite components whose processed fragments will not associate with MHC molecules on the surface of an antigen presenting cell, or by interfering with the antigen processing and presentation machinery of the ceU6. Thus, polymorphisms at loci critical to these processes could minimize the impact of pathogens that exploit such evasion strat© 1989, Elsevier Science Publishers Ltd. (UK) 01694707/89/$03.50
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B o x I. T h e M H C G e n e s of M i c e a n d H u m a n s
Simplified, comparative linkage maps of the MHC of mice (H-2) and humans (HLA). Both contain pseudogenes or genes whose functional significance is unknown, and these are excluded from the diagram. Genes for some of the components of the complement system, and some enzymes, are closely linked to, or embedded within, the HHC region, and these are also excluded. Class I HHC molecules (shaded boxes) appear on the surface of most nucleated cells, and ;ire found non-covalently associated with a small protein, 132microglobulin. Processed fragments of antigen, eg. from viruses in an infected cell, are thought to locate in the 'Bjorkman groove' of the class I molecules, and this complex constitutes the recognition unit for a specific T-cell receptor. Class II molecules (open boxes) are found as nonMouse Chromosome 17
H-2
I
K I---~
covalently linked heterodimers on the cell membrane. Class II molecules are thought to present processed antigen to T-cells in a groove distal to the membrane, similar to the 'Bjorkman groove' of class I molecules. Class II molecules have a more limited tissue distribution than those of class I, but appear constitutively on several tissues and many cells of the immune system, and can be induced in others under conditions of immunological or infection stress. In mice, Aa and AI3 chains preferentially associate, as do Ea and E~, although AJEI3 and AI~E~ associationsmight occur. In humans,there are more classII molecules, and more genes for 13-than s-chains, although preferential pairing probably occurs with the appropriate s-chain. 13-chainstend to be more polymorphic than cL-chains.
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egies. An individual within a polymorphic population would be at an advantage if a hypothetical pathogen is only successful in those individuals that bear a particular allele. This lowers the chance of being infected in the first place, and the pathogen's population might be constrained to below its threshold level7. The range of pathogens, and their antigenic shifts, drifts and intrinsic wtriabilities would maintain polymorphism at critical loci. The prediction is, therefore, that genetically homogeneous populations of animals would be highly susceptible to eradication by transmissible diseases, and this is supported by evidence from natural occurrences and human interference in wild populations 8. Polymorphism might, therefore, be advantageous, but there is likely to be a limit to the number of/VIHC proteins that an individual can advantageously express. This is because each addition to the repertoire would widen the range of antigens to which immunological tolerance will have to operate 9']°. There is also the probabil-
[~1~
B
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ity that a given allele might simultaneously confer resistance to one pathogen, but susceptibility to another. Parasites that can circumvent host polymorphisms would have a selective advantage, and the bonus of being a member of a polymorphic population also applies to them. This acts as a selective pressure to favour multiple alleles in parasite genes coding for molecules interacting with host tissues. That is another story (see Ref. 11).
Polymorphismsand pathogens The most polymorphic locus known in man is that coding for glucose 6phosphate dehydrogenase (G6PD), for which more than 300 alleles are known. That genetic variability at this locus is related to infection is suggested by the fact that some alleles are more frequent in malarious areas than can be accounted for by mutation alone. The obvious possibility is that certain alleles are associated with a degree of resistance to Plasmodium infection, as is the sickle cell trait. The human MHC does not have as
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Molecular
2
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7
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Fig. I. Kinetics of antigen recognition in Anisakis simplex infection, and restricted recogn~on in humans, ffZSl]-Iabelled parasite culture supernatant (ES) from third-stage larvae of A. simplex was immunoprecipitoted w/th serum from a normal rabbit (track 2) and from a rabbit 7, 2 I, 28, 35, 42, 58, 63 and 142 days after oral infection with A. simplex (tracks .3-10, rest~ectively). Track I I shows an immunoprecipitation with normal human serum, and track 12 with serum from an infected human. Note that the latter serum fails to precipitate some of the major components. Immunoprecipitotes were analysed on 5-25% gradient SDS-PAGE gels. Track I was loaded with the antJgen used for the immunoprec/p/totion. The relative molecular masses are given in kDa. (Seealso Ref. 25,)
Fig. 2. Heterogeneity of responsivenessof humans infected with Ascaris lumbricoides, ffzsI]-Iabelled ES material from lung-stage larvae of A. suum was immunoprecipitated with serum from infected people, and anal~ed by SDS-PAGF_The tracks are molecular mass markers (track M), ffZSl].labelled F.S material (track R), an immunoprecipitat~ with serum from an uninfected human (track N), and serum from infected people in Nigeria (numbered tracks). These data are courtesyof Eleanor M.Fraser, using sera donated by Celia Holland and David Crompton. This effect has also been observedin sera from infected people in India 31 and St Lucia (sera courtesyof Don Bundy).
many alleles per locus as G6PD, but recombination within the complex can provide a greater number of genotypes (or 'haplotypes'). There are 19, 39, 8, 17, 14, 3 and 6 recognized specificities at the HLA-A, B, C, D, DR, DP, DQ loci, respectively, and many are as yet uncharacterized (Box 1). A further important point is that certain pairs of alleles do not occur in ratios that can be predicted from their gene frequencies if it is assumed that recombination between them has reached equilibrium. This 'linkage disequilibrium' is presumably due to residual founder effects, recent population mixings, and/or certain combinations of alleles having a selective advantage or disadvantage with respect to infections. Another consideration is that certain alleles, or haplotypes, have a reproductive cost related to the immune system, such as autoimmunity, maternal/ foetal incompatibility, and hypersensitivity to certain environmental mat-
anticipated that MHC effects are unlikely to be seen. But, as we shall see, they do occur, from the level of immune control of infection as a whole to the specificity of antibody responses.
Nematodes Serum antibody responses are the most accessible manifestation of the immune repertoire, and can be used to follow the kinetics of responses to particular antigens as well as to estabfish limitations to an individual's immune response. For nematodes, the choice of antigens used in immunoassays has in the past decade moved away from worm homogenates to the materials that parasites express on their surfaces and/or release into culture supernatant in vitro; the latter are known as excretory/secretory materials (ES). Surface and secreted materials can have common components 14,15, and the choice of which to use usually depends on supply and ease ofhand!ing. ES material has extra e r i a l s 12,13. significance since it is known to contain a It is relatively easy to conceive of an asso- variety of biologically active materials, for ciation between MHC alleles and the example anti-clotting factors 16, proimmune control of relatively simple agents teinases 17-19, acetylcholinesterase20,21 , such as viruses. MHC-restricted responses superoxide dismutase 22, and lipases23, to a single component of a virus could have which are presumably relevant to parasite far-reaching consequences for the success nutrition, tissue penetration and immune of infection in an individual host. How- evasion. The logic behind the choice of ever, with large and antigenically complex these materials for experimentation is that parasites, such as nematodes, it might be they are the sites of direct interaction
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between host and parasite and as such, are accessible to immune, effector mechanisms. Internal components of parasites are not irrelevant to the immunobiology of infections, since they will elicit an Lmmune response during moulting and following the killing and degradation of parasites in tissues. Release of such antigens might trigger immunopathological manifestations of infection, for example the Mazzotti reaction in onchocerciasis, or Loeffler's syndrome in ascariasis. Nematode infections are renowned for such hypersensitivity responses, and it is vital to identify which parasite components will provoke adverse reactions in the context of infection, vaccination or chemotherapy. Another significance of internal antigens is that they could be used to provide informarion on whether parasites are being killed m vivo, either by the immune system or following drug treatment.
Restricted immune recognition of nematode antigens The simplest fo~a of restricted responses would be detected during the development of immunity. Strains of mice have been found to differ in the time after infection at which they mount a detectable antibody response to the surface antigens of Trichinella spiralis, allahough eventually all the surface (glyco)proteins are recognized 24. Sequential recognition of this kind has also been noted for ES materials, and an example of this is given in Fig. 1 for infection of rabbits with Anisakis simplex. The major part of the normal antibody response to this nematode appears to be focused on secreted antigens, rather than internal components 2s, but Anisakis can penetrate gastrointestinal tissue in humarls, and when this is modelled by parenteral infection of experimental animals, many more of the parasite's somatic materials become subject to an antibody response. This is presumably because worms that are normally confined to the gut lumen can be expelled without exposing the immune system to worm somatic antigens, in contrast to tissue infections in which parasites are kiUed and disintegrate in situ. The combination of kinetics and specificity would therefore provide an estimate of the age and the extent of certain infections. However, the usefulness of this would be limited by the ability of a given host to respond to the set of antigens with which it is presented by the parasite. This brings us back to genetic control of the
319
specificity of the immune response, what it means, how it can be interpreted, and how it arises. There are now several examples of restricted antibody specificity in both chronic and repeated nematode infections which possibly have a genetic basis. These include, Nippostrongylus brasiF~ensis in the rat z6, Heligmosomoides dubius27 and Trichuris muris in the mouse 2s, a s well as Necator americanus29, Trichinella spiralis3°, Ascaris lumbricoides31 (Fig. 2), and Brugia malayi32in humans. In the case of Brug/a infections, there appears to be sonte loose correlation between antibody recognition patterns and infection status. It remains to be seen, though, whether this is due to a link between disease and immune specificity, or whether both are controlled by the same or linked genetic loci. In other words, which is cause, and which is effect, or do the recognition patterns have any relevance at all? It has been argued that the analysis must be taken to the level of the individual antigenic determinants (epitopes) on parasite antigens in order to establish which factors reflect or determine the outcome of infection ~2. Experience with T- and B-cell responses to viral antigens 33-42 indicates that this would be a worthwhile strategy but increases our need for an understanding of genetic control of immune specificity. The phenomenon of restricted antigen recognition could also be valuable in identifying protective antigens. If a particular treatment rendered an individual immune to a pathogen, but only elicited a response Molecular
R
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Fig. 3. Heterogeneity of antigen recogn/t/on in vaccination and natural infection with the cattle lungworm Dicryocaulus viviparus. [IZSl]labelled ES material from adult D. viviparus (R) was immunoprec/pitoted with serum from cattle that had been vaccinated with the irradiated larval vaccine, 'Dictol" and then experimentally infected with normal larvae (N), or challenged with normal larvae (V), or experimentally infected with serum from infections with normal larvae that had been drug-abbreviated after i$ days (D) or from fmld cases of acute Iongworm disease (F). (Data from CBr/ttan et al., unpublished.)
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to some of the antigens given, it could be argued that these 'immune-selected' antigens should be considered as candidates for a vaccine. Anti-nematode vaccines are at present few in number and relatively crude, but the development of the next generation of vaccines by, for example, using recombinant virus vectors, will require the identification of one or a few crucial antigens. For instance, the vaccine against bovine lungworm, Dictyocaulus viviparus, is highly effective, but animals that have been vaccinated and then challenged with normal parasites respond heterogeneously (Fig. 3; C. Britton et al., unpublished). While this cannot directly identify protective antigens, the process of elimination would greatly simplify the search. It must not be forgotten that a protective antigen for one individual might not be so for another, and it has been argued that responses to certain specificities might even divert a host's response away from the successful control of infection 43. The immune control of experimental nematode infections is known to be influenced by several genes (reviewed in Ref. 44), but the best characterized are those linked to the MHC. For instance, in experimental infections with T. spiralis in the mouse, the H-2 complex influences both the time-course and the magnitude of intestinal and muscle burdens of parasites 45'46. Because the MHC restricts the immune repertoire, it makes sense to Table I. M H C haplotypes of commonly used strains of mice Strain C57BL/I 0 BALB/b
H-2 haplotype a b b
DBAz BALB/c BI0.D2
d d d
CBA C3H/He BALB/k BI0.BR
k k k k
NIH BI0.G
q q
SJL
s
BIO.S
s
aA haplotype is a set of genetic determinants located on a single chromosome. The above strains are inbred and therefore homozygous for all MHC genes.The single letter designations mean that the combination of alleles expressed by mice of a given hapIotype are unique to that hapIotype. Recombinant haplotypes are those in which a genetic crossover has occurred within the MHC. An example would be a mouse that had the H-2b allele of the H-2K protein, and the H-2q allele of the H-2D protein, encoded on a single homologous chromosome. The rules for the naming of recombinant and other haplotypes are given in Refi 5.
investigate its effect on the specificity of the immune response and the chain of events between antigen exposure and effector mechanisms. MHC control of antibody response specificity to helminths was first described for chronic infections of mice with Schistostrma mansoni 47. However, the source of antigen used was in vitro translates of whole adult worm mRNA, which presumably represented mainly somatic antigens. But ES or surface antigens are more likely to be relevant to immunity in vivo. The nematode infection most amenable to immunogenetic analysis of antigen recognition has proved to be Ascaris in rodents because the antibody response is limited in an MHC-dependent manner. This is not unique toAscarifl 8, but it is, so far, the clearest demonstration of MHC restriction among helminths.
Ascaris as a model for genetic control The ES materials of the tissue-invading larval stages of Ascaris are subject to a substantial antibody response, and the composition of ES alters markedly as the larvae migrate to the lungs 48'49. Humans, in areas endemic for Ascaris lumbricoides, react heterogeneously to ES antigens of larvae of this species 31 (Fig. 2). This can be modelled in inbred mice in which specificity of the antibody response to these stage-specific antigens differs radically from strain to strain. An example of this is illustrated in Fig. 4, showing an analysis of the components precipitated from radio-iodinated ES of the infective larvae ofA. suum.The points to note are (1) that no strain recognizes all the components of ES, (2) that there is sequential recognition of the antigens with time, (3) that only animals with identical MHC haplotypes have identical profiles, and (4) that when tertiary infections seem to be similar between different haplotypes, the primary response patterns distinguish them (compare H-2 k and H-2b). So both the kinetics of antigen recognition and the mature recognition profiles can be characteristic of each MHC haplotype. To confirm that repertoire restriction can properly be ascribed to the MHC, it is necessary to analyse the response patterns of mice that are congenic for the H-2 region. That is, mice having the same genetic background, but differing at H-2 loci, and vice versa. Such an analysis has confirmed H-2 control of the antibody repertoire to Ascaris; mice of the same MHC, but different genetic backgrounds
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N IH H - 2q R
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Molecular mass
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14
responded identically (Fig. 5), and those with the same background but disparate MHC responded differently (Fig. 6). (For a primer on the MHC of the mouse, and use of selected strains in immunogenetics, see Ref. 27; see also Table 1.) A direct correspondence between immune specificity and effector mechanisms is, however, unlikely to be simple because the strains that react identically to Ascaris infection in terms of antigen recognition have been found :in the past to differ in a number of other immunological responses to the parasite. For example, strains with the same H-2 can differ markedly in IgE responses (see below), eosinophilia s2, and the success with which Ascaris larvae reach the lungs following infection 53. One point which later proved to be of significance was that :mice of only one haplotype, H-2 s, responded to an ES antigen of 14 kDa. This protein can be purified in relative abundance from the body fluid of adult worms ~Lnd has permitted deeper insight into MHC control. During infection, BALB/c mice; do not respond to this molecule but will readily respond
a
b
c
Fig. 4 (left). MHC-restricted recognition of ESantigens of Ascaris suum infective larvae in the mouse. Serum from a range of Ascaris-infected mouse strains was reacted with radiolabelled ES material from infective larvae of the parasite, and analysed by SDS-PAGE. The strain of mice used and their MHC (H-2) haplotypes are given above the triplets of tracks. Serum was sampled before infection (a), after one infection (b), and after three infections (c). The reference track (R) gives the profile of the iodinated antigen used in the assay. (Data from Ref. 50. ©Blackwell Scientific Publications.) Fig. 5 (above). Mice of the same MHC haplotype have the same antigen recognition profiles in response to Ascaris suum infection, regardless of genetic background. The recognition profiles of H-2 Kstrains of mice are shown here, assayed against the ES antigens of the lung-stage larva of the parasite. Radio-iodinated ES material (reference antigen, track R) was immunoprecipitated with normal serum from each strain (a), and serum token from mice 28 days after infection (b), and 14 days after the last of three infections (c). (Data from Ref. 5 I. ©American Association of Immunologists.)
when the 14 kDa protein is given in adjuvant 51. Thus, the MHC restriction only operates in the context of infection. This would have relevance to vaccination because it might be possible to elicit a response in an individual who would otherwise be a non-responder. However, it leaves the conundrum of how infection fails to elicit a response to this antigen, even in the face of repeated infections, when there is apparently no absolute bartier to a response. This emphasizes the point that the immune response to particular parasite antigens differs radically according to how they are presented to the host, and that the biological relevance of response restriction can only be properly understood in infection. Non-responsiveness to a given antigen could have several causes, including the induction of suppressor circuits 54, the failure of antigen-MHC interaction 3'4, or tolerance to MHC-antigen complexes 1°. It remains to be seen which mechanism operates for Ascaris infection, but something can be said about one aspect of suppression. One of the mouse class II MHC molecules, I-E, has been associated
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Molecular mass
M
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Fig. 6. Mice with a common genetic background respond according to their MHC haplotype. The recognition profiles of congenic strains of mice on the BALB background are shown here, assayed against the ES antigens of the lung-stage larva of the parasite. The strains were: BALB/c (H-2°), BALB/b (H-2b), and BALB/k (H-2k). Radio-iodinated ES material (track R) was immunoprecipitated with normal serum from each strain (a), and 14 days after the last of three infections (c). (Data from Ref. 5 I. ©American Association of Immunologists.)
Fig. 7. Complex inheritance patterns of antigen recognition in Ft hybrids. A schematic consensus of recognition patterns in BALB/c and SJL parents, and their Fi hybrid offspring. The immunoprecipitations were performed using serum from mice that had been infected with Ascaris on three occasions. The density of tane and width of the bands represents the strength of recognition. (Adapted from Ref. 5 I.)
with the suppression of responses to certain defined antigens 55'56, and the H-2 k allele is associated with susceptibility to infection with T. spiralis 57. However, some strains of mouse are defective in cell surface expression of the I-E heterodimer 58'59, and can be used to test the supposed suppressor activity of I-E. The H-2 s haplotype is among these, but release from I-E-associated suppression does not necessarily explain the responsiveness of strains bearing this haplotype, because those of H-2 b and H-2 q are also I-E-negative, yet do not respond.
Consequences of heterozygosity Inbred experimental animals are homozygous in the MHC, which is a very rare event in most wild mammalian species. Animals that are heterozygous at all MHC loci are more representative, and this situation can be modelled by using F] hybrids of inbred strains. When such hybrids are infected with Ascaris 51 (Fig. 7), they are found to have unexpected antigen recognition patterns, which cannot be predicted from the parental phenotypes. Even more unexpectedly, one ES component was subject to a response in both parents, but only faintly in the Fl. These effects might seem paradoxical, but can be accommodated within the 'cross-tolerance' hypothesis 9'6°, by which certain antigen-MHC combinations resemble self determinants and are therefore eliminated from the immune repertoire. If this were the explanation, then it would be important to study the regu-
lation of responses to the separate epitopes of a given antigen species, which would only make sense in an immunogenetic context. Whatever proves to be the case, these kinds of phenomena are fundamental to the interaction of an individual's immune system with parasite antigens. M H C and allergic reactions One of the hallmarks of infection with multicellular parasites is hypersensitivity elicited either by the infection itself, or by intervention. Several allergens from nematodes have been described 61 and among these Ascaris has yielded more than any other. The antigen that re-enters the story here is the 14 kDa protein, which is potently allergenic, and which appears to resemble a previously described allergen of Ascaris, 'Allergen A', in its molecular size, isoelectric point and amino acid composition 62,63 (J.F.Christie et al., unpublished). The 14 kDa molecule is allergenic to infected mice (Table 2), and the IgE response to it is H2-restricted. This might provide a direct link between the MHC, immune effector mechanisms and immunopathology and prompts one to ask whether the differential recognition of the 14 kDa in infected humans (Fig. 2) relates to hypersensitivity and/or a predisposition to a particular magnitude of infection. Relatively few infected humans respond to this molecule, which encourages the search for genetic correlates with its immune recognition. The specificity of the IgE response to parasite allergens can be under genetic
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control, but the use of defined strains of rodents can also shed light on the control of the level of the IgE re,'sponse. For example, SJL mice cannot mount an IgE response to the 14 kDa allergen, unlike B 10. S mice, which are identical at the H-2 (Table 2). This presents us with a system whereby separate genetic influences on hypersensitivity response to a parasitic infection can be studied under biologically meaningful conditions.
Is the MHC important in nematode infections? Does MHC-restricted antigen/allergen recognition have any bearing on immunity to nematode infections in humans? It is too early to make any conclusions as yet, but epidemiological work on this is gathering momentum, and HLAassociated effects have already been seen. For instance, a recent preliminary survey in the Caribbean has revealed an association between certain MHC alleles and a tendency to high or low infection with Ascaris lumbricoides and Trichuris trichiura64. Such findings are of potential importance to targeted chemotherapy (should MHC typing ever become cheap enough), but the implications must go beyond these particular nematodiases. Epidemiological studies will be essential to characterize further the role of the MHC, but we will remain dependent on experimental models to understand the processes involved. This prompts us to ask whether the MHC effects on Ascaris infection in rodents are: typical. The very nature of the immune response is such that the MHC is bound to have some effect on the selection of antigens to which an individual responds. However, there are examples in which selective recognition appears not to occur 24, or in which the MHC is thought to have: a subordinate role in repertoire selection28. MHC control may not, therefore, be universal, but too few nematode infections have been studied to generalize. Another consideration is the relewance of unnatural host-parasite combinations, which are increasingly being used to focus work on parasites of humans in laboratory hosts. Ascaris in mice is, of course, a case in point. On the other hand, we have recently found that MHC restriction applies as exquisitely to Nippostrongylus brasiliensis antigens in infections in rats, a natural combination, as it does to the Ascaris-mouse model (M.W. Kennedy and A.J. Blair, unpublished). The differ-
323
Table 2. H-2-restricted IgE antibody responses t o the 14 kDa antigen of AscariP S t r a i n of m o u s e
BI0 B I 0.D2 BI0.BR B I 0.G BI0.S SJL
H-2 haplotype
b d k q s s
PCA titre P u r i f i e d 14 k D a ABF antigen 0 256 0 256 0 >512 0 256 128 512 0 16
algE was assayedin a passivecutaneous anaphylaxis assay(PCA) in which serum from mice of the strains listed was tested in rats. The assaywas carried out against two sources of allergen: the purified 14 kDa and the body fluid of adult worms (ABF) from which the 14 kDa was obtained. The assaywith ABF was carried out in parallel as a positive control to ensure that the mice used as serum donors were able to produce an IgE response to other allergens of the parasite. The SJL mice are included to show control of the amplitude of the IgE response by non-H-2 genes. Hence, H-2 controls the specificity of the response, but other genes regulate the level of the responses k
ences between the various studies may lie in whether or not a given parasite has a tissue migratory phase, or in the strength of the immune stimulus in a particular case. Certainly, before MHC restriction can be eliminated, careful attention will have to be paid to the kinetics of immune responses, the dose of parasites used, and the class of antibody assayed. £he MHC is emerging as a gene complex of great interest in helminth parasitology. The understanding of its pivotal position in the immune response is advancing rapidly in other areas of immunology, and will provide considerable insight into the co-evolution of parasite antigens and host immune recognition systems. This will probably become clearest for the antigenically shifting and drifting viruses but there are, as we have seen, already good reasons to study it further in parasitic helminths.
Vice versa? The other side of the coin is that parasitic helminths themselves could be polymorphic. The degree of genetic variability in parasitic nematodes is comparable tO65, and perhaps less than 66, the average for free-living invertebrates; however, such estimates have come mainly from screening for alleles in internal, 'housekeeping' isoenzymes, and not the components that constitute the host-parasite interface. A glimpse at the evolutionary forces involved has already come from the unexpectedly rapid rate of evolution of the active sites of proteinase inhibitors of mammalian tissue 67. This has been taken as a sign of past adaptations to counter modifications in the enzymes produced by pathogens. It will be important in the future to look at polymorphism in the materials that
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Acknowledgements Our work is supported by The Wellcome Trust, Medical Research Council, World Health Organization, and the Scottish Home and Health Department. I am very much indebted to Fiona McMonaglefor help with the artwork.
living parasites expose to their hosts in order to understand the mutual adaptations undergone between them, and how this affects host variability in responsiveness and immunopathology. Of immediate concern is the use of simple antigen preparations for vaccination. Lastly, if host-parasite interface molecules from both partners are polymorphic and determine the success of infection, might it be that the genetic composition of parasites within an individual is not representative of the parasite population as a whole? That is, do hosts select the parasites that reach maturity within them? References
1 Townsend, A. and McMichael, A. (1987)Nature 329, 482-483 2 Davis, M.M. and Bjorkman, P.J. (1988) Nature 334, 395-402 3 Allen,P.M. etal. (1987)Nature327, 713-715 4 Allen, P.M. (1987)Immunol. Today 8,270-273 5 Klein, J. (1986) Natural History of the Major Histocompatibiliry Complex John Wiley & Sons 6 Nagy, Z.A. etal. (1989) lmmunol. Today 10, 132-138 7 Anderson, R.M. (1988) in Genetics of Resistance to Bacterial and Parasitic Infection (Wakelin, D. and Blackwell,J.M., eds), pp 233-263, Taylor & Francis 80'Brien, S.J. and Evermann, J.F. (1988) Trends Ecol. Evol. 3,254-259 9 Matzinger, P. (1981)Nature 292,497-501 10 Vidovic, D. and Matzinger, P. (1988) Nature 336, 222-225 11 Arnot, D.; McCutchan, T.F., Good, M.F. and Miller, L.H. [Debate] (1989) Parasitology Today 5, 138-146 12 Tiwari, J.L. and Terasaki, P.I. (1985) HLA and Disease Associations Springer-Verlag 13 Thomson, G. (1986) in Handbook of Experimental Immunology (Vol. 3) (Weir, D.M., ed.), pp 102.1-102.12, BlackwellScientificPublications 14 Smith, H.V. et al. (1981) Mol. Biochem. Parasitol. 4, 183-193 15 Maizels, R.M., de Savigny, D. and Ogilvie, B.M. (1984)Parasitelmmunol. 6, 23-37 16 Hotez, P.J. and Cerami, A. (1983)J. Exp. Med. 157, 1594-1603 17 Hotez, P.J. et al. (1985)J. Biol. Chem. 260, 7343-7348 18 Robertson, B.D. etal.(1989)Exp. Parasitol. 69, 30-36 19 Knox, D.P. and Kennedy, M.W. (1988) Mol. Biochem. P arasitol. 28,207-216 20 Rothwell, T.L.W., Ogilvie, B.M. and Love, R.J. (197 3) lnt. J. P arasitol. 3,599--608 21 Rathanr, S. et al. (1987) Mol. Biochem. Parasitol. 26, 257-265 22 Roades, M.L. (1983)Exp. Parasitol. 56, 41-54 23 Thorsen, R.E. (1953)Am.J. Hyg. 58, 1-15 24 Jungery, M. and Ogilvie, B.M. (1982)J. Immunol. 129, 83%843 25 Kennedy, M.W. et aL (1988)Mol.Biochem. Parasitol. 31, 35--46 26 Qureshi, F. et al. (1987)Mol. Biochem. Parasitol. 24, 137-145 27 Wakelin, D. (1985)Parasitology Today 1, 17-23 28 Else, K. andWakelin, D. (1988) Parasitelmmunol. 11, 77-90 29 Pritchard, D.I. et al. (1986) Parasite 1mmunol. 8, 359-367 30 Almond, N.M. et al. (1986) Trop. Med. Parasitol. 37, 381-384 31 Hasweli-Elkins, M.R. et al. Parasite lmmunol. (in press) 32 Maizels, R.M. et al. (1987) in Filariasis (Ciba Foun-
dation Symposium 127), pp 18%199, John Wiley & Sons 33 Mills, K.H.G., Skehel, J.J. and Thomas, D.B. (1986) Eur. J. Immunol. 16,276-280 34 Mills, K.H.G., Skehel, J.J. and Thornas, D.B. (1986) J. Exp. Med. 163, 1477-1490 35 Mills, K.H.G. (1986)Immunol. Today 7,260-263 36 Morrison, R.P. etal. (1987)Nature329, 72%731 37 Wiley, D.C. and Skehel, J.J. (1987) Annu. Rev. Biochem. 56,365-394 38 Thomas, D.B. et al. (1987) Eur. J. lmmunol. 17, 133--136 39 Mills, K.H.G. et al. (1988) J. Immunol. 140, 4083-4090 40 Barnett, B.C. et al. (1989) Eur. J. Immunol. 19, 515-528 41 Graham, C.M. et al. (1989) Eur. J. Immunol. 19, 523-552 42 Burt, D.S. etal.J. Exp. Med. (in press) 43 Anders, R.F. (1986) Parasite lmmunol. 8, 52%539 44 Wakelin, D. (1988) Genetics of Resistance to Bacterial and Parasitic Infection Taylor & Francis 45 Wakelin, D. and Donachie, A.M. (1983) Immunology 48,343-350 46 Wassom, D.L. et al. (1984) Immunology 51,625-631 47 Kee, K.C. etal. (1986) Parasitelmmunol. 8,565-574 48 Kennedy, M.W. and Qureshi, F. (1986) Immunology 58, 515-522 49 Kennedy, M.W. etal. (1987) Clin. Exp. Immunol. 67, 20-30 50 Kennedy, M.W. et al. (1987) Parasite Immunol. 9, 26%273 51 Tomlinson, L.A.T. etal.J. Immunol. (in press) 52 Vadas, M.A. (1982)J. Immunol. 128,691-695 53 Mitchell, G.F. et al. (1976) Int. Arch. Allergy Appl. Immunol. 52, 64-78 54 Sercarz, E.E. etal. (1978) Immunol. Rev. 39, 108-136 55 Baxevanis, C.N., Nagy, Z.A. and Klein, J. (1981) Proc. Natl Acad. Sci. USA 78, 3809-3813 56 Oliveira, D.B.G. et al. (1985) Immunogenetics 22, 16%175 57 Wassom, D.L., Krco, C.J. and David, C.S. (1987) Immunol. Today 8, 39-43 58 Murphy, D.B. et al. (1980)Proc. Natl A cad. Sci. USA 77, 5404-5408 59 Jones, P.P., Murphy, D.B. and McDevitt, H.O. (1981)Immunogenetics 12, 321-337 60 Schwartz,R.H. (1978)Scand.J. Immunol. 7, 3-10 61 Jarrett, E.E.E. and Miller, H.R.P. (1982) Prog. Allergy 31,178-233 62 Ambler, J. et al. (1973) J. Immunol. Methods 2, 315-323 63 Ambler, J. etal. (1973)Immunochemistry 10, 815-820 64 Bundy, D.A.P. (1988)Philos. Trans. R. Soc. London, Ser. B 321,405-420 65 Nadler, S.A. (1987)Parasitology Today 3,154-155 66 Bullini, L. etal. (1986) Evolution40, 437-440 67 Hill, R.E. and Hastie, N.D. (1987)Nature 236, 96-99
Letters to the Editor Parasitology Today welcomes letters to the editor. Please addressletters to: Dr Caroline Ash
Parasitology Today ElsevierTrends Journals 68 Hills Road Cambridge CB2 I LA, UK and mark clearly whether they are intended for publication.
Parasitology Today, vol. 5, no. I O, 1989 62 Burnham, W.R., Reeve, R.R. and Finch, R. (1980) Gut21, 1097-1099 63 Sargeaunt, P.G., Williams, J.E. and Grene, J.D. (1978) Trans. R. Soc. Trop. Med. Hyg. 27, 519-521 64 Goldmeier,D. etal. (1986) Lancet 1,641-644 65 Nguyen-Dinh, P. et al. (1987) Third International Conference on AIDS (Abstr.)
66 Collins, W.E. et al. (1971) Trans. R. Soc. Trop. Med. Hyg. 65,43-58 67 Arnot, D. (1989) Parasitology Today 5, 138-142 68 Hiatt, R.A. et al. (1979)J. Infect. Dis. 139,659 69 Shapiro, S.Z. and Pearson, T.W. (1986)Parasite Antigem: Towards New Strategies for Vaccines (Pearson, T.W., ed.), p. 225, Marcel Dekker
Genetic Control of the Immune Repertoire in Nematode Infections M.W. Kennedy I
Wellcome Laboratories for Experimental Parasitology University of Glasgow Bearsden GlasgowG61 I QH, UK
Mammals vary considerably, both within and between species, in the way in which their innate and adaptive immune systems respond to infections. An understanding of the processes involved in such variability will not only contribute to explaining heterogeneity in susceptibility and pathology, but will also be relevant to vaccination. This will be particularly important for the new generation of vaccines that are likely to be composed of one or a few cloned or synthesized antigens. For helminth infections, this could have particular relevance to hypersensitivity responses. The adaptive immune response is fundamentally constrained by the genetic constitution of an individual, and the need to avoid reactivity to self. This will have important implications for the dynamic relationship between host defences and parasite evasion mechanisms at both physiological and evolutionary levels. In this review, Malcolm Kennedy examines the genetic control of the specificity of the immune response to nematode infections, and in particular, the role of the major histocompatibility complex. The adaptive immune system has the capacity to respond to an almost inconceivable array of antigens, including those that had never been encountered until the advent of synthetic chemistry. Despite this, there are strict limitations which mean that every individual probably has its own unique immunological repertoire. There are two principal constraints. First is the requirement for tolerance to an individual's own tissues, to harmless or beneficial organisms such as those occurring on the skin or in the alimentary tract, and to potentially antigenic materials inhaled or present in food. Second, an immune response to most antigens depends on the successful presentation of processed antigen to lymphocytes, and their subsequent activation. The cell surface proteins encoded by the major histocompatibility gene complex (MHC), originally discovered as a barrier to tissue transplantation, are at the heart of this process. The MHC comprises a set of closely linked genes in all species so far investigated. Its structure and function are best understood in humans and mice, in which the gene complexes are known as the HLA and H-2, respectively (see Box 1). The products of the class I region are involved
in direct cell-mediated killing of, for example, virus-infected cells. The class II region is primarily associated with antigen presentation and cooperation between T- and B-lymphocytes in the induction of an antibody response. It is thought that a response depends not only on whether an individual's MHC molecules have an affinity for given processed fragments of an antigen, but also on the manner in which the fragments are presented to T-cells 1-4. Both of these functions are regulated by class II-associated immune response (Ir) genes 5.
The advantage of polymorphism Since there are materials to which certain individuals cannot, or must not, respond, this leaves loop-holes in the immune repertoire that pathogens can exploit to their advantage. This could be achieved by mimicking host molecules, by the selection of parasite components whose processed fragments will not associate with MHC molecules on the surface of an antigen presenting cell, or by interfering with the antigen processing and presentation machinery of the ceU6. Thus, polymorphisms at loci critical to these processes could minimize the impact of pathogens that exploit such evasion strat© 1989, Elsevier Science Publishers Ltd. (UK) 01694707/89/$03.50
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B o x I. T h e M H C G e n e s of M i c e a n d H u m a n s
Simplified, comparative linkage maps of the MHC of mice (H-2) and humans (HLA). Both contain pseudogenes or genes whose functional significance is unknown, and these are excluded from the diagram. Genes for some of the components of the complement system, and some enzymes, are closely linked to, or embedded within, the HHC region, and these are also excluded. Class I HHC molecules (shaded boxes) appear on the surface of most nucleated cells, and ;ire found non-covalently associated with a small protein, 132microglobulin. Processed fragments of antigen, eg. from viruses in an infected cell, are thought to locate in the 'Bjorkman groove' of the class I molecules, and this complex constitutes the recognition unit for a specific T-cell receptor. Class II molecules (open boxes) are found as nonMouse Chromosome 17
H-2
I
K I---~
covalently linked heterodimers on the cell membrane. Class II molecules are thought to present processed antigen to T-cells in a groove distal to the membrane, similar to the 'Bjorkman groove' of class I molecules. Class II molecules have a more limited tissue distribution than those of class I, but appear constitutively on several tissues and many cells of the immune system, and can be induced in others under conditions of immunological or infection stress. In mice, Aa and AI3 chains preferentially associate, as do Ea and E~, although AJEI3 and AI~E~ associationsmight occur. In humans,there are more classII molecules, and more genes for 13-than s-chains, although preferential pairing probably occurs with the appropriate s-chain. 13-chainstend to be more polymorphic than cL-chains.
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egies. An individual within a polymorphic population would be at an advantage if a hypothetical pathogen is only successful in those individuals that bear a particular allele. This lowers the chance of being infected in the first place, and the pathogen's population might be constrained to below its threshold level7. The range of pathogens, and their antigenic shifts, drifts and intrinsic wtriabilities would maintain polymorphism at critical loci. The prediction is, therefore, that genetically homogeneous populations of animals would be highly susceptible to eradication by transmissible diseases, and this is supported by evidence from natural occurrences and human interference in wild populations 8. Polymorphism might, therefore, be advantageous, but there is likely to be a limit to the number of/VIHC proteins that an individual can advantageously express. This is because each addition to the repertoire would widen the range of antigens to which immunological tolerance will have to operate 9']°. There is also the probabil-
[~1~
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ity that a given allele might simultaneously confer resistance to one pathogen, but susceptibility to another. Parasites that can circumvent host polymorphisms would have a selective advantage, and the bonus of being a member of a polymorphic population also applies to them. This acts as a selective pressure to favour multiple alleles in parasite genes coding for molecules interacting with host tissues. That is another story (see Ref. 11).
Polymorphismsand pathogens The most polymorphic locus known in man is that coding for glucose 6phosphate dehydrogenase (G6PD), for which more than 300 alleles are known. That genetic variability at this locus is related to infection is suggested by the fact that some alleles are more frequent in malarious areas than can be accounted for by mutation alone. The obvious possibility is that certain alleles are associated with a degree of resistance to Plasmodium infection, as is the sickle cell trait. The human MHC does not have as
A
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Molecular
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Fig. I. Kinetics of antigen recognition in Anisakis simplex infection, and restricted recogn~on in humans, ffZSl]-Iabelled parasite culture supernatant (ES) from third-stage larvae of A. simplex was immunoprecipitoted w/th serum from a normal rabbit (track 2) and from a rabbit 7, 2 I, 28, 35, 42, 58, 63 and 142 days after oral infection with A. simplex (tracks .3-10, rest~ectively). Track I I shows an immunoprecipitation with normal human serum, and track 12 with serum from an infected human. Note that the latter serum fails to precipitate some of the major components. Immunoprecipitotes were analysed on 5-25% gradient SDS-PAGE gels. Track I was loaded with the antJgen used for the immunoprec/p/totion. The relative molecular masses are given in kDa. (Seealso Ref. 25,)
Fig. 2. Heterogeneity of responsivenessof humans infected with Ascaris lumbricoides, ffzsI]-Iabelled ES material from lung-stage larvae of A. suum was immunoprecipitated with serum from infected people, and anal~ed by SDS-PAGF_The tracks are molecular mass markers (track M), ffZSl].labelled F.S material (track R), an immunoprecipitat~ with serum from an uninfected human (track N), and serum from infected people in Nigeria (numbered tracks). These data are courtesyof Eleanor M.Fraser, using sera donated by Celia Holland and David Crompton. This effect has also been observedin sera from infected people in India 31 and St Lucia (sera courtesyof Don Bundy).
many alleles per locus as G6PD, but recombination within the complex can provide a greater number of genotypes (or 'haplotypes'). There are 19, 39, 8, 17, 14, 3 and 6 recognized specificities at the HLA-A, B, C, D, DR, DP, DQ loci, respectively, and many are as yet uncharacterized (Box 1). A further important point is that certain pairs of alleles do not occur in ratios that can be predicted from their gene frequencies if it is assumed that recombination between them has reached equilibrium. This 'linkage disequilibrium' is presumably due to residual founder effects, recent population mixings, and/or certain combinations of alleles having a selective advantage or disadvantage with respect to infections. Another consideration is that certain alleles, or haplotypes, have a reproductive cost related to the immune system, such as autoimmunity, maternal/ foetal incompatibility, and hypersensitivity to certain environmental mat-
anticipated that MHC effects are unlikely to be seen. But, as we shall see, they do occur, from the level of immune control of infection as a whole to the specificity of antibody responses.
Nematodes Serum antibody responses are the most accessible manifestation of the immune repertoire, and can be used to follow the kinetics of responses to particular antigens as well as to estabfish limitations to an individual's immune response. For nematodes, the choice of antigens used in immunoassays has in the past decade moved away from worm homogenates to the materials that parasites express on their surfaces and/or release into culture supernatant in vitro; the latter are known as excretory/secretory materials (ES). Surface and secreted materials can have common components 14,15, and the choice of which to use usually depends on supply and ease ofhand!ing. ES material has extra e r i a l s 12,13. significance since it is known to contain a It is relatively easy to conceive of an asso- variety of biologically active materials, for ciation between MHC alleles and the example anti-clotting factors 16, proimmune control of relatively simple agents teinases 17-19, acetylcholinesterase20,21 , such as viruses. MHC-restricted responses superoxide dismutase 22, and lipases23, to a single component of a virus could have which are presumably relevant to parasite far-reaching consequences for the success nutrition, tissue penetration and immune of infection in an individual host. How- evasion. The logic behind the choice of ever, with large and antigenically complex these materials for experimentation is that parasites, such as nematodes, it might be they are the sites of direct interaction
Parasitology Today, vol. 5, no. I O, 1989
between host and parasite and as such, are accessible to immune, effector mechanisms. Internal components of parasites are not irrelevant to the immunobiology of infections, since they will elicit an Lmmune response during moulting and following the killing and degradation of parasites in tissues. Release of such antigens might trigger immunopathological manifestations of infection, for example the Mazzotti reaction in onchocerciasis, or Loeffler's syndrome in ascariasis. Nematode infections are renowned for such hypersensitivity responses, and it is vital to identify which parasite components will provoke adverse reactions in the context of infection, vaccination or chemotherapy. Another significance of internal antigens is that they could be used to provide informarion on whether parasites are being killed m vivo, either by the immune system or following drug treatment.
Restricted immune recognition of nematode antigens The simplest fo~a of restricted responses would be detected during the development of immunity. Strains of mice have been found to differ in the time after infection at which they mount a detectable antibody response to the surface antigens of Trichinella spiralis, allahough eventually all the surface (glyco)proteins are recognized 24. Sequential recognition of this kind has also been noted for ES materials, and an example of this is given in Fig. 1 for infection of rabbits with Anisakis simplex. The major part of the normal antibody response to this nematode appears to be focused on secreted antigens, rather than internal components 2s, but Anisakis can penetrate gastrointestinal tissue in humarls, and when this is modelled by parenteral infection of experimental animals, many more of the parasite's somatic materials become subject to an antibody response. This is presumably because worms that are normally confined to the gut lumen can be expelled without exposing the immune system to worm somatic antigens, in contrast to tissue infections in which parasites are kiUed and disintegrate in situ. The combination of kinetics and specificity would therefore provide an estimate of the age and the extent of certain infections. However, the usefulness of this would be limited by the ability of a given host to respond to the set of antigens with which it is presented by the parasite. This brings us back to genetic control of the
319
specificity of the immune response, what it means, how it can be interpreted, and how it arises. There are now several examples of restricted antibody specificity in both chronic and repeated nematode infections which possibly have a genetic basis. These include, Nippostrongylus brasiF~ensis in the rat z6, Heligmosomoides dubius27 and Trichuris muris in the mouse 2s, a s well as Necator americanus29, Trichinella spiralis3°, Ascaris lumbricoides31 (Fig. 2), and Brugia malayi32in humans. In the case of Brug/a infections, there appears to be sonte loose correlation between antibody recognition patterns and infection status. It remains to be seen, though, whether this is due to a link between disease and immune specificity, or whether both are controlled by the same or linked genetic loci. In other words, which is cause, and which is effect, or do the recognition patterns have any relevance at all? It has been argued that the analysis must be taken to the level of the individual antigenic determinants (epitopes) on parasite antigens in order to establish which factors reflect or determine the outcome of infection ~2. Experience with T- and B-cell responses to viral antigens 33-42 indicates that this would be a worthwhile strategy but increases our need for an understanding of genetic control of immune specificity. The phenomenon of restricted antigen recognition could also be valuable in identifying protective antigens. If a particular treatment rendered an individual immune to a pathogen, but only elicited a response Molecular
R
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D
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67.0 43.0 30.0.,. 21.1 14.4
Fig. 3. Heterogeneity of antigen recogn/t/on in vaccination and natural infection with the cattle lungworm Dicryocaulus viviparus. [IZSl]labelled ES material from adult D. viviparus (R) was immunoprec/pitoted with serum from cattle that had been vaccinated with the irradiated larval vaccine, 'Dictol" and then experimentally infected with normal larvae (N), or challenged with normal larvae (V), or experimentally infected with serum from infections with normal larvae that had been drug-abbreviated after i$ days (D) or from fmld cases of acute Iongworm disease (F). (Data from CBr/ttan et al., unpublished.)
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to some of the antigens given, it could be argued that these 'immune-selected' antigens should be considered as candidates for a vaccine. Anti-nematode vaccines are at present few in number and relatively crude, but the development of the next generation of vaccines by, for example, using recombinant virus vectors, will require the identification of one or a few crucial antigens. For instance, the vaccine against bovine lungworm, Dictyocaulus viviparus, is highly effective, but animals that have been vaccinated and then challenged with normal parasites respond heterogeneously (Fig. 3; C. Britton et al., unpublished). While this cannot directly identify protective antigens, the process of elimination would greatly simplify the search. It must not be forgotten that a protective antigen for one individual might not be so for another, and it has been argued that responses to certain specificities might even divert a host's response away from the successful control of infection 43. The immune control of experimental nematode infections is known to be influenced by several genes (reviewed in Ref. 44), but the best characterized are those linked to the MHC. For instance, in experimental infections with T. spiralis in the mouse, the H-2 complex influences both the time-course and the magnitude of intestinal and muscle burdens of parasites 45'46. Because the MHC restricts the immune repertoire, it makes sense to Table I. M H C haplotypes of commonly used strains of mice Strain C57BL/I 0 BALB/b
H-2 haplotype a b b
DBAz BALB/c BI0.D2
d d d
CBA C3H/He BALB/k BI0.BR
k k k k
NIH BI0.G
q q
SJL
s
BIO.S
s
aA haplotype is a set of genetic determinants located on a single chromosome. The above strains are inbred and therefore homozygous for all MHC genes.The single letter designations mean that the combination of alleles expressed by mice of a given hapIotype are unique to that hapIotype. Recombinant haplotypes are those in which a genetic crossover has occurred within the MHC. An example would be a mouse that had the H-2b allele of the H-2K protein, and the H-2q allele of the H-2D protein, encoded on a single homologous chromosome. The rules for the naming of recombinant and other haplotypes are given in Refi 5.
investigate its effect on the specificity of the immune response and the chain of events between antigen exposure and effector mechanisms. MHC control of antibody response specificity to helminths was first described for chronic infections of mice with Schistostrma mansoni 47. However, the source of antigen used was in vitro translates of whole adult worm mRNA, which presumably represented mainly somatic antigens. But ES or surface antigens are more likely to be relevant to immunity in vivo. The nematode infection most amenable to immunogenetic analysis of antigen recognition has proved to be Ascaris in rodents because the antibody response is limited in an MHC-dependent manner. This is not unique toAscarifl 8, but it is, so far, the clearest demonstration of MHC restriction among helminths.
Ascaris as a model for genetic control The ES materials of the tissue-invading larval stages of Ascaris are subject to a substantial antibody response, and the composition of ES alters markedly as the larvae migrate to the lungs 48'49. Humans, in areas endemic for Ascaris lumbricoides, react heterogeneously to ES antigens of larvae of this species 31 (Fig. 2). This can be modelled in inbred mice in which specificity of the antibody response to these stage-specific antigens differs radically from strain to strain. An example of this is illustrated in Fig. 4, showing an analysis of the components precipitated from radio-iodinated ES of the infective larvae ofA. suum.The points to note are (1) that no strain recognizes all the components of ES, (2) that there is sequential recognition of the antigens with time, (3) that only animals with identical MHC haplotypes have identical profiles, and (4) that when tertiary infections seem to be similar between different haplotypes, the primary response patterns distinguish them (compare H-2 k and H-2b). So both the kinetics of antigen recognition and the mature recognition profiles can be characteristic of each MHC haplotype. To confirm that repertoire restriction can properly be ascribed to the MHC, it is necessary to analyse the response patterns of mice that are congenic for the H-2 region. That is, mice having the same genetic background, but differing at H-2 loci, and vice versa. Such an analysis has confirmed H-2 control of the antibody repertoire to Ascaris; mice of the same MHC, but different genetic backgrounds
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N IH H - 2q R
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Molecular mass
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a
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mass
67
45
14
responded identically (Fig. 5), and those with the same background but disparate MHC responded differently (Fig. 6). (For a primer on the MHC of the mouse, and use of selected strains in immunogenetics, see Ref. 27; see also Table 1.) A direct correspondence between immune specificity and effector mechanisms is, however, unlikely to be simple because the strains that react identically to Ascaris infection in terms of antigen recognition have been found :in the past to differ in a number of other immunological responses to the parasite. For example, strains with the same H-2 can differ markedly in IgE responses (see below), eosinophilia s2, and the success with which Ascaris larvae reach the lungs following infection 53. One point which later proved to be of significance was that :mice of only one haplotype, H-2 s, responded to an ES antigen of 14 kDa. This protein can be purified in relative abundance from the body fluid of adult worms ~Lnd has permitted deeper insight into MHC control. During infection, BALB/c mice; do not respond to this molecule but will readily respond
a
b
c
Fig. 4 (left). MHC-restricted recognition of ESantigens of Ascaris suum infective larvae in the mouse. Serum from a range of Ascaris-infected mouse strains was reacted with radiolabelled ES material from infective larvae of the parasite, and analysed by SDS-PAGE. The strain of mice used and their MHC (H-2) haplotypes are given above the triplets of tracks. Serum was sampled before infection (a), after one infection (b), and after three infections (c). The reference track (R) gives the profile of the iodinated antigen used in the assay. (Data from Ref. 50. ©Blackwell Scientific Publications.) Fig. 5 (above). Mice of the same MHC haplotype have the same antigen recognition profiles in response to Ascaris suum infection, regardless of genetic background. The recognition profiles of H-2 Kstrains of mice are shown here, assayed against the ES antigens of the lung-stage larva of the parasite. Radio-iodinated ES material (reference antigen, track R) was immunoprecipitated with normal serum from each strain (a), and serum token from mice 28 days after infection (b), and 14 days after the last of three infections (c). (Data from Ref. 5 I. ©American Association of Immunologists.)
when the 14 kDa protein is given in adjuvant 51. Thus, the MHC restriction only operates in the context of infection. This would have relevance to vaccination because it might be possible to elicit a response in an individual who would otherwise be a non-responder. However, it leaves the conundrum of how infection fails to elicit a response to this antigen, even in the face of repeated infections, when there is apparently no absolute bartier to a response. This emphasizes the point that the immune response to particular parasite antigens differs radically according to how they are presented to the host, and that the biological relevance of response restriction can only be properly understood in infection. Non-responsiveness to a given antigen could have several causes, including the induction of suppressor circuits 54, the failure of antigen-MHC interaction 3'4, or tolerance to MHC-antigen complexes 1°. It remains to be seen which mechanism operates for Ascaris infection, but something can be said about one aspect of suppression. One of the mouse class II MHC molecules, I-E, has been associated
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Molecular mass
M
R
BALB/c BALB/b BALB/k
Holecular
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~
4 1 ~
25.5
~
..............................
225
~
1 1 8 ~
25.5 1 4 m
Fig. 6. Mice with a common genetic background respond according to their MHC haplotype. The recognition profiles of congenic strains of mice on the BALB background are shown here, assayed against the ES antigens of the lung-stage larva of the parasite. The strains were: BALB/c (H-2°), BALB/b (H-2b), and BALB/k (H-2k). Radio-iodinated ES material (track R) was immunoprecipitated with normal serum from each strain (a), and 14 days after the last of three infections (c). (Data from Ref. 5 I. ©American Association of Immunologists.)
Fig. 7. Complex inheritance patterns of antigen recognition in Ft hybrids. A schematic consensus of recognition patterns in BALB/c and SJL parents, and their Fi hybrid offspring. The immunoprecipitations were performed using serum from mice that had been infected with Ascaris on three occasions. The density of tane and width of the bands represents the strength of recognition. (Adapted from Ref. 5 I.)
with the suppression of responses to certain defined antigens 55'56, and the H-2 k allele is associated with susceptibility to infection with T. spiralis 57. However, some strains of mouse are defective in cell surface expression of the I-E heterodimer 58'59, and can be used to test the supposed suppressor activity of I-E. The H-2 s haplotype is among these, but release from I-E-associated suppression does not necessarily explain the responsiveness of strains bearing this haplotype, because those of H-2 b and H-2 q are also I-E-negative, yet do not respond.
Consequences of heterozygosity Inbred experimental animals are homozygous in the MHC, which is a very rare event in most wild mammalian species. Animals that are heterozygous at all MHC loci are more representative, and this situation can be modelled by using F] hybrids of inbred strains. When such hybrids are infected with Ascaris 51 (Fig. 7), they are found to have unexpected antigen recognition patterns, which cannot be predicted from the parental phenotypes. Even more unexpectedly, one ES component was subject to a response in both parents, but only faintly in the Fl. These effects might seem paradoxical, but can be accommodated within the 'cross-tolerance' hypothesis 9'6°, by which certain antigen-MHC combinations resemble self determinants and are therefore eliminated from the immune repertoire. If this were the explanation, then it would be important to study the regu-
lation of responses to the separate epitopes of a given antigen species, which would only make sense in an immunogenetic context. Whatever proves to be the case, these kinds of phenomena are fundamental to the interaction of an individual's immune system with parasite antigens. M H C and allergic reactions One of the hallmarks of infection with multicellular parasites is hypersensitivity elicited either by the infection itself, or by intervention. Several allergens from nematodes have been described 61 and among these Ascaris has yielded more than any other. The antigen that re-enters the story here is the 14 kDa protein, which is potently allergenic, and which appears to resemble a previously described allergen of Ascaris, 'Allergen A', in its molecular size, isoelectric point and amino acid composition 62,63 (J.F.Christie et al., unpublished). The 14 kDa molecule is allergenic to infected mice (Table 2), and the IgE response to it is H2-restricted. This might provide a direct link between the MHC, immune effector mechanisms and immunopathology and prompts one to ask whether the differential recognition of the 14 kDa in infected humans (Fig. 2) relates to hypersensitivity and/or a predisposition to a particular magnitude of infection. Relatively few infected humans respond to this molecule, which encourages the search for genetic correlates with its immune recognition. The specificity of the IgE response to parasite allergens can be under genetic
Parasitology Today, vol. 5, no. 10, 1989
control, but the use of defined strains of rodents can also shed light on the control of the level of the IgE re,'sponse. For example, SJL mice cannot mount an IgE response to the 14 kDa allergen, unlike B 10. S mice, which are identical at the H-2 (Table 2). This presents us with a system whereby separate genetic influences on hypersensitivity response to a parasitic infection can be studied under biologically meaningful conditions.
Is the MHC important in nematode infections? Does MHC-restricted antigen/allergen recognition have any bearing on immunity to nematode infections in humans? It is too early to make any conclusions as yet, but epidemiological work on this is gathering momentum, and HLAassociated effects have already been seen. For instance, a recent preliminary survey in the Caribbean has revealed an association between certain MHC alleles and a tendency to high or low infection with Ascaris lumbricoides and Trichuris trichiura64. Such findings are of potential importance to targeted chemotherapy (should MHC typing ever become cheap enough), but the implications must go beyond these particular nematodiases. Epidemiological studies will be essential to characterize further the role of the MHC, but we will remain dependent on experimental models to understand the processes involved. This prompts us to ask whether the MHC effects on Ascaris infection in rodents are: typical. The very nature of the immune response is such that the MHC is bound to have some effect on the selection of antigens to which an individual responds. However, there are examples in which selective recognition appears not to occur 24, or in which the MHC is thought to have: a subordinate role in repertoire selection28. MHC control may not, therefore, be universal, but too few nematode infections have been studied to generalize. Another consideration is the relewance of unnatural host-parasite combinations, which are increasingly being used to focus work on parasites of humans in laboratory hosts. Ascaris in mice is, of course, a case in point. On the other hand, we have recently found that MHC restriction applies as exquisitely to Nippostrongylus brasiliensis antigens in infections in rats, a natural combination, as it does to the Ascaris-mouse model (M.W. Kennedy and A.J. Blair, unpublished). The differ-
323
Table 2. H-2-restricted IgE antibody responses t o the 14 kDa antigen of AscariP S t r a i n of m o u s e
BI0 B I 0.D2 BI0.BR B I 0.G BI0.S SJL
H-2 haplotype
b d k q s s
PCA titre P u r i f i e d 14 k D a ABF antigen 0 256 0 256 0 >512 0 256 128 512 0 16
algE was assayedin a passivecutaneous anaphylaxis assay(PCA) in which serum from mice of the strains listed was tested in rats. The assaywas carried out against two sources of allergen: the purified 14 kDa and the body fluid of adult worms (ABF) from which the 14 kDa was obtained. The assaywith ABF was carried out in parallel as a positive control to ensure that the mice used as serum donors were able to produce an IgE response to other allergens of the parasite. The SJL mice are included to show control of the amplitude of the IgE response by non-H-2 genes. Hence, H-2 controls the specificity of the response, but other genes regulate the level of the responses k
ences between the various studies may lie in whether or not a given parasite has a tissue migratory phase, or in the strength of the immune stimulus in a particular case. Certainly, before MHC restriction can be eliminated, careful attention will have to be paid to the kinetics of immune responses, the dose of parasites used, and the class of antibody assayed. £he MHC is emerging as a gene complex of great interest in helminth parasitology. The understanding of its pivotal position in the immune response is advancing rapidly in other areas of immunology, and will provide considerable insight into the co-evolution of parasite antigens and host immune recognition systems. This will probably become clearest for the antigenically shifting and drifting viruses but there are, as we have seen, already good reasons to study it further in parasitic helminths.
Vice versa? The other side of the coin is that parasitic helminths themselves could be polymorphic. The degree of genetic variability in parasitic nematodes is comparable tO65, and perhaps less than 66, the average for free-living invertebrates; however, such estimates have come mainly from screening for alleles in internal, 'housekeeping' isoenzymes, and not the components that constitute the host-parasite interface. A glimpse at the evolutionary forces involved has already come from the unexpectedly rapid rate of evolution of the active sites of proteinase inhibitors of mammalian tissue 67. This has been taken as a sign of past adaptations to counter modifications in the enzymes produced by pathogens. It will be important in the future to look at polymorphism in the materials that
Parasitology Today, vol. 5, no. I O, 1989
324
Acknowledgements Our work is supported by The Wellcome Trust, Medical Research Council, World Health Organization, and the Scottish Home and Health Department. I am very much indebted to Fiona McMonaglefor help with the artwork.
living parasites expose to their hosts in order to understand the mutual adaptations undergone between them, and how this affects host variability in responsiveness and immunopathology. Of immediate concern is the use of simple antigen preparations for vaccination. Lastly, if host-parasite interface molecules from both partners are polymorphic and determine the success of infection, might it be that the genetic composition of parasites within an individual is not representative of the parasite population as a whole? That is, do hosts select the parasites that reach maturity within them? References
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