Journal of Infection (1992) 25, Supplement z, 11-26

REVIEW Chlamydial

vaccinesmfuture

trends

M i c h a e l E. Ward

Department of Microbiology, Southampton University Medical School, Level C, South Laboratory and Pathology Block, Southampton General Hospital, Southampton S09 4XY, U.K.

Introduction In many industrialised countries, Chlamydia trachomatis is the major cause of sexually transmitted disease. Although these infections are asymptomatic and thus insidious, they are far from trivial. Infants born through a chlamydial infected cervix have around a 33% probability of developing neonatal chlamydial conjunctivitis or pneumonia. Ascending infection from the endocervix commonly gives rise to endometritis, salpingitis, pelvic inflammatory disease and, ultimately, infertility. T h e prevalence of infertility following one to three attacks of symptomatic, laparoscopically proven salpingitis was 9-52~/o, despite treatment 1 with C. trachomatis the main pathogen. This underestimates the real impact of chlamydial infection on fertility, as many cases of upper genital tract chlamydial infection go unrecognised. In parts of sub-Saharan Africa, around 60 % of infertile women have indicators of pelvic infection. 2 Moreover, in this region some 60% of gynaecology consultations are for infertility. In T h e Gambia and other African countries infertility with tubal obstruction is strongly associated with serological evidence of past chlamydial or gonococcal infection. 3' 4 Chlamydial infection may also cause tubal dysfunction rather than obstruction, resulting in ectopic pregnancy. Ectopic pregnancy too is associated with prior chlamydial or gonococcal infection 5 and its incidence is increasing worldwide. 6 In men, chlamydial occlusion of the epididymis may also result in infertility, although the full extent of the problem has yet to be established. In industrialised countries, the costs of chlamydial infection to health services are enormous. In the U.S.A. for example, the annual cost of treating pelvic inflammatory disease (PID) was conservatively estimated to be $2.8 billion. By the year 2ooo the estimated cost will be $8 billion, assuming no increase in the incidence of P I D and related complications. 7 Comparable figures have been calculated for Sweden. s T h e social cost should also be remembered, with infertile women sometimes rejected by their husbands or even, in the developing world, ostracised by their communities. Clearly urgent preventive measures need to be taken against chlamydial infection. Improvements in the diagnosis and treatment of these infections, the tracing of infected partners and health education all have a role to play, but it seems unlikely that this will be sufficient. What is really required is a prophylactically effective anti-chlamydial vaccine. Reasons for believing that o x 6 3 - 4 4 5 3 / 9 2 / S i o o i I + I6 $03.00/0

© 1992 T h e British Society for the Study of Infection

12

M.E. WARD

a chlamydial vaccine might be realistic include the low incidence of chlamydial disease in individuals frequently exposed to chlamydial infection; the fact that active trachoma decreases with age and the observation that vaccination of primates or m a n with whole chlamydiae leads to serovar specific immunity. A chlamydial vaccine might have the added bonus that it might also help control trachoma, 9 an ocular infection due to C. trachomatis which is one of the world's major causes of preventable blindness. This paper reviews progress towards the development of a chlamydial vaccine in the context of the unique biology of Chlamydia and the challenge which this poses to the i m m u n e system. The biology of c h l a m y d i a l infection T h e genus Chlamydia consists of three species; C. trachomatis responsible for h u m a n oculogenital disease; C. psittaci which is primarily a pathogen of animals but may cause atypical p n e u m o n i a and endocarditis in m a n ; and C. pneumoniae, a newly recognised species causing h u m a n respiratory disease. Like the Rickettsia, Chlamydia are obligate intracellular bacterial pathogens in eukaryotic cells. However, they differ from Rickettsia in having a unique developmental cycle. 10.11 Infection of the host cell is initiated by the elementary body (EB), a 'spore like' structure adapted for survival and transit between host cells. T h e chlamydial adhesins which p r o m o t e chlamydial attachment have yet to be unambiguously defined. T h e host cell receptor p r o m o t i n g chlamydial adhesion may involve phosphatidyl ethanolamine a n d / o r asialo G M I. Entry of infectious EB requires the active participation of the host cell. Endocytosis involves both microfilament-dependent and independent mechanisms, depending on both the infecting strain and the m o d e of presentation to the host cell (reviewed in Ward and Clarke, I99o11). Once endocytosed, live EB, at least of C. psittaci, prevent endosomal acidification or fusion with lysosomes. T h e endocytosed EB develops within a host cell endosome into the larger, fragile reticulate body (RB). T h e RB divides by binary fission, replicating within an expanding endosome, the chlamydial inclusion. Each of these RB eventually differentiates to produce one or more EB 1° by processes we do not yet understand. T h e important point here is that, at the cellular level, Chlamydia must definitely be considered cytotoxic pathogens. One of the key areas of future research will be to try and define the functions of the host cell and inclusion membranes. It is clear that Chlamydia must in some way modify transport properties across host cell membranes in order to gain both the nutrients and high energy molecules ( A T P and G T P ) which they require. It is unlikely Chlamydia leave this to chance. It is known that chlamydial lipopolysaccharide a n d / o r exoglycolipid become inserted into the host cell membranes. Such antigen is exported in copious quantity from the host cell and is the basis of some of the diagnostic chlamydial antigen detection techniques. T h e chlamydial exoglycolipid is poorly characterised but appears to differ from the chlamydial lipopolysaccharide in being gulose rich, a feature which it shares with some alginates. One of the effects of m e m b r a n e incorporated chlamydial antigen may be to rigidify the cytoplasmic m e m b r a n e of infected cells. H o w this might affect chlamydial function remains to be assessed.

Future of chlamydial vaccines

13

Chlamydial evolution Given the uniqueness of the chlamydial developmental cycle it is relevant to ask 'how has this arisen in evolution ?' T h e fashionable view once was that as C. trachomatis and C. psittaci share only about Io % homology on total D N A genomic hybridisation, then these organisms were examples of convergent evolution from divergent sources. T o me, this has never made much sense, because of the uniqueness of chlamydial biology and the fact that these organisms are totally different from any other bacteria. However, once Mike Carter and co-workers in this laboratory succeeded in obtaining the first sequence for the M O M P gene of C. pneumoniae, 12 it became possible to construct a computer taxonomic map for Chlamydia based on M O M P gene sequences. M O M P is an ideal protein from this point of view because it has large conserved regions essential for chlamydial replication yet also has four variable regions characteristic, in the case of C. trachomatis, of each serovar. T h e computer program used was developed for plant taxonomy, drawing a taxonomic tree which clearly distinguishes between C. psirtaci, C. pneumoniae and C. trachomatis, supporting species designation for C. pneumoniae. Remarkably, the computer program was powerful enough to divide C. trachomatis into its two main immunological subgroups, the C complex and the B complex. T h e resulting dendrogram shows quite clearly that all these organisms have arisen by divergent evolution, from a common, unknown ancestor. 12 Chlamydial EB and RB lack the peptidoglycan strengthening layer in the outer envelope characteristic of most other bacteria, a feature which makes Chlamydia similar to the primitive, free-living Archaebacteria. Future studies in chlamydial evolution will include further developing the M O M P gene dendrogram and incorporating into it the M O M P genes of some of the possible precursor organisms.

Productive, inapparent and persistent infection T h e development cycle so far described is an example of productive chlamydial infection, whereby the host cell releases large numbers of infectious Chlamydia. This is not the whole story. Lee and Moulder showed back in I98O that it was possible, under certain conditions, to maintain chlamydial infection in a cryptic form in tissue culture cells so that the infection could be passed from cell to cell by serial passage over many months. Only sporadically were there episodes of productive infection in which chlamydial inclusions were produced and infection occurred. A clinical counterpart to this in vitro model is seen in trachoma, arguably the world's major cause of preventable blindness. In many ways trachoma serves as a microcosm for the multiplicity of infections which C. trachomatis can produce. In trachoma, just as in PID, the most serious complications are the result of chlamydial-induced scar formation. However, it is much easier to examine somebody's eyes than, say, the upper female genital tract. In our trachoma studies we collaborate with David Mabey, a friend and scientific colleague who, for several years, was a clinician at the U.K. Medical Research Council Tropical Disease Unit located in T h e Gambia in West Africa. Just as

14

M.E. WARD

in the in vitro model, trachoma in children is characterised by intermittent periods of clinical activity and chlamydial shedding. Laboratory assays specific for chlamydial antigen or D N A may be positive, even when viable organisms cannot be recovered. 13 Active trachoma is essentially a childhood disease, finished by about age I5. T h e complications due to ocular scarring do not really become evident until about age 40. In between there is a mysterious period when individuals are obviously exposed to infection, are apparently protected from active disease, and rarely shed viable chlamydiae. Nevertheless, ocular scarring continues to progress. 14 Using a prototype chlamydial enzyme immunoassay antigen detection test on longitudinal, whole village studies in T h e Gambia we obtained, for the first time, quantitative evidence that clinically inapparent, trachomatous infection does indeed occur, especially in the very young and in adults with scarring disease. 9' 1~'15 Indeed ocular carriage of chlamydial antigen, in patients from w h o m viable C. trachomatis is only rarely isolated, was significantly associated with conjunctival scarring) 5 This has subsequently been confirmed by P C R (Hayes et al., submitted). In Manchester, Steve Campbell a n d colleagues have suggested that a key factor in persistent or inapparent chlamydial infection is mucosal turnover. T r a u m a to the genital tract or a coincident gonococcal infection is t h o u g h t to stimulate mucosal turnover, perhaps triggering conversion of an existing cryptic persistent infection into symptomatic, productive infection. 1~ In the genital tract, infection is evidently maintained during menses despite sloughing of the endometrial lining. It is known that the leading lamella of endometrial cells are capable of picking up and transporting bacterial particles (phagokinesis) including Chlamydia. Perhaps infected cells from endometrial glandular stubs migrate, spreading infection onto the d e n u d e d stroma following menses or trauma. ~6 This does not explain why C. trachomatis infection is generally restricted to glandular epithelial cells in vivo. A major area for future chlamydial research is going to be to define the role of persistent infection in the epidemiology of clinical chlamydial infections. This will require careful comparison of viability-dependent assays of infection with viability independent methods, including quantitative chlamydial antigen detection and PCR. I m m u n o l o g i c a l r e q u i r e m e n t s o f a c h l a m y d i a l vaccine

H i d d e n within the cell, Chlamydia are relatively inaccessible to the host's i m m u n e . s y s t e m . Antibody binding to the EB envelope might inhibit chlamydial uptake by receptor blockade or by modifying EB surface charge and hydrophobicity. In the upper genital tract the fallopian tubes are capable of responding to infection with sIgA antibody and the epithelia possess the necessary receptors for secretory component. 17 Hydrophilic slgA would likely be more effective than I g G for blocking chlamydial attachment to the host cell. However, because chlamydial infection is primarily restricted to ocular and genital epithelia, there have to be adequate levels of effective antibody in genital secretions if chlamydiae are to be neutralised before cell entry. This will be difficult to achieve. Dimeric (sIgA) or pentameric (IgM) antibody would probably be more effective than m o n o m e r i c I g G for cross-linking the

Future of chlamydial vaccines

15

surface of the interiorized EB, thereby blocking the restructuring of the outer envelope essential for differentiation. Opsonisation of EB by the phagocytic cells which patrol the genital epithelia would probably be most effectively achieved by IgG, not sIgA. This effect would be enhanced if complement fixed at the surface of the EB is important in protection. Complement is likely to be present following the initiation of infection once the acute inflammatory response is underway. IgG antibodies are also likely to be critical if antibody dependent cytotoxic (ADCC) attack by lymphocytes on chlamydial infected cells is important. Unfortunately, although chlamydial antigen is exported from infected cells, there is still no clear evidence that it is freely exposed for immune attack at the aqueous surface (reviewed in Ward, 19881o). Such antigens would be mandatory for targeting chlamydial infected cells for A D C C or To attack. T h e potential role of N K cells or T gamma delta cells in restricting chlamydial infections to the mucosal surface deserves further study. I m m u n o p a t h o l o g i c a l reactions to c h l a m y d i a l vaccines

Paradoxically, even though genital rather than ocular chlamydial infections are of greatest importance in the developed world, most funding until recently has gone into developing a trachoma vaccine. Empirical attempts in the I96OS to prevent trachoma by vaccination of human subjects with crude whole C. trachornatis resulted at best in short-lived protection which was serovar specific and associated with local (tear) antibody responses (reviewed in Schachter and Dawson, I97818). T h e r e are at least I5 different serovars of C. trachomatis, of which four (A, B, Ba and C) are particularly associated with trachoma although they may rarely cause genital tract infection. Unfortunately in some subjects the disease was made worse when a vaccinated individual subsequently became naturally infected with C. trachomatis of a different serovar to the immunising strain. T h e presumed immunopathological mechanism was genus specific and associated with damaging cell-mediated immune responses. T h e major complications of both ocular and genital chlamydial infections involve scarring damage, occurring mainly in individuals repeatedly exposed to the infection. In the pig-tailed macaque, primary inoculation is associated with a self-limited salpingitis with little residual damage, whereas repeated inoculation leads to adnexal adhesions and hydrosalpinx formation. 19 Presumably individuals repeatedly challenged are likely to have immunologically committed T-cells capable of producing gamma interferon, tumour necrosis factor etc. on interaction with chlamydial antigen. In vitro gamma interferon is chlamydiastatic, stimulating tryptophan breakdown and thus depriving replicating RB of an essential amino acid. 2° T u m o u r necrosis factor and recombinant interleukin I are also chlamydiastatic but seem to operate by a different mechanism which involves prostaglandin E2 elevation. In fact, there is some evidence that the susceptibility of host cells to chlamydial infection and replication is under cyclic nucleotide and prostaglandin regulatory controls ~ a subtlety which still has to be worked out. T h e net result can be a persistent, non-productive form of chlamydial infection. U n d e r these

I6

M.E. WARD

circumstances chlamydiae may secrete enhanced amounts of a 57 to 6o kDa heat shock protein 2~'23 similar to groEL type heat shock proteins in Escherichia coli and Mycobacteria. Cell-mediated i m m u n e responses to this protein in a guinea-pig model of inclusion conjunctivitis due to C. psittaci have been implicated in the development of damaging chronic inflammatory responses. ~ It is not yet clear if this ultimately leads to scarring. Inhibition of chlamydial development by gamma interferon or cytokines may be the mechanism by which persisting, clinically inapparent chlamydial infection occurs in vivo (see above). In trachoma, immunoregulatory mechanisms may be compromised by the fact that, as we have recently shown, chlamydial infection results in the aberrant production of class II M H C antigens at the surface of ocular epithelia) 5 Again, this probably reflects the local production of gamma interferon and cytokines secondary to chlamydial induced inflammation. T h e function of class II M H C is to process antigen for Th-cells. In diseases like thyroiditis, immunoregulatory defects and even a u t o i m m u n e attack have been associated with aberrant class II expression on infected epithelial cells. T w o considerations for chlamydial vaccine design arise out of the possible role for T-cells. Firstly, both circulating and mucosal antibody responses to chlamydial protein antigens are undoubtedly going to be T-cell dependent and considerable progress has recently been made in characterising m u r i n e T helper epitopes of M O M P ) 6' 27 Cytotoxic T-cells or NK-cells may well be important in restricting and controlling established infection. Secondly, it is imperative that the T-cell responses liable to induce scarring damage (cicatricial disease) are avoided. In m a n this means it is going to be necessary to channel vaccine production through appropriate Th-subsets. For both these considerations, the present-day emphasis is to define some chlamydial antigen capable of generating a protective, but not damaging, i m m u n e response. Vaccine design and m u c o s a l i m m u n i t y in the genital tract

A key problem of chlamydial vaccine design is how to achieve effective mucosal immunity. T h e classic m e t h o d of inducing specific sIgA antibody is by the oral presentation of particulate, replicating antigen directly to the gut or other epithelial surface, providing a persisting antigenic stimulus via Mcells to the c o m m o n mucosal i m m u n e system. 28 Immunologically committed IgA plasmablasts traffic between the gut, lung, breasts and perhaps other glandular or mucosal organs, perhaps explaining (i) why live C. trachomatis serovar Lz organisms administered orally or intranasally protect mice against vaginal challenge and (ii) why only live chlamydiae fed to guinea-pigs are able to elicit i m m u n i t y to chlamydial challenge (reviewed in Taylor-Robinson and Ward, I98929). Live replicating Chlamydia vaccines are one way of achieving antigenic persistence but they are unacceptable in m a n because of the risk of adverse delayed hypersensitivity reactions. However, genetically engineered, live attenuated virus or bacterial vaccine vectors offer an alternative mechanism by which a persistent antigenic stimulus could be achieved. T h i s presupposes that mucosal i m m u n i t y in the genital tract, which is hardly understood, is like that in the gut. In reality the genital tract has no obvious homologue to the enteric M-cell, and comparatively few intramural lymphoid follicles for

Future of chlamydial vaccines

I7

antigen processing. T h e vagina is relatively anergic immunologically, explaining why female infertility due to sperm antibody is a rare event! Much vaginal antibody is probably derived as a result of the transudation of serum IgG and IgA via glandular lacunae across the endometrial stroma, a° Parenteral routes may therefore prove very important for stimulating mucosal immunity in the genital tract. Is a v a c c i n e f e a s i b l e ?

T h e fact that repeated genital or ocular chlamydial infections are commonplace might suggest there can be little hope of producing an effective chlamydial vaccine. T h e question arises whether chlamydial vaccine development is a dream or reality. It is worth summarising some of the evidence that protective immunity to chlamydial infection is eventually produced following repeated exposure. (i) T h e r e is a lower than expected incidence of chlamydial infection in individuals frequently exposed to the organism, e.g. prostitutes, at This is not true of other organisms which are sexually transmitted and whose prevalence is related to the n u m b e r of sexual partners, e.g. Trichomonas vaginalis. (2) Active ocular chlamydial infection is primarily restricted to children suggesting that immunity develops with age. I4,~2 (3) H u m a n trial and studies in other primates show that short-term protective immunity to C. trachomatis can be produced by vaccination with whole Chlamydia (see above). (4) Antibodies directed against the chlamydial major outer membrane protein ( M O M P ) neutralise chlamydial infection of host cells in vivo and in vitro.a3.34 M O M P is exposed at the surface of the infectious chlamydial EB, forming a pore in the outer membrane controlling nutrient ingress 35 and perhaps, in part, chlamydial differentiation. M O M P may also be involved in chlamydial adhesion to the host cell 26 although no definite chlamydial adhesins or receptors have yet been unambiguously identified. 1°,11 (5) Sequencing studies 36-3s show that, although M O M P is the basis of limited serotype variation, it is nevertheless antigenically stable. Prospects for the development of a chlamydial vaccine have recently been reviewed. 29 MOMP

and chlamydial

vaccine development

T h e only protective antigen which has been unambiguously identified is the chlamydial major outer membrane protein, M O M P . This protein, discovered independently by two groups in the U.S.A. 39,4° and one in the U.K. 41 represents the majority of the surface exposed protein of the infectious EB. Stephens et al. 37'42 succeeded in the challenging task of initial cloning and sequencing of the gene encoding the M O M P of C. trachomatis serovar L2 after a n u m b e r of false starts by other workers. This was shortly followed by the M O M P sequence for serovar L I. Comparative sequencing of M O M P from different C. trachomatis serovars 87 gave important insight into the structure of the protein. Essentially, M O M P is an outer envelope, pore-forming protein of around 40 kDa characterised by four variable sequences (VS I to IV) with five intervening constant regions of conserved structure and function. High-level

18

M.E. WARD

expression of the M O M P in E. coli was first achieved in Southampton by Pickett et al. 43 This was of considerable practical importance. Chlamydia trachomatis is difficult and expensive to grow on a large scale; it would have been a horrendous task to produce adequate quantities of M O M P for vaccine studies without the availability of recombinant protein. It had been known for some time that M O M P is an immunodominant antigen, carrying epitopes at the EB surface which determine the serovar specificity of C. trachomatis. Some I5 serovars of C. trachomatis have been internationally recognised using the classic (but crude) microimmunofluorescence test. Amino acid variations arise within and around the serovar specific M O M P epitopes. 35 Chlamydiologists derive considerable, if perverse, pleasure from semantic arguments concerning when a variant genotype becomes a new serovar[ This philosophical question is likely to lead to reappraisal of the serological classification of C. trachomatis as more sequence data become available. Immunologically related serovars are classified within the same serocomplex because they share a common, sub-species specific M O M P epitope. T h e r e are two main sero-complexes, confusingly given the same letters (B and C) as two of the serotypes (to further confound non-specialists) ] At least two further epitopes on M O M P carry C. trachomatis species and even Chlamydia genus specificities. At the present time, partial (variable regions) or complete nucleotide sequences are available for M O M P of all the classic 15 serovars of C. trachomatis. Sequences are also currently available for five strains of C. psittaci and for two strains of C. pneumoniae (see Carter et al. 12 for references to M O M P sequences). T h e importance of these M O M P B cell epitopes is that the serovar and sub-species (sero-complex) epitopes are exposed at the surface of the EB and thus accessible for immune attack. Monoclonal antibody to these epitopes neutralises chlamydial infection for certain (not all) tissue culture cells and in vivo for the primate eye. 3~ Relatively large amounts of antibody are required for neutralisation, although complement may enhance its efficacy. T h e next step was to define, as precisely as possible, the amino acid sequence of the critical binding region of the M O M P epitopes. Likely epitopic regions were first crudely defined by M O M P sequence comparison. Practical epitope mapping was then achieved conventionally using either synthetic, freesolution peptides ~ or genetically engineered beta-galactosidase fusion proreins 45 spanning the theoretical epitopes. Both of these approaches are timeconsuming and failed to give single amino acid resolution of the critical antibody binding sites. T h e approach in this laboratory 46-4s was to map antibody binding sites (epitopes) to single amino acid resolution using a radical new technique of solid phase peptide synthesis. Solid-phase peptide synthesis has been around for a long time. However, a modification of the technique first described by Geysen et al., 49 uses cyclotronirradiated polythene 'pegs' for the peptide synthesis. T h e peptides are synthesised by F M O C chemistry in situ on pegs arranged in a I2 × 8 matrix corresponding to an E L I S A tray. Thus, the solid phase peptides can be readily tested for antibody binding by EIA. T h e revolutionary feature of this technique was that it became facile to synthesise many thousands of peptides both simultaneously and cheaply. For this reason Wayne Conlan and myself

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Fig. I. Reactivity of the serovar L I M O M P - s p e c i f i c monoclonal antibody 5F9 With synthetic peptide analogues (mimotopes) of its critical binding site, D A V P . Hexapeptides were prepared by F M O C chemistry and assayed u s i n g the Pepscan (Geysen) technique. At position I, the aspartate residue (D) was exchanged for each of the other possible, naturally occurring, L-amino acids• A m i n o acids at other positions were held constant. T h i s strategy was repeated as s h o w n at position 2, 3 and 4; replacing respectively the alanine (A), valine (V) and proline (P) of the natural epitope. N and M at positions 5 and 6, present in the natural epitope, were kept constant t h r o u g h o u t . T h e experiment shows that a n u m b e r of amino acids can substitute for the D or P at positions I and 4, b u t the alanine and valine at positions 2 a n d 3 are essential. T h i s simple rule was used to design the experiment s h o w n in Fig. 2.

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20

M.E. WARD

quickly adopted the m e t h o d in I987 as soon as it became commercially available. At its simplest, a series of peptide fragments six amino acids in length and each overlapping by one amino acid are synthesised along the region of the putative serovar specific epitope, then assayed by E L I S A for their ability to react with neutralising monoclonals. T h u s , the critical binding domain for the serovar L I specific epitope was found to be D A V P , the single letter shorthand code for the amino acids aspartate, alanine, valine and proline. Moreover two different L I serotype specific antibodies, one from Atlanta and the other from Southampton, both recognised precisely the same contact peptides indicating this is a generally recognised epitope. ~° If any of these amino acids were missing from the peptide, antibody failed to bind. Using this powerful approach it was possible in just a few weeks to define, to single amino acid resolution, the binding sites of antibody to serotype, sub-species, species and genus-specific epitopes of serovar L I M O M P . 46 This approach has since been extended to other serovars, 35 representing, we believe, the highest resolution m a p p i n g of chlamydial M O M P B cell epitopes yet achieved. T h e interaction of the antibody paratope with chlamydial epitopes shows considerable subtlety. T h u s , an N terminal shift in critical binding site of just one amino acid was sufficient to prevent monoclonal antibody binding to the subspecies epitope on the surface of viable EB. 48 Loss of binding requirement for a single amino acid in the same region enabled an anti-peptide monoclonal antibody to react with useful, broadened species specificity with the subspecies-specific epitope at the surface of chlamydial EM. 48 T h i s indicates the potential for engineering and targeting antibodies to the chlamydial surface by using peptide immunogens. Unfortunately there is evidence that surface exposure of some critical epitopes is reduced as immature EB m e t a m o r p h o s e into fully infectious EB. 47 Epitopes at the surface of chlamydial EB show considerable topographic complexity. T h u s , we have recently demonstrated that variable segments II and IV (containing the serovar and subspecies-specific epitopes), are spanned by a single monoclonal antibody paratope. 5° Parts of VS II and IV thus form discrete, linear contact regions within a discontinuous epitope. This is to be expected given that the antibody paratope covers an area of several h u n d r e d square ~ngstr6ms. 48 Alterations in the binding constants for select regions within the discontinuous epitope will drastically affect antibody specificity. Once the critical amino acids had been identified for each epitope, it was possible, by synthesising all possible replacement amino acids at each position, to define the amino acids acceptable for antibody binding 46'~s a task which would have been hopelessly laborious using a conventional peptide synthesiser. T h e hitherto unpublished result is shown in Fig. I. For serotype Li-specific antibody binding to D A V P , the alanine (A) and valine (V) were absolutely irreplaceable, whereas limited substitution was possible for the aspartate (D) and proline (P) (Fig. I). Retaining the essential alanine and valine residues, it proved possible to construct ' m i m o t o p i c ' hexameric peptides which reacted with the L I serotype-specific antibody m u c h stronger (I3-fold higher OD) than with the native peptide (Fig. 2), even t h o u g h the remaining four amino acids were foreign to the natural M O M P sequence. T h e likely explanation is that short chain peptides, being inherently flexible, only approximate to the

Future of chlamydial vaccines

21

Multiple amino acid replacement LI type epitope~-x--~MAb 5F9 14 12 I0 8

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ii

D S A V P N

Fig. 2. Experiment showing the binding of the same monoclonal antibody (5F9) to mimotopic peptides in the Pepscan technique. The alanine-valine minimal essential binding site found in the previous experiment was retained throughout. Multiple amino acids at other positions in the epitope K D A V P N M were then changed to test a number of structural theories. The horizontal line shows the reactivity of the natural epitopic sequence, arbitrarily assigned a value of I on the vertical scale. Retaining the essential alanine and valine residues, it proved possible to construct 'mimotopic' hexameric peptides which reacted with 5F9, a monoclonal antibody produced by immunisation with native serovar L I EB, much stronger (I3-fold higher OD) than with the native peptide. One explanation is that short chain peptides, being inherently flexible, only approximate to the homologous constrained amino acid sequence within the native protein. However, some of the mimotopic peptides may fortuitously produce conformations which more closely resemble the three-dimensional structure of the native sequence internally imaged by the F(ab) 2 of the monoclonal antibody. Such mimotopic peptides, with their greater conformadonal fidelity, could be important for serodiagnosis and vaccine development (see text). (Both figures hitherto unpublished experiments of Conlan and Ward performed in 1988.) *Reactivity of I = OD of 0.22.

homologous, constrained, amino acid sequence within the native protein. By contrast mimotopic peptides may fortuitously produce conformations which more closely resemble the three-dimensional structure of the native sequence as imaged by the corresponding antibody. We are currently exploring whether, conversely, mimotopic peptides used as vaccines are able to generate antibody which binds more effectively to constrained MOMP at the surface of Chlamydia than the native peptide. If this proves true, mimotopic peptides would be a major advance in vaccine design, as well as forming the basis for novel serodiagnostic tests. Another approach is to use recombinant M O M P (rMOMP) itself. Immunisation of rabbits with r M O M P results in an antibody response almost entirely directed against the variable, surface exposed, neutralising regions on M O M P . 51 We assume the strong immunogenicity of these regions on the recombinant protein may be because of the functional juxtaposition of good T~-epitopes, or, more likely, because of the high mobility of these surface loops on the chlamydial surface. The strong association of protein antigenicity with surface mobility has been noted for model proteins like myohaemerythrin.49

22

M.E. WARD

Epitope m a p p i n g and neutralisation data for C. trachomatis have been primarily derived from studies of m u r i n e monoclonal antibodies. Clearly antigens must be immunogenic in m a n if they are to make successful vaccines. Antibody responses to proteins are generally T - d e p e n d e n t and require the cooperation of class Ii M H C restricted Th-lymphocytes. T h e relationship between individual class II haplotypes and responsiveness to individual epitopes is still largely unpredictable, notwithstanding the computer algorithms which have been published for this purpose. However, using the Geysen procedure in conjunction with overlapping hexameric and decameric peptides of M O M P , it was a simple matter to define the epitopic specificity of polyclonal h u m a n serum antibodies, thereby pragmatically determining the proportion of individuals in a target population able to respond to a (neutralising) epitope of interest. T h u s , in the case of trachoma in the Gambian village of Jali, we were able to establish conservatively that some 60 % of individuals had serum antibody to the serovar-specific epitope and the subspecies-specific epitope on M O M P of a serovar B strain circulating in the village 52 (Kajbaf et al., in preparation). Both these epitopes are associated with neutralisation. I assume that in the natural infection these antibodies offer little protection because they are present only at low levels at the relevant epithelial surfaces. T h e importance of this observation is that immunogenetic factors in man, such as M H C Class II haplotype, are unlikely to restrict significantly the immunogenicity of these epitopes once they have been engineered into an appropriate vaccine. A n i m a l m o d e l s for vaccine d e v e l o p m e n t

Animal models are regrettably essential for testing vaccine efficiency and to study the immunopathological basis of chlamydial disease. N o n - h u m a n primate models are relevant but expensive and scarce for the large-scale breeding experiments necessary to ascertain the consequences of chlamydial infection for fertility or blindness. T h e guinea-pig can be used for studying experimental ocular and genital C. psittaci infections, but is generally refractory to infection with C. trachornatis. T h e mouse offers a consistent model of genital tract infection using h u m a n genital tract isolates of C. trachomatis. 53'~4 This model is particularly useful because it permits statistically adequate studies of chlamydial colonisation and sequelae, including salpingitis and infertility. T h e plentiful availability of good immunological markers in the mouse makes this animal valuable for studies of genital tract chlamydial infection. I m p a i r m e n t o f o v u m transport occurs as early as 8 days after intrabursal inoculation of C. trachornatis into C57HeJ mice, with spread to the uninoculated contra-lateral tube by 28 days after infection. Once egg transport is impaired the effect is long-lasting, with mice 260 days after challenge still affected. 5~ T h e most striking pathological change on field emission scanning electron microscopy was mucus congestion at day 8 accompanied by tubal oedema and the loss of ciliated epithelial cells from the oviduct lumen. M u c u s accumulation may be a major factor leading to tubal blockage. Hydrosalpinx is a c o m m o n outcome of chlamydial infection in the mouse model, as it is in women. Oviducts collected I6O-26o days after chlamydial infection had an apparently normal epithelial surface, with only

Future of chlamydial vaccines

23

patchy signs of epithelial disorganisation and apparently normal ciliary activity. Nevertheless, ovum transport remained impaired. This situation is analogous to that of women with tubal occlusion in whom surgical restoration of tubal patency often fails to achieve fertility and there is a high frequency of subsequent ectopic pregnancy. 5G'57 In collaboration with Maureen Tuffrey, we have recently begun to use the mouse model for vaccine development studies (Tuffrey et al., in pressSS). To summarise, mice immunised parenterally with serovar L1 r M O M P on alum adjuvant showed a slight, but insignificant, decrease in the number of animals colonised following subsequent challenge with a serovar F genital tract isolate of C. trachomatis. Salpingitis was also less severe in vaccinated than in control animals. These findings correlated with the presence of specific IgG in the circulation and cervical washes of test animals. In mice vaccinated with r M O M P via the Peyer's patches of the gut, in an attempt to stimulate the common mucosal immune system, there was a marked reduction in the number of chlamydiae being shed from the vaginas of immunised mice coupled with a reduction in the duration of colonisation. Unfortunately this did not reduce the severity of subsequent salpingitis or infertility. Although the magnitude of these effects was small, it seems likely that reduction of chlamydial colonisation following local immunisation with r M O M P requires the local mucosal immune system whereas reduction in disease severity can be achieved by parenteral immunisation; either effect might be appropriate for controlling h u m a n infertility and ectopic pregnancy due to Chlamydia. There are several ways in which this heterotypic protective immunity might be enhanced. Purification of the r M O M P under milder conditions than we employed so as to preserve conformational epitopes might be crucial for protection. In the guinea-pig model, native M O M P extracted from C. psittaci using octyl glucoside rather than the more denaturing SDS shows better protection. ~9 T h e use of more potent adjuvants or of insoluble r M O M P to trigger a more sustained immune response might also be crucial. M O M P might also be incorporated into live vectors or microencapsulated to enhance the common mucosal immune system. Concluding remarks Studies of chlamydial immunochemistry at the molecular level really only began in earnest in I98I with the identification of M O M P . Chlamydia are problem organisms to work with, being difficult to grow in any quantity and lacking a transformation or other system for their genetic manipulation. Progress over the last I o years has been remarkable, including the identification of at least one protective antigen and the delineation of its B-cell epitopes to single amino acid resolution. T h e fact that r M O M P is capable of conferring modest protection in the mouse is encouraging for future vaccine development but the search must continue for other protective antigens. T h e r e are still a number of proteins of unknown function which have been identified in the chlamydial outer envelope. In vaccine development the emphasis is switching to different methods of targeting the immune response. T h e problem of stimulating long-term mucosal immunity in the genital tract is substantial; we know so little about genital tract immunology. Progress on chlamydial and

24

M.E. WARD

o t h e r g e n i t a l t r a c t v a c c i n e s will b e d e p e n d e n t o n , a n d s t i m u l a t e d e v e l o p m e n t s in, m u c o s a l a n d r e p r o d u c t i v e i m m u n o l o g y . T h e g r e a t l y e n h a n c e d u n d e r s t a n d i n g o f t h e c e l l u l a r r o u t e s o f a n t i g e n p r o c e s s i n g a n d p r e s e n t a t i o n , t h e role o f i m m u n o m o d u l a t o r s a n d t h e a d v e n t o f S C I D m o u s e v a r i a n t s will b e c r u c i a l to t h e d e v e l o p m e n t o f g e n i t a l t r a c t v a c c i n e s a n d t h e u n d e r s t a n d i n g o f i m m u n i t y to c h l a m y d i a l i n f e c t i o n s . (I am profoundly grateful to the Edna McConnell Clark Foundation (New York), the Swedish Agency for Research Cooperation ( S A R E C , Stockholm), the Wessex Medical T r u s t (Southampton) and the H u m a n Reproduction P r o g r a m m e of the W o r l d Health Organisation (Geneva) for their support of chlamydial research in Southampton.)

References I. Westr6m L, M~rdh P-A. Chlamydia salpingitis. Br Med Bull I983; 39: I45-I5O. z. Cates W, Farley T M M , Rowe PJ World-wide patterns of infertility: is Africa different ? Lancet I985; ii: 596-598. 3. Mabey DCW, Ogbaselassie G, Robertson JN, Heckels JE, Ward ME. Tubal infertility in the Gambia: chlamydial and gonococcal serology in women with tubal occlusion compared with pregnant controls. Bull World Health Organization I985; 63 : I Io7-I I I3. 4. Robertson JN, Ward ME. Gonococcal and chlamydial infection in infertility and ectopic pregnancy. Contemp Rev Obstet Gynaecol I988; I: 60-66. 5. Robertson JN, Hogston P, Ward ME. Gonococcal and chlamydial antibodies in ectopic and intrauterine pregnancy. Br J Obstet Gynaecol I988; 95: 7I 1-716. 6. Chow WH, Daling J, Cares W, Greenberg RS Epidemiology of ectopic pregnancy. Epidemiol Rev I987; 9: 70-94. 7. Washington AE, Johnson RE, Sanders LL. C. trachomatis infections in the United States : what are they costing us? J . 4 M A I987; e57: 2070-2073. 8. WHO Guidelines for the prevention of genital chlamydial infections. Copenhagen : Published by World Health Organization Regional Office for Europe, I99O. 9. Ward ME, Hawkins JD, Shahani A. Evaluation of trachoma control strategies using a computerised simulation. In: Bowie W e t al., Eds. Fhlamydial infections. Cambridge: Cambridge University Press I99O: 591-594, Io. Ward ME. The chlamydial developmental cycle. In: Barron A, Ed., The Microbiology of chlamydia. Baco Raton, FL: CRC Press, z988: 7z-97. I I. Ward ME, Clarke, IN. New perspectives in chlamydial biology and development. In: Bowie W et al., Eds. Chlamydial infections. Cambridge: Cambridge University Press, I99O: 3-I4. I2. Carter MW, AI-Mahdawi SAH, Giles, IG, Trehame JD, Ward, ME, Clarke, IN. C. pneumoniae : nucleotide sequence and taxonomic value of the major outer membrane protein of IOL-2o7. J Gen Microbiol I99I ; I37: 465-475. 13. Schachter J, Moncada J, Dawson CR et al. Non-culture methods for diagnosing chlamydial infection in patients with trachoma: a clue to the pathogenesis of the disease ? J Infect Dis I988; I58:I347-I352. x4. Ward ME, Bailey R, Lesley A, Kajbaf M, Robertson JN, Mabey DCW. Persisting inapparent chlamydial infection in a trachoma endemic community in The Gambia. Scand J Infect Dis I99o: 69 (Suppl.): I37-I48. 15. Mabey DCW, Robertson JN, Ward ME. Detection of ChIamydia trachomatis by enzyme immunoassay in patients with trachoma. Lancet I987; ii: z49I-I492. I6. Campbell S, Richmond SJ, Haynes P, Gump D, Yates P, Allen TD. An in vitro model of Chlamydia trachomatis infection in the generation phase of the human endometrial cycle. J Gen Microbiol I988; I34: 2077-2087. 17. Kuteh WH, Kuteh CC, Blackwell RE, Cart BR, Gore H, Mestecky J. Secretory immune system of the female reproductive tract II. Local immune system in normal and infected fallopian tube. Fert Steril I99o; 54: 51-55. I8. Schachter I, Dawson C. Chlamydial infections. Littleton, MA: PSG Publishing, I98L 19. Patton DL, Kuo C-C, Wang SP, Halbert SA. Distal tubal obstruction induced by repeated

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20. 2i. 22. 23. 24. 25. 26. 27. 28. 29. 30. 3 I.

3~. 33. 34. 3536.

37. 38. 39.

25

Chlamydia trachomatis salpingeal infections in pig-tailed macaques. J Infect Dis I987; x55 : I292-I299. Byrne GI. The host cell, host immune responses, and the intraceUular growth of Chlamydia. In Moulder JW, Ed. Intracellular parasitism. Boca Raton, FL: CRC Press, I989 : 35-49. Ward ME, Salari HS. Control mechanisms governing the infectivity of Chlamydia trachomatis for Hela cells: modulation by cyclic nucleotides, prostaglandins and calcium. J Gen Microbiol I982; I I 2 : 639-65o. Morrison RP, Belland RJ, Lyng K, Caldwell HD. Chlamydial disease pathogenesis. The 57-kDa chlamydial hypersensitivity antigen is a stress response protein. J Exp Med I989; I7o: I27I-I283. Morrison RP, Lyng K, Caldwell HD. Chlamydial disease pathogenesis. Ocular hypersensitivity elicited by a genus-specific 57-kDa protein..7 Exp Med I989; I69: 663-675. Watkins NG, Hadlow WJ, Moos AB, Caldwell HD. Ocular delayed hypersensitivity: a pathogenetic mechanism of chlamydial conjunctivitis in guinea-pigs. Proc Natl Acad Sci USA I982; 83: 7480-7484. Mabey DCW, Bailey RL, Dunn D et al. Expression of MHC class iI antigens by conjunctivial epithelial cells in trachoma implications concerning the pathogenesis of blinding disease. J Clin Path I99I; 44: 285-289. Su H, Zhang Y-X, Barrera O, Watkins NG, Caldwell HD. Differential effect of trypsin on infectivity of Chlamydia trachomatis: loss of infectivity requires cleavage of major outer membrane protein variable domains II & IV. Infect Immun I988; 56: 2o94-2Ioo. Allen JE, Beatty PR, Stephens RS. Recombinant fusion proteins define T cell antigenic sites on the major outer membrane protein of C. trachomatis. In: Bowie W e t al., Eds. Chlamydial infections. Cambridge: Cambridge University Press, i99o : IoI-IO4. Allardyce RA, Bienenstock J. The mucosal immune system in health and disease with an emphasis on parasitic infection. Bulletin of the World Health Organization I984; 62: 7-25. Taylor-Robinson D, Ward ME. Immunity to chlamydial infections and the outlook for vaccination. In : Meheus A, Spier R, Eds. Vaccines for sexually transmitted diseases. Oxford : Butterworths, I989: 78-94. Parr EL, Parr MB. Anti-bacterial IgA and IgG in mouse uterine luminal fluid, vaginal washings and serum. J Reprod Immunol I988; x3: 65-72. Laga M, Manoka AT, Nzila N, Ryder R, Behets F, van Dyck E, Piot, P. Genital chlamydial infection among prostitutes in Kinshasa. Prevalance, incidence, risk factors and interaction with HIV. In: Bowie W e t al., Eds. Chlamydial infections. Cambridge: Cambridge University Press, I99O: 584-587. Treharne JD, The Community epidemiology of trachoma. Rev Infect Dis I985; 7: 760-764. Lucero ME, Kuo CC. Neutralization of Chlamydia trachomatis cell culture infection by serovar-specific monoclonal antibodies. Infect Immun I985; 5o: 595-597. Zhang Y-X, Stewart S, Joseph T, Taylor HR, Caldwell HD. Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of C. trachomatis. J Immunol I987; x38: 575-58L Bavoil P, Ohlin A, Schachter J. Role of disulfide bonding in outer membrane structure and permeability in Chlamydia trachomatis. Infect Immun I984; 44: 479-485. Hayes LJ, Pickett MA, Conlan JW, Ferris S, Everson JS, Ward ME, Clarke IN. The major outer membrane proteins of Chlamydia trachomatis serovars A and B; intra-serovar amino acid changes do not alter specificities of serovar- and C subspecies-reactive antibody binding domains. J Gen Microbiol I99o; x36: I559-I566. Stephens RS, Sanchez-Pescador R, Wagar EA, Inuoye C, Urdea MS. Diversity of Chlamydia trachomatis major outer membrane protein genes. J Bacteriol I987; I69: 3879-3885. Yuan Y, Zhang Y-X, Watkins NG, Caldwell HD. Nucleotide and deduced amino acid sequences for the four variable domains of the major outer membrane proteins of the I5 Chlamydia trachomatis serovars. Infect Immun I989; 57 : Io40-Io49. Hatch TP, Vance DW, A1-Hossainey E. Identification of a major envelope protein in Chlamydia spp. J Bacteriol I98I ; r46 : 426-429.

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4o. Caldwell HD, Kromhout J, Schachter J. Purification and partial characterisation of the major outer membrane protein of Ghlamydia trachomatis. Infect Immun x98I; 3x:

ZI6Z-II76. 4 I. Salari SH, Ward ME. Polypeptide composition of Chlamydia trachomatis. J Gen Microbiol I98I; I23: I97-2o7. 42. Stephens RS, Inouye CJ, Wagar EA. A species-specific major outer membrane protein domain. In: Oriel D et al., Eds. Chlamydial infections. Cambridge: Cambridge University Press, 1986; IIO-II3. 43. Pickett MA, Ward ME, Clarke IN. High-level expression and epitope localisation of the major outer membrane protein from Chlamydia trachomatis serovar LI. Mol Microbiol I988; 2: 681-685. 44. Stephens RS, Wagar EA, Schoolnik GK. High-resolution mapping of serovar-specific and common antigenic determinants of the major outer membrane protein of Chlamydia trachomatis. J Exp Med 1988; 167: 817-83I. 45. Baehr W, Zhang Y-X, Joseph T, Su H, Nano FE, Everett KDE, Caldwell HD. Mapping antigenic domains expressed by Chlamydia trachomatis major outer membrane protein (MOMP) genes. Proc Natl Acad Sci USA I988; 85: 4000-40o4. 46. Colan JW, Clarke IN, Ward ME. Epitope mapping with solid phase peptides : identification of type-, subspecies-, species, and genus- reactive antibody binding domains on the major outer membrane protein of Chlamydia trachomatis. Mol Microbiol I988; 2: 673-679. 47. Colan JW, Ferris S, Clarke IN, Ward ME. Surface-exposed epitopes on the major outer membrane protein of Chlamydia trachomatis defined with peptide antisera. J Gen Microbiol I989; x35: 3219-3228. 48. Conlan JW, Kajbaf M. Clarke IN, Chantler S, Ward ME. The major outer membrane protein of Chlamydia trachomatis: critical binding site and conformation determine the specificity of antibody binding to viable chlamydiae. Mol Microbiol I989; 3: 3n-318. 49. Geysen HM, Meloen RH, Barteling SJ. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc Natl Acad Sci USA I984; 8I: 3998-4oo2. 5o. Conlan JW, Persson K, Newha11 WJ, Ward ME. Mapping of discontinuous epitopes on the major outer membrane protein of Chlamydia trachomatis using synthetic peptides and rnonoclonal antibodies. IN: Bowie E et al., Eds. Chlamydial infections, Cambridge: Cambridge University Press, I99o: 81-84. 5I. Conlan JW, Ferris S, Clarke IN, Ward ME. Isolation of recombinant fragments of the major outer membrane protein of Chlamydia trachomatis: their potential as subunit vaccines. J Gen Microbiol 1990; 36: 2o 13-2020. 52. Ward ME, Conlan JW, Clarke IN. Prospects for chlamydial vaccine development. Proc European Soc Chlamydia Bologna I988: I2I-I23. 53- Tuffrey M, Falder P, Gale J, Taylor-Robinson D. Salpingitis in mice induced by human strains of Chlamydia trachomatis. Br J Exp Pathol 1986; 67: 6o5-616. 54. Tuffrey M, Falder P, Gale J, Quinn R, Taylor-Robinson D. Infertility in mice infected genitally with a human strain of Chlamydia trachomatis. J Reprod Fertil I986 ; 78 : 251-26o. 55. Tuffrey M, Alexander F, Inman C, Ward ME. Correlation of infertility with altered tubal morphology and function in mice with salpingitis induced by a human genital-tract isolate of Chlamydia trachomatis. J Reprod Fertil I99O; 88:295-305. 56. Zamberletti D, Fedele L, Vercellini P, Acaia B, Candiani G. The significance of scanning electron microscopy findings from the endosalpinx in tubal pregnancies. Acta Eur Fertil 1983; I4: 57-65. 57. Tam PPL, Mao KR, Mac-Moune Lai F. The ultrastructural changes of the mucosa of blocked fallopian tubes. Br J Obstet Gynaecol I988; 95:802-807 58. Tuffrey M, Alexander I, Conlan W, Woods C, Ward M. Hetrotypic protection of mice against chlamydial salpingitis and colonisation of the lower genetial tract with a human serovar F isolate of C. trachomatis by prior immunisation with recombinant serovar L I major outer membrane protein. J Gen Microbiol I992, in press. 59. Batteiger BE, Rank RG, Soderberg LSF. Immunization of guinea-pigs with isolated chlamydial outer membrane proteins. In: Bowie W e t al., Eds. Chlamydial infections. Cambridge: Cambridge University Press, I99O: 265-268.