- Email: [email protected]
Vaccines against Chlamydia: approaches and progress
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Vaccines against Chlamydia: approaches and progress Andrew J. Stagg
Infections of the eye and genital tract with the bacterium Chlamydia trachomatis are a major cause of morbidity worldwide and are costly to treat. Development of a vaccine capable of protecting against infection or severe disease presents special challenges but would be the most effective long-term option for control of chlamydial disease. Progress has been made in understanding protective and pathological immune mechanisms in these infections, and a number of potential vaccine candidates have been developed. INFECTION with Chlamydia trachomatis is a major cause of sexually transmitted genital tract disease worldwide and of ocular disease in the developing world (Table 1). In 1990, an estimated 1.5–6.7 million individuals worldwide were visually impaired as a result of trachoma1. Genital tract infection can be asymptomatic or cause mild disease, but in up to 40% of infected women the organism spreads into the upper genital tract and is a major cause of pelvic inflammatory disease (PID), with consequences that include infertility and ectopic pregnancy. It has recently been estimated that 80 000 women in the UK alone suffer from these serious consequences of chlamydial infection every year, costing nearly £60 million to treat2. If left untreated, both ocular and genital tract chlamydiosis are chronic inflammatory conditions with disease severity being related to persistent or repeated infection. Pathology is immunologically mediated3 but the exact mechanism is unclear. Here, I outline the challenges to the development of chlamydial vaccines based on our understanding of the immunology of chlamydiosis, and describe current approaches and future prospects for vaccine development in the light of these challenges.
(1.0 mm), metabolically active, reticulate bodies (RBs). RBs divide by binary fission within the growing inclusion and undergo a second differentiation step to yield new infectious EBs, which are released from the host cell. There are four species within the genus Chlamydia. C. psittaci and C. pecorum are important veterinary pathogens; C. pneumoniae causes acute respiratory infections in humans and has recently been linked with heart disease as a result of its detection in atherosclerotic lesions3. However, C. trachomatis is the major human pathogen and the development of vaccines for control of infections by this species is the focus of this review. C. trachomatis can be subdivided into biovars based on the host species and anatomical site that are infected. The biovars are further subdivided into serologically distinguishable variants termed serovars (Table 1), which arise from variation in the 40 kDa major outer membrane protein (MOMP). This variation is clustered in four surface-exposed variable regions (VSI–VSIV) separated by five conserved regions. MOMP is oligomeric and might have porin activity5. An involvement in attachment to host cells has also been proposed5 and it might be at this level that restriction of serovars to particular sites of infection (Table 1) occurs.
The Chlamydia genus Chlamydia are obligate intracellular Gram-negative bacteria with a unique life cycle (reviewed in Ref. 4). They exist in two major forms (Fig. 1): the small (200–300 nm) metabolically inactive elementary body (EB) is the infectious form; EBs are taken up by host cells into membrane-bound inclusions in which they reorganize into larger Andrew J. Stagg PhD Research Fellow Antigen Presentation Research Group, Imperial College School of Medicine at Northwick Park Hospital, Watford Road, Harrow, Middlesex, UK HA1 3UJ. Tel: +44 181 869 3428 Fax: +44 181 869 3532 e-mail: [email protected]
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Why do we need a vaccine? Antibiotic therapy effectively eliminates chlamydial infection but does not always affect established pathology; the presence of asymptomatic infection makes control by treatment of symptomatic individuals alone unlikely to be successful. However, screening for asymptomatic infection, followed by treatment of infectious individuals, could provide an alternative to vaccination for control of sexually transmitted infection in the developed world6, but are likely to prove too costly to be practicable in the developing world. Computer modelling suggests that even a partially efficacious chlamydial vaccination programme would rapidly reduce the prevalence of genital infection7. This approach is potentially less costly than a screening programme and could thus be extended to infections in the developing world. However, chlamydial infections present a number of challenges that need to be overcome before an effective vaccine can be developed. These are discussed below.
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The need to avoid immunopathology
Table 1. Diseases caused by C. trachomatis Biovara
Host
Disease
Serovar
Host cells
Ocular/genital Humans
Ocular: trachoma; conjunctivitis
A–C
Epithelial
Humans
Genital: urethritis; cervicitis; salpingitis; PID; reactive arthritis; epididymitis; perihepatitis
D–K
Epithelial
L1, L2, L3
Epithelial; macrophages
Ocular: ophthalmia neonatorum LGV
Humans
Lymphogranuloma venereum
MoPn
Mice
Pneumonitis
a
LGV, lymphogranuloma venereum; MoPn, mouse pneumonitis agent.
Epithelial
Because pathology in severe chlamydial disease is immunologically mediated4, there is a danger that vaccination might prime an inappropriate immune response and increase the severity of subsequent disease. This appears to have occurred in some clinical trials in the 1960s and 1970s, in which crude preparations of whole organisms were used in an attempt to vaccinate against trachoma (reviewed in Ref. 12). The findings are still debated but they appeared to show that the whole-cell vaccine increased the severity of disease in those who subsequently became infected, although it did reduce the incidence of disease in some circumstances. Similar observations have been made experimentally in monkey models of ocular chlamydial infection and in a mouse model of genital tract infection.
The need to define protective immune mechanisms The need to improve on protection conferred by natural infection
Immune mechanisms that protect the human host from chlamydial infection are not well characterized and this has been an obstacle to vaccine design. Recent studies with knockout mice carrying disrupted genes for cytokines17,22,23 or their receptors16 have begun to
Repeated chlamydial infections are common but a degree of protective immunity does seem to develop. The incidence of genital infection falls with age in a way that is unlikely to be attributable to changes in sexual behaviour, and reinfection is more likely to be detected when a longer time interval has elapsed since initial infection, suggesting a short-term protective effect8. In female sex workers in Kenya, re-infection is often caused by a different strain from that identified in the previous infection9. One explanation for this is the development of strainspecific immunity – an interpretation supported by early vaccine trials for trachoma (see below). Severe disease is associated with the A31 major histocompatibility complex (MHC) class I allele and, independently, a low CD4+ T-cell count (among HIV-seropositive individuals), suggesting a modifying effect of the immune response on disease outcome10. In ocular infection, active trachoma with shedding of large numbers of organisms is largely confined to childhood. Individuals with severe conjunctival scarring have lower cell-mediated immune responses than exposed controls or patients recovering from disease; this is consistent with a role for these responses in clearing infection11. Therefore, a degree of immunity is conferred by infection but it is shortlived and serovar-specific. An effective Figure 1. Transmission electron micrograph of a Buffalo Green Monkey Kidney cell 48 h after infection vaccine will need to increase the level of with Chlamydia trachomatis serovar L1. EB (elementary bodies), RB (reticulate bodies) and intermediate protection and confer immunity to all forms are evident within the inclusion. serovars.
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reveal the complexity of the host response to chlamydial infection. The relative contributions of antibody and cell-mediated mechanisms to control of infection and the most appropriate choice for immunoprophylaxis are still a matter of debate. Figures 2 and 3 illustrate potential protective mechanisms.
Humoral responses Specific antibodies might bind Chlamydia during the extracellular stages of its lifecycle (Fig. 2). In humans, anti-chlamydial IgA is associated with reduced shedding of the bacterium in women with chlamydial cervicitis (see Ref. 4 for references), and the development of serovar-specific immunity9 suggests the involvement of antibody. Studies in the guinea pig have also indicated a role for antibody in limiting primary genital infection13 and in a panel of knockout mice
Lumen
a
Epithelium
Stroma
Transcytosis
IgG
Plasma cell sIgA
b
Figure 2. Potential protective humoral immune mechanisms in chlamydial infection. Elementary bodies (EB, black circles) and reticulate bodies (RB, clear circles) are shown during a cycle of infection in genital tract epithelium (top, initiation of infection; bottom, release of new infectious progeny). Dimeric secretary immunoglobulin A (sIgA), locally produced and transcytosed into the lumen, and serum-derived or locally produced IgG might (a) prevent infection by neutralization or (b) reduce shedding of organisms.
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with disrupted b2-microglobulin (b2m), MHC class II or CD4 genespecific vaginal antibody correlated with the resolution of genital infection14. However, antibody responses are neither required to resolve primary genital chlamydial infections in mice15 nor sufficient to control primary infection or reinfection in mice with a disrupted interferon g (IFN-g) receptor gene16. Antibodies might contribute to control of secondary infection, but cell-mediated mechanisms again play the dominant role15,17. High concentrations of monoclonal antibodies (mAbs) to surfaceexposed epitopes on MOMP can neutralize infectivity in vitro and in vivo in animal models18. Thus it might be theoretically possible, but practically very difficult, to develop a vaccine that generates and sustains high levels of neutralizing antibody at the appropriate mucosal surface and thus block initial infection (Fig. 2a). The secondary infection experiments discussed above15–17 suggest that recall of a memory antibody response primed by vaccination is unlikely to control infection. Indeed, delayed exposure to antibodies might even enhance infectivity19. On a more positive note, the development of an antibody response might be sufficient to reduce shedding, which should block transmission and/or reduce the spread of the organism into the upper genital tract (Fig. 2b). A ‘backpack’ model in which hybridomas secreting mAbs to MOMP are implanted in syngeneic mice, shows only marginal effects on colonization of the genital tract, but reduced ascending infection and upper genital tract pathology20. A vaccine that blocks ascending infection of the genital tract and prevents PID would be a worthwhile achievement.
Cell-mediated responses Cell-mediated immunity can potentially control chlamydial infection in a number of ways (Fig. 3). Presentation of chlamydial antigen by antigen-presenting cells (APCs) (Fig. 3a) or by the infected cells themselves (Fig. 3b) might result in local activation of T cells and the production of cytokines that inhibit chlamydial growth or activate macrophages to scavenge extracellular bacteria (Fig. 3c). Knockout mice lacking MHC class II antigens and hence CD4+ T cells cannot resolve genital infection14, demonstrating the critical importance of these cells to the immune response to Chlamydia. These mice fail to make a T-cell-dependent antibody response to Chlamydia but, as discussed above, this is unlikely to account for their susceptibility. The inability to generate a T-helper 1 (Th1) response and the consequent reduced production of cytokines such as IFN-g and tumour necrosis factor a (TNF-a) is likely to be important. IFN-g is cytotoxic for Chlamydia-infected cells21 and can also inhibit growth of some strains by upregulating host-cell production of the enzyme indoleamine 2,3-deoxygenase, leading to cellular depletion of the essential amino acid tryptophan. IFN-g also induces nitric oxide synthase, and the nitric oxide produced inhibits chlamydial growth. Knockout mice lacking IFN-g or its receptor display delayed bacterial clearance after primary genital infection with C. trachomatis16,17,22,23, but they eventually control infection, indicating the presence of compensatory mechanisms such as the production of TNF-a17. Although IFN-g knockouts can acquire immunity to reinfection17,23 this was not the case in the IFN-g receptor knockouts. One possible explanation for this is the presence of additional ligands that can partially compensate for the absence of IFN-g by signalling through the IFN-g receptor. Thus, cytokines, especially those signalling through the IFN-g and TNF-a receptors, appear to be important contributors to clearance of the
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bacteria. Unfortunately, IFN-g and TNF-a are also implicated in the immunopathology that is a lasting consequence of chlamydial infection24,25. As well as being activated by APCs, effector CD4+ and CD8+ T cells might directly recognize infected cells (Fig. 3b). MHC class II restricted-CD4+ T cells are not known to recognize infected cells, but it is theroretically possible because epithelial cells can express MHC class II antigens following infection. Also, co-culture of epithelial cells with Chlamydia-specific CD4+ T cells inhibits bacterial growth by an unknown mechanism26. By contrast, human and murine CD8+ cytotoxic T cells (CTLs) are known to recognize Chlamydia-infected cells27–29. In addition to mediating cell lysis, CD8+ T cells are an important source of IFN-g. Nevertheless, b2m knockout mice, which lack CD8+ T cells, can successfully control genital chlamydial infection14. These studies neither exclude a contributory role for CTLs in the intact immune response nor preclude the possibility that a vaccine capable of stimulating a potent CTL response could confer protection. However, such a vaccine might do more harm than good because certain MHC class I alleles correlate with PID in humans10 and monkeys30, and with trachoma in humans31. The generation of Th1 cells and cytokines such as IFN-g plays an important role in controlling chlamydial infection, but recent experiments suggest additional complexity. Mouse strains can differ in susceptibility to genital infection despite making predominately Th1 responses32, and early bacterial clearance appears to depend on an IL-12-dependent but IFN-g-independent mechanism22. In mice, the ability to clear Chlamydia from the upper genital tract is associated with rapid recruitment of APCs into the tissue (A. Stagg et al., unpublished); such cells might be an important source of IL-12.
Lumen
Epithelium
Stroma
DC
Th1 CD4+
a Th1 CD4+ IFN-γ TNF-α
CD8+
CD8+
b
Cytolysis
Macrophage activation
c TNF-α NO TNF-α
Exploiting the immune response to Chlamydia As explained above, the immune response to Chlamydia is both complex and flexible. It is not yet clear which is the most appropriate response to target for immunoprophylaxis, or whether it will be possible to confer protection while avoiding immunopathology. Much of our understanding of cell-mediated immunity in chlamydial infection is based upon mouse models; little is known about the response in humans. Given that infection in humans runs a chronic course and appears to produce only short-term, serovar-specific immunity, the possibility that the organism has evolved mechanisms to block or evade the protective mechanisms that have been identified in mice warrants further investigation. A vaccine aimed at reducing the damage associated with severe disease might be a more realizable goal than one aimed at preventing infection. However, the possibility remains that any vaccine that is only partially effective might exacerbate disease; rapid and effective clearance of the organism might be the best protection against pathology. Nevertheless, attempts have been made to design vaccines that fully protect against chlamydiosis. These are described below. For the current discussion it is assumed that broadly similar approaches to vaccine development can be applied to ocular and genital infection; this is reasonable given the similarities in immunology but has not been firmly established.
near future, because of the risk of immunopathology, and because the large-scale production of pure chlamydiae is extremely difficult. The genomes of C. trachomatis and C. pneumoniae* have now been sequenced so, in the long term, it might be possible to engineer modified organisms that do not induce pathology. However, this approach can only be speculated upon at present and would require a huge research effort before its feasibility can be assessed.
Whole-organism vaccines
Subunit vaccines
In animal models, vaccines based upon whole organisms can confer a degree of protection against C. trachomatis infection. For instance, intranasal immunization of mice with viable organisms confers significant protection against colonization of the genital tract. However, whole-organism vaccination is unlikely to be attempted again in the
Recent research has focused on the development of vaccines based on MOMP, which does not appear to induce immunopathology. Inevitably, there is a lag between the definition of protective immune
Mφ NO
Figure 3. Potential protective cellular immune mechanisms in chlamydial infection. T cells, originally activated in lymphoid tissue draining the infection site by migratory dendritic cells (DC) and homing back to the tissue might be locally inactivated to produce cytokines [such as interferon g (IFN-g) and tumour necrosis factor a (TNF-a)] that (a) inhibit chlamydial growth or (c) activate macrophages. Alternatively they might directly recognize infected cells (b) and lyse them.
*http://www.genset.fr/
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Glossary Biovar – Subclassification of Chlamydia trachomatis isolates based upon the host species infected and the type of disease produced. Elementary body (EB) – One of two predominant chlamydial forms, EBs are the stage in the organism’s life cycle adapted to extracellular survival. They are resistant to osmotic and mechanical stress, have a condensed chromosome and appear metabolically inactive. The EB is the infectious chlamydial particle and mediates attachment to and invasion of target host cells. Immunodominance – The ability of one antigen or epitope in a mixture to elicit the stongest immune response in vivo. Major outer membrane protein (MOMP) – A 40 kDa, cysteine-rich chlamydial envelope protein comprising 60% of the EB’s surface protein. It is encoded by the omp1 gene, which is developmentally regulated during the chlamydial life cycle. Pelvic inflammatory disease (PID) – Inflammation of the female genital tract above the level of the uterine cervix. Inflammation can be confined to the endometrium (endometritis) or spread to the fallopian tubes (salpingitis), and can also involve the ovaries (salpingooophoritis). Further spread may involve the peritoneum (peritonitis) or the liver (Fitz–Hugh Curtis syndrome). PID is almost always associated with spread of microbes from the vagina or cervix into the upper genital tract. Long-term consequences include infertility and ectopic pregnancy, and can occur even when chlamydial PID is asymptomatic. Reticulate body (RB) – The vesicle-bounded, metabolically active form in the life-cycle of Chlamydia. They are more friable than EBs and are not infectious but undergo division by binary fission. As they do so the vesicle enlarges, often to occupy a large proportion of the cell; this structure is termed an inclusion. RBs revert to EBs, which are released from the host cell to continue the infectious process. Serovar – Serologically distinguishable variants that, in the case of C. trachomatis, arise from variation in MOMP gene sequences. T-helper 1 (Th1) cells – A CD4+ T-helper cell population characterized by the cytokines that they produce (interferon g, tumour necrosis factors a and b and interleukin 2). They tend to be involved in cell-mediated immunity.
mechanisms and the incorporation of these ideas into vaccine candidates, and we can expect our current knowledge of immune responses to chlamydiae to influence the design of future vaccines. To date, most MOMP-based vaccine candidates are designed to elicit protective antibody responses, although recent studies (see above) suggest that this approach is unlikely to be successful. MOMP is immunodominant for serological responses. Protective, serovar-specific antibodies recognize epitopes within VSI and VSII; species-specific and subspecies-specific epitopes are located in VSIV; and VSIII does not appear to contain important antibody recognition sites. This means that a vaccine protecting against all strains would have to contain variable regions from several different serovars. Regions of MOMP that stimulate T cells, in humans as well as in mice, have now been identified33–36. Although its dominance in cellmediated responses has not been established, many regions of 170
MOMP contain T-cell epitopes and many different MHC molecules can be used for recognition of MOMP epitopes. Furthermore, regions containing T-cell epitopes tend to be located in conserved rather than variable regions. These observations suggest that it should be possible to construct subunit vaccines containing relatively small, defined regions of MOMP capable of stimulating T-cell responses against all serovars in most or all of a population with different human leukocyte antigen (HLA) haplotypes. With the rationale that protection would be mediated by T-celldependent antibody production, two groups have engineered chimeric peptides in which variable segments containing B-cell epitopes are linked to regions containing one or more T-cell epitopes. The peptide produced by Su and Caldwell37 contained VSI of serovar A and induced antibodies to this sequence in six different mouse strains with disparate MHC haplotypes, indicating that the T-cell epitope(s) could be recognized in the context of multiple MHC alleles. However, the fine specificity of the antibody response differed between mouse strains: although sera from all strains recognized VSI, only two strains developed antibodies that recognized whole bacteria, and only these two sera had significant neutralization titres. Congenic mice revealed that the fine specificity of the antibodies raised to the vaccine was under the control of genes outside the MHC. Zhong and colleagues38 incorporated several variable segments into a single peptide with the intention of protecting against the multiple serovars. They increased antibody titres by incorporating polymers of the peptides into a lipid-based hydrophobic core. However, the antibody titres to whole organisms were again several orders of magnitude lower than titres to the peptide; neutralizing titres were lower still. These studies with chimeric peptides illustrate the importance of generating antibodies to conformation-dependent epitopes if neutralization is to be achieved. They also highlight the confounding factor of genetically determined variation in host response. These are challenges to designing peptide-based vaccines that elicit antibody production. Future vaccines might need to include structural features of the native B-cell epitope, as well as the correct linear sequence. Another potential problem arises from MOMP variants within the same serovar; these are small deviations from the prototype sequence but can be sufficient to allow escape from neutralization by antibodies39. MOMP and a number of these immunogenic subunits have been evaluated in animal models for their ability to confer protection against infection or disease but to date they have been at best partially effective (Table 2). This is despite the stimulation of both mucosal and serum antibody responses40–43. Some protection was also obtained by immunization with MOMP-derived peptide in the absence of antibody production44. There are several possible explanations for the poor protection conferred by these candidate vaccines: (1) Although immunogenic, the vaccine candidates might fail to stimulate protective immune mechanisms. Perhaps the antibody responses generated at mucosal surfaces are of insufficient titre or an inappropriate subclass, or perhaps they are not directed against protective (possibly conformation-dependent) epitopes. Alternatively, they might not stimulate the protective cell-mediated mechanisms revealed by recent knockout experiments (see above). Although systemic T-cell responses were observed in some studies, nothing is known about the responses stimulated at the site of infection. The chimeric peptide containing VSIV developed by Caldwell and colleagues42 can induce high titres of neutralizing antibody in
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Table 2. Protection conferred by MOMP-based vaccines in animal models of chlamydial infectiona Immunogenicity
Protection
Vaccine
Host and site of infection
Challenge organism (route)
Route of immunization (adjuvant)
Specific antibody
CMI
Colonization
Disease
Ref.
Partially purified (OGP) MOMP C. psittaci GPIC
Guinea pig GT
C. psittaci GPIC (ivag)
Subcutaneous (Freunds)
IgG: serum; mucosal IgA: mucosal
+
Reduced
ND
40
Recombinant 1/2–3/4 MOMP (serovar L1)
Mouse GT
Serovar F (iu)
Systemic (aluminium hydroxide gel) Mucosal (via Peyer’s patches)
IgG: serum; mucosal IgA: mucosal IgG: mucosal IgA: mucosal
ND
No effect
Reduced
41
ND
Reduced
No effect
41
P11 synthetic peptide (Conserved 12mer)
Mouse GT
Serovar F (iu)
Intradermal (aqueous solution)
None
+
Shedding delayed
Slightly delayed
44
Chimeric peptide (Th epitope + VSIV serovar A)
Mouse GT
Serovar D (ivag/iu)
Subcutaneous (aluminium hydroxide gel)
IgG: serum; mucosal No IgA
ND
No effect
ND
42
Partially purified (OGP) MOMP (serovar C)
Monkey eye
Serovar C (ocular)
Oral (cholera toxin) or systemic (Freunds)
IgG: serum; mucosal IgA: mucosal
+
Reduced
No effect
43
a
Abbreviations: CMI, cell-mediated immunity (assessed by in vitro proliferative responses of spleen or lymph node cells); GPIC, guinea pig inclusion conjunctivitis agent; GT, genital tract; iu, intrauterine; ivag, intravaginal; ND, not determined; OGP, octyl-b-D glucopyranoside.
the mouse strain used for the protection studies, but parenteral immunization failed to induce IgA responses in either serum or genital tract secretions. Mucosal immunization may be required. The importance of route of delivery is also illustrated by the finding that different routes have differing effects upon colonization and disease41. In mice, intra-nasal immunization appears particularly effective at inducing long-term memory responses at mucosal sites, and might be the route of choice in future studies. (2) MOMP might not be the best antigen to use in a subunit vaccine. MOMP was the antigen of choice for first-generation subunit vaccines because it is immunodominant for antibody responses, but perhaps other components of the bacterium will prove to be more potent stimulators of protective cell-mediated mechanisms. Much remains to be learnt about the specificity of Chlamydia-reactive T cells, particularly in humans. A comparison of responses of T cells from infected individuals with those from exposed but uninfected individuals, for instance in trachoma-endemic regions, might be informative. Similarly, the fine specificity of response in mice with different susceptibilities to chlamydial disease might be informative. The DNA vaccine approach (see below) would permit rapid screening of chlamydial libraries for antigens that are protective in such models.
(3) The models used might not have been optimized for the detection of protective immunity. For instance, it might be difficult to demonstrate protective effects with a vaccine against genital infection when subsequent challenge involves direct inoculation of relatively large numbers of organisms into the upper genital tract41,44. Intrauterine challenge maximizes pathology and could overwhelm a weakly protective response, or bypass mechanisms that would operate in the cervix or vagina. In this context, the partial protection against intrauterine challenge, obtained by intradermal injection of a MOMP peptide in simple solution44, might be more impressive than it appears. Models of ascending infection employing small challenge doses to mimic the insidious nature of natural infection might be a more reasonable test of protective effects.
Anti-idiotypic vaccines Another approach to chlamydia vaccines is anti-idiotypic (anti-Id) antibodies. A monoclonal anti-Id that mimics the chlamydial exoglycolipid antigen GLXA – a poorly characterized antigen secreted by infected cells – has been produced by immunization of mice with a mAb to GLXA (Ref. 45). Administered subcutaneously in alumina adjuvant, the anti-Id conferred 50–90% protection against colonization 171
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of mouse conjunctivas and reduced disease severity. Unfortunately, it did not protect against genital tract infection (M. Tuffrey, pers. commun.).
Improved delivery of chlamydial vaccines There is considerable scope for improved delivery of MOMP-based vaccines. Microbial delivery vectors are being explored: hybrid polio viruses expressing MOMP VSI and VSIV have been produced and shown to be highly immunogenic in rabbits46; and MOMP epitopes have been expressed in attenuated strains of Salmonella47. More recently, a number of groups have begun to develop ‘naked’ DNA vaccines: intramuscular injection of mice with a plasmid containing the MOMP gene confers significant protection against infection of the respiratory tract by the MoPn biovar of C. trachomatis48. Recovery of the bacteria was reduced >100-fold and morbidity reduced. These results are at least as good as those obtained with MOMP protein and synthetic peptides (Table 2), and early experience suggests that intranasal administration of the DNA construct is even more effective (B. Brunham, pers. commun.). Intramuscular injection induced both cell-mediated immunity and serum antibodies recognizing native EBs. The protective component of the response remains unidentified but, if it proves to be neutralizing antibody, it might be necessary to incorporate several MOMP genes into a DNA vaccine to confer protection against multiple C. trachomatis serovars; T-cell mechanisms might be predicted to be more crossreactive. Attempts to protect mice against genital infection using DNA vaccines have so far proved unsuccessful (L.M. de la Maza, pers. commun.). This might be due to technical reasons, such as the construct used or the vaccination protocol, but could also reflect a real difference in protective mechanisms at different mucosal sites. In the future it might be possible to modulate mucosal immunity by administrating cytokines, chemokines or hormones; genes for such immunomodulators could be incorporated into DNA vaccines.
Summary Major advances have been made in our understanding of the complex immune response that develops in response to chlamydial infection. Although there are still areas of debate, this progress will inform fu-
The outstanding questions • Can protective immune mechanisms identified in mouse models be applied to human infection? • Can similar vaccine approaches be applied to genital and ocular infection? • Will carefully chosen vaccine components avoid immunopathology or is the type of response invoked of central importance? • Is MOMP the best candidate for a subunit vaccine and are conformational epitopes essential for protection? • Can alternative delivery systems (e.g. DNA vaccines) improve the efficacy of chlamydial vaccines and can the local mucosal immune response be modulated to favour protective mechanisms? • Would vaccines that protect against severe disease rather than infection be a more achievable goal? • Can systems for genetic manipulation of Chlamydia be developed and what will sequencing of the genome reveal?
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ture attempts at rational vaccine design, leading to an improvement upon the protection obtained with early subunit vaccines. If the optimism that an effective vaccine can be developed is to be fulfilled, delivery systems capable of stimulating effective mucosal responses, particularly T-cell responses, are likely to be required. Acknowledgements. I thank Stella Knight and Maureen Tuffrey for critical reading of the manuscript. The work at Imperial College was supported by The Arthritis and Rheumatism Council, the Medical Research Council and the European Union. I apologize to those chlamydiologists whose work is not fully cited owing to space constraints.
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located on the major outer membrane protein of Chlamydia trachomatis, J. Immunol. 138, 575–581 Peterson, E.M., Cheng, X., Motin, V. and de la Maza, L.M. (1997) Effect of immunoglobulin G isotype on the infectivity of Chlamydia trachomatis in a mouse model of intravaginal infection, Infect. Immun. 65, 2693–2699 Cotter, T.W. et al. (1995) Protective efficacy of major outer membrane-specific immunoglobulin A (IgA) and IgG monoclonal antibodies in a murine model of Chlamydia trachomatis genital tract infection, Infect. Immun. 63, 4704–4714 Byrne, G.I. et al. (1988) g-interferon-mediated cytotoxicity related to murine Chlamydia trachomatis, Infect. Immun. 56, 2023–2027 Perry, L.L., Feilzer, K. and Caldwell, H.D. (1997) Immunity to Chlamydia trachomatis is mediated by T helper 1 cells through IFN-g-dependent and independent pathways, J. Immunol. 158, 3344–3352 Cotter, T.W. et al. (1997) Dissemination of Chlamydia trachomatis chronic genital tract infection in gamma interferon gene knockout mice, Infect. Immun. 65, 2145–2152 Beatty, W.L., Byrne, G.I. and Morrison, R.P (1993) Morphological and antigenic characterization of interferon g-mediated persistent Chlamydia trachomatis infection in vitro, Proc. Natl. Acad. Sci. U. S. A. 90, 3998–4002 Conway, D.J. et al. (1997) Scarring trachoma is associated with polymorphism in the tumor necrosis factor a (TNF-a) gene promoter and with elevated TNF-a levels in tear fluid, Infect. Immun. 65, 1003–1006 Igietseme, J.U., Wyrick, P.B., Goyeau, D. and Rank, R.G. (1994) An in vitro model for immune control of chlamydial growth in polarized epithelial cells, Infect. Immun. 62, 3528–3535 Starnbach, M.N., Bevan, M.J. and Lampe, M.F. (1994) Protective cytotoxic T lymphocytes are induced during murine infection with Chlamydia trachomatis, J. Immunol. 153, 5183–5189 Rasmussen, S.J., Timms, P., Beatty, P.R. and Stephens, R.S. (1996) Cytotoxic-Tlymphocyte-mediated cytolysis of L cells persistently infected with Chlamydia spp., Infect. Immun. 64, 1944–1949 Holland, M.J. et al. (1997) Synthetic peptides based on Chlamydia trachomatis antigens identify cytotoxic T lymphocyte responses in subjects from a trachoma-endemic population, Clin. Exp. Immunol. 107, 44–49 Lichtenwalner, A.B. et al. (1997) Evidence of genetic susceptibility to Chlamydia trachomatis-induced pelvic inflammatory disease in the pig-tailed macaque, Infect. Immun. 65, 2250–2253 Conway, D.J. et al. (1996) HLA Class I and II polymorphisms and trachomatous scarring in a Chlamydia trachomatis-endemic population, J. Infect. Dis. 174, 643–646 Darville, T. et al. (1997) Mouse strain-dependent variation in the course and outcome of chlamydial genital tract infection is associated with differences in host response, Infect. Immun. 65, 3065–3073 Su, H., Morrison, R.P, Watkins, N.G. and Caldwell, H.D. (1990) Identification and characterization of T helper epitopes of the major outer membrane protein of Chlamydia trachomatis, J. Exp. Med. 172, 203–212 Allen, J.E., Locksley, R.M. and Stephens, R.S (1991) A single peptide from the major outer membrane protein of Chlamydia trachomatis elicits T cell help for the production of antibodies to protective determinants, J. Immunol. 147, 674–679 Stagg, A.J. et al. (1993) Primary T-cell responses to the major outer membrane protein of Chlamydia trachomatis, Immunology 79, 1–9 Ortiz, L. et al. (1996) Chlamydia trachomatis major outer membrane protein (MOMP) epitopes that activate HLA class II-restricted T cells from infected humans, J. Immunol. 157, 4554–4567 Su, H. and Caldwell, H.D. (1992) Immunogenicity of a chimeric peptide corresponding to T helper and B cell epitopes of the Chlamydia trachomatis major outer membrane protein, J. Exp. Med. 175, 227–235 Zhong, G., Toth, I., Reid, R. and Brunham, R.C. (1993) Immunogenicity evalu-
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ation of a lipidic amino acid-based synthetic peptide vaccine for Chlamydia trachomatis, J. Immunol. 151, 3728–3736 Lampe, M.F., Wong, K.G., Kuehl, L.M. and Stamm, W.E. (1997) Chlamydia trachomatis major outer membrane protein variants escape neutralization by both monoclonal antibodies and human immune sera, Infect. Immun. 65, 317–319 Batteiger, B.E., Rank, R.G., Bavoil, P.M. and Soderberg, L.S.F. (1993) Partial protection against genital reinfection by immunization of guinea-pigs with isolated outer-membrane proteins of the chlamydial agent of guinea-pig inclusion conjunctivitis, J. Gen. Microbiol. 139, 2965–2972 Tuffrey, M. et al. (1992) Heterotypic protection of mice against chlamydial salpingitis and colonization of the lower genital tract with the human servar F isolate of Chlamydia trachomatis by prior immunization with recombinant serovar L1 major outer-membrane protein, J. Gen. Microbiol. 138, 1707 Su, H., Parnell, M. and Caldwell, H.D. (1995) Protective efficacy of a parenterally administered MOMP-derived synthetic oligopeptide vaccine in a murine model of Chlamydia trachomatis genital tract infection: serum neutralizing IgG antibodies do not protect against chlamydial genital tract infection, Vaccine 13, 1023–1032 Campos, M. et al. (1995) A chlamydial major outer membrane protein extract as a trachoma vaccine candidate, Invest. Ophthalmol. Vis. Sci. 36, 1477–1491 Knight, S.C. et al. (1995) A peptide of Chlamydia trachomatis shown to be a primary T-cell epitope in vitro induces cell-mediated immunity in vivo, Immunology 84, 8–15 Whittum-Hudson, J.A. et al. (1996) Oral immunization with an anti-idiotypic antibody to the exoglycolipid antigen protects against experimental Chlamydia trachomatis infection, Nat. Med. 2, 1116–1121 Murdin, A.D., Su, H., Klein, M.H. and Caldwell, H.D. (1995) Poliovirus hybrids expressing neutralization epitopes from variable domains I and IV of the major outer membrane protein of Chlamydia trachomatis elicit broadly crossreactive C. trachomatis-neutralizing antibodies, Infect. Immun. 63, 1116–1121 Hayes, L.J. et al. (1991) Chlamydia trachomatis major outer membrane protein epitopes expressed as fusions with LamB in an attenuated aroA strain of Salmonella typhimurium; their application as potential immunogens, J. Gen. Microbiol. 137, 1557–1564 Zhang, D.J. et al. (1997) DNA vaccination with the major outer-membrane protein gene induces immunity to Chlamydia trachomatis (mouse pneumonitis infection), J. Infect. Dis. 176, 1035–1040
New features on the international ImMunoGeneTics database… Protein displays of human immunoglobulin and T-cellreceptor variable regions, descriptions of mutations and allele alignments. 3-D representations of immunoglobulin and T-cell-receptor variable regions. DNA plots for the analysis of human immunoglobulin and T-cell-receptor rearranged sequences. All freely available at http://imgt.cnusc.fr:8104
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Vaccines against Chlamydia: approaches and progress Andrew J. Stagg
Infections of the eye and genital tract with the bacterium Chlamydia trachomatis are a major cause of morbidity worldwide and are costly to treat. Development of a vaccine capable of protecting against infection or severe disease presents special challenges but would be the most effective long-term option for control of chlamydial disease. Progress has been made in understanding protective and pathological immune mechanisms in these infections, and a number of potential vaccine candidates have been developed. INFECTION with Chlamydia trachomatis is a major cause of sexually transmitted genital tract disease worldwide and of ocular disease in the developing world (Table 1). In 1990, an estimated 1.5–6.7 million individuals worldwide were visually impaired as a result of trachoma1. Genital tract infection can be asymptomatic or cause mild disease, but in up to 40% of infected women the organism spreads into the upper genital tract and is a major cause of pelvic inflammatory disease (PID), with consequences that include infertility and ectopic pregnancy. It has recently been estimated that 80 000 women in the UK alone suffer from these serious consequences of chlamydial infection every year, costing nearly £60 million to treat2. If left untreated, both ocular and genital tract chlamydiosis are chronic inflammatory conditions with disease severity being related to persistent or repeated infection. Pathology is immunologically mediated3 but the exact mechanism is unclear. Here, I outline the challenges to the development of chlamydial vaccines based on our understanding of the immunology of chlamydiosis, and describe current approaches and future prospects for vaccine development in the light of these challenges.
(1.0 mm), metabolically active, reticulate bodies (RBs). RBs divide by binary fission within the growing inclusion and undergo a second differentiation step to yield new infectious EBs, which are released from the host cell. There are four species within the genus Chlamydia. C. psittaci and C. pecorum are important veterinary pathogens; C. pneumoniae causes acute respiratory infections in humans and has recently been linked with heart disease as a result of its detection in atherosclerotic lesions3. However, C. trachomatis is the major human pathogen and the development of vaccines for control of infections by this species is the focus of this review. C. trachomatis can be subdivided into biovars based on the host species and anatomical site that are infected. The biovars are further subdivided into serologically distinguishable variants termed serovars (Table 1), which arise from variation in the 40 kDa major outer membrane protein (MOMP). This variation is clustered in four surface-exposed variable regions (VSI–VSIV) separated by five conserved regions. MOMP is oligomeric and might have porin activity5. An involvement in attachment to host cells has also been proposed5 and it might be at this level that restriction of serovars to particular sites of infection (Table 1) occurs.
The Chlamydia genus Chlamydia are obligate intracellular Gram-negative bacteria with a unique life cycle (reviewed in Ref. 4). They exist in two major forms (Fig. 1): the small (200–300 nm) metabolically inactive elementary body (EB) is the infectious form; EBs are taken up by host cells into membrane-bound inclusions in which they reorganize into larger Andrew J. Stagg PhD Research Fellow Antigen Presentation Research Group, Imperial College School of Medicine at Northwick Park Hospital, Watford Road, Harrow, Middlesex, UK HA1 3UJ. Tel: +44 181 869 3428 Fax: +44 181 869 3532 e-mail: [email protected]
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Why do we need a vaccine? Antibiotic therapy effectively eliminates chlamydial infection but does not always affect established pathology; the presence of asymptomatic infection makes control by treatment of symptomatic individuals alone unlikely to be successful. However, screening for asymptomatic infection, followed by treatment of infectious individuals, could provide an alternative to vaccination for control of sexually transmitted infection in the developed world6, but are likely to prove too costly to be practicable in the developing world. Computer modelling suggests that even a partially efficacious chlamydial vaccination programme would rapidly reduce the prevalence of genital infection7. This approach is potentially less costly than a screening programme and could thus be extended to infections in the developing world. However, chlamydial infections present a number of challenges that need to be overcome before an effective vaccine can be developed. These are discussed below.
Copyright ©1998 Elsevier Science Ltd. All rights reserved. 1357 - 4310/98/$19.00 PII: S1357-4310(98)01232-5
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The need to avoid immunopathology
Table 1. Diseases caused by C. trachomatis Biovara
Host
Disease
Serovar
Host cells
Ocular/genital Humans
Ocular: trachoma; conjunctivitis
A–C
Epithelial
Humans
Genital: urethritis; cervicitis; salpingitis; PID; reactive arthritis; epididymitis; perihepatitis
D–K
Epithelial
L1, L2, L3
Epithelial; macrophages
Ocular: ophthalmia neonatorum LGV
Humans
Lymphogranuloma venereum
MoPn
Mice
Pneumonitis
a
LGV, lymphogranuloma venereum; MoPn, mouse pneumonitis agent.
Epithelial
Because pathology in severe chlamydial disease is immunologically mediated4, there is a danger that vaccination might prime an inappropriate immune response and increase the severity of subsequent disease. This appears to have occurred in some clinical trials in the 1960s and 1970s, in which crude preparations of whole organisms were used in an attempt to vaccinate against trachoma (reviewed in Ref. 12). The findings are still debated but they appeared to show that the whole-cell vaccine increased the severity of disease in those who subsequently became infected, although it did reduce the incidence of disease in some circumstances. Similar observations have been made experimentally in monkey models of ocular chlamydial infection and in a mouse model of genital tract infection.
The need to define protective immune mechanisms The need to improve on protection conferred by natural infection
Immune mechanisms that protect the human host from chlamydial infection are not well characterized and this has been an obstacle to vaccine design. Recent studies with knockout mice carrying disrupted genes for cytokines17,22,23 or their receptors16 have begun to
Repeated chlamydial infections are common but a degree of protective immunity does seem to develop. The incidence of genital infection falls with age in a way that is unlikely to be attributable to changes in sexual behaviour, and reinfection is more likely to be detected when a longer time interval has elapsed since initial infection, suggesting a short-term protective effect8. In female sex workers in Kenya, re-infection is often caused by a different strain from that identified in the previous infection9. One explanation for this is the development of strainspecific immunity – an interpretation supported by early vaccine trials for trachoma (see below). Severe disease is associated with the A31 major histocompatibility complex (MHC) class I allele and, independently, a low CD4+ T-cell count (among HIV-seropositive individuals), suggesting a modifying effect of the immune response on disease outcome10. In ocular infection, active trachoma with shedding of large numbers of organisms is largely confined to childhood. Individuals with severe conjunctival scarring have lower cell-mediated immune responses than exposed controls or patients recovering from disease; this is consistent with a role for these responses in clearing infection11. Therefore, a degree of immunity is conferred by infection but it is shortlived and serovar-specific. An effective Figure 1. Transmission electron micrograph of a Buffalo Green Monkey Kidney cell 48 h after infection vaccine will need to increase the level of with Chlamydia trachomatis serovar L1. EB (elementary bodies), RB (reticulate bodies) and intermediate protection and confer immunity to all forms are evident within the inclusion. serovars.
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reveal the complexity of the host response to chlamydial infection. The relative contributions of antibody and cell-mediated mechanisms to control of infection and the most appropriate choice for immunoprophylaxis are still a matter of debate. Figures 2 and 3 illustrate potential protective mechanisms.
Humoral responses Specific antibodies might bind Chlamydia during the extracellular stages of its lifecycle (Fig. 2). In humans, anti-chlamydial IgA is associated with reduced shedding of the bacterium in women with chlamydial cervicitis (see Ref. 4 for references), and the development of serovar-specific immunity9 suggests the involvement of antibody. Studies in the guinea pig have also indicated a role for antibody in limiting primary genital infection13 and in a panel of knockout mice
Lumen
a
Epithelium
Stroma
Transcytosis
IgG
Plasma cell sIgA
b
Figure 2. Potential protective humoral immune mechanisms in chlamydial infection. Elementary bodies (EB, black circles) and reticulate bodies (RB, clear circles) are shown during a cycle of infection in genital tract epithelium (top, initiation of infection; bottom, release of new infectious progeny). Dimeric secretary immunoglobulin A (sIgA), locally produced and transcytosed into the lumen, and serum-derived or locally produced IgG might (a) prevent infection by neutralization or (b) reduce shedding of organisms.
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with disrupted b2-microglobulin (b2m), MHC class II or CD4 genespecific vaginal antibody correlated with the resolution of genital infection14. However, antibody responses are neither required to resolve primary genital chlamydial infections in mice15 nor sufficient to control primary infection or reinfection in mice with a disrupted interferon g (IFN-g) receptor gene16. Antibodies might contribute to control of secondary infection, but cell-mediated mechanisms again play the dominant role15,17. High concentrations of monoclonal antibodies (mAbs) to surfaceexposed epitopes on MOMP can neutralize infectivity in vitro and in vivo in animal models18. Thus it might be theoretically possible, but practically very difficult, to develop a vaccine that generates and sustains high levels of neutralizing antibody at the appropriate mucosal surface and thus block initial infection (Fig. 2a). The secondary infection experiments discussed above15–17 suggest that recall of a memory antibody response primed by vaccination is unlikely to control infection. Indeed, delayed exposure to antibodies might even enhance infectivity19. On a more positive note, the development of an antibody response might be sufficient to reduce shedding, which should block transmission and/or reduce the spread of the organism into the upper genital tract (Fig. 2b). A ‘backpack’ model in which hybridomas secreting mAbs to MOMP are implanted in syngeneic mice, shows only marginal effects on colonization of the genital tract, but reduced ascending infection and upper genital tract pathology20. A vaccine that blocks ascending infection of the genital tract and prevents PID would be a worthwhile achievement.
Cell-mediated responses Cell-mediated immunity can potentially control chlamydial infection in a number of ways (Fig. 3). Presentation of chlamydial antigen by antigen-presenting cells (APCs) (Fig. 3a) or by the infected cells themselves (Fig. 3b) might result in local activation of T cells and the production of cytokines that inhibit chlamydial growth or activate macrophages to scavenge extracellular bacteria (Fig. 3c). Knockout mice lacking MHC class II antigens and hence CD4+ T cells cannot resolve genital infection14, demonstrating the critical importance of these cells to the immune response to Chlamydia. These mice fail to make a T-cell-dependent antibody response to Chlamydia but, as discussed above, this is unlikely to account for their susceptibility. The inability to generate a T-helper 1 (Th1) response and the consequent reduced production of cytokines such as IFN-g and tumour necrosis factor a (TNF-a) is likely to be important. IFN-g is cytotoxic for Chlamydia-infected cells21 and can also inhibit growth of some strains by upregulating host-cell production of the enzyme indoleamine 2,3-deoxygenase, leading to cellular depletion of the essential amino acid tryptophan. IFN-g also induces nitric oxide synthase, and the nitric oxide produced inhibits chlamydial growth. Knockout mice lacking IFN-g or its receptor display delayed bacterial clearance after primary genital infection with C. trachomatis16,17,22,23, but they eventually control infection, indicating the presence of compensatory mechanisms such as the production of TNF-a17. Although IFN-g knockouts can acquire immunity to reinfection17,23 this was not the case in the IFN-g receptor knockouts. One possible explanation for this is the presence of additional ligands that can partially compensate for the absence of IFN-g by signalling through the IFN-g receptor. Thus, cytokines, especially those signalling through the IFN-g and TNF-a receptors, appear to be important contributors to clearance of the
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bacteria. Unfortunately, IFN-g and TNF-a are also implicated in the immunopathology that is a lasting consequence of chlamydial infection24,25. As well as being activated by APCs, effector CD4+ and CD8+ T cells might directly recognize infected cells (Fig. 3b). MHC class II restricted-CD4+ T cells are not known to recognize infected cells, but it is theroretically possible because epithelial cells can express MHC class II antigens following infection. Also, co-culture of epithelial cells with Chlamydia-specific CD4+ T cells inhibits bacterial growth by an unknown mechanism26. By contrast, human and murine CD8+ cytotoxic T cells (CTLs) are known to recognize Chlamydia-infected cells27–29. In addition to mediating cell lysis, CD8+ T cells are an important source of IFN-g. Nevertheless, b2m knockout mice, which lack CD8+ T cells, can successfully control genital chlamydial infection14. These studies neither exclude a contributory role for CTLs in the intact immune response nor preclude the possibility that a vaccine capable of stimulating a potent CTL response could confer protection. However, such a vaccine might do more harm than good because certain MHC class I alleles correlate with PID in humans10 and monkeys30, and with trachoma in humans31. The generation of Th1 cells and cytokines such as IFN-g plays an important role in controlling chlamydial infection, but recent experiments suggest additional complexity. Mouse strains can differ in susceptibility to genital infection despite making predominately Th1 responses32, and early bacterial clearance appears to depend on an IL-12-dependent but IFN-g-independent mechanism22. In mice, the ability to clear Chlamydia from the upper genital tract is associated with rapid recruitment of APCs into the tissue (A. Stagg et al., unpublished); such cells might be an important source of IL-12.
Lumen
Epithelium
Stroma
DC
Th1 CD4+
a Th1 CD4+ IFN-γ TNF-α
CD8+
CD8+
b
Cytolysis
Macrophage activation
c TNF-α NO TNF-α
Exploiting the immune response to Chlamydia As explained above, the immune response to Chlamydia is both complex and flexible. It is not yet clear which is the most appropriate response to target for immunoprophylaxis, or whether it will be possible to confer protection while avoiding immunopathology. Much of our understanding of cell-mediated immunity in chlamydial infection is based upon mouse models; little is known about the response in humans. Given that infection in humans runs a chronic course and appears to produce only short-term, serovar-specific immunity, the possibility that the organism has evolved mechanisms to block or evade the protective mechanisms that have been identified in mice warrants further investigation. A vaccine aimed at reducing the damage associated with severe disease might be a more realizable goal than one aimed at preventing infection. However, the possibility remains that any vaccine that is only partially effective might exacerbate disease; rapid and effective clearance of the organism might be the best protection against pathology. Nevertheless, attempts have been made to design vaccines that fully protect against chlamydiosis. These are described below. For the current discussion it is assumed that broadly similar approaches to vaccine development can be applied to ocular and genital infection; this is reasonable given the similarities in immunology but has not been firmly established.
near future, because of the risk of immunopathology, and because the large-scale production of pure chlamydiae is extremely difficult. The genomes of C. trachomatis and C. pneumoniae* have now been sequenced so, in the long term, it might be possible to engineer modified organisms that do not induce pathology. However, this approach can only be speculated upon at present and would require a huge research effort before its feasibility can be assessed.
Whole-organism vaccines
Subunit vaccines
In animal models, vaccines based upon whole organisms can confer a degree of protection against C. trachomatis infection. For instance, intranasal immunization of mice with viable organisms confers significant protection against colonization of the genital tract. However, whole-organism vaccination is unlikely to be attempted again in the
Recent research has focused on the development of vaccines based on MOMP, which does not appear to induce immunopathology. Inevitably, there is a lag between the definition of protective immune
Mφ NO
Figure 3. Potential protective cellular immune mechanisms in chlamydial infection. T cells, originally activated in lymphoid tissue draining the infection site by migratory dendritic cells (DC) and homing back to the tissue might be locally inactivated to produce cytokines [such as interferon g (IFN-g) and tumour necrosis factor a (TNF-a)] that (a) inhibit chlamydial growth or (c) activate macrophages. Alternatively they might directly recognize infected cells (b) and lyse them.
*http://www.genset.fr/
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Glossary Biovar – Subclassification of Chlamydia trachomatis isolates based upon the host species infected and the type of disease produced. Elementary body (EB) – One of two predominant chlamydial forms, EBs are the stage in the organism’s life cycle adapted to extracellular survival. They are resistant to osmotic and mechanical stress, have a condensed chromosome and appear metabolically inactive. The EB is the infectious chlamydial particle and mediates attachment to and invasion of target host cells. Immunodominance – The ability of one antigen or epitope in a mixture to elicit the stongest immune response in vivo. Major outer membrane protein (MOMP) – A 40 kDa, cysteine-rich chlamydial envelope protein comprising 60% of the EB’s surface protein. It is encoded by the omp1 gene, which is developmentally regulated during the chlamydial life cycle. Pelvic inflammatory disease (PID) – Inflammation of the female genital tract above the level of the uterine cervix. Inflammation can be confined to the endometrium (endometritis) or spread to the fallopian tubes (salpingitis), and can also involve the ovaries (salpingooophoritis). Further spread may involve the peritoneum (peritonitis) or the liver (Fitz–Hugh Curtis syndrome). PID is almost always associated with spread of microbes from the vagina or cervix into the upper genital tract. Long-term consequences include infertility and ectopic pregnancy, and can occur even when chlamydial PID is asymptomatic. Reticulate body (RB) – The vesicle-bounded, metabolically active form in the life-cycle of Chlamydia. They are more friable than EBs and are not infectious but undergo division by binary fission. As they do so the vesicle enlarges, often to occupy a large proportion of the cell; this structure is termed an inclusion. RBs revert to EBs, which are released from the host cell to continue the infectious process. Serovar – Serologically distinguishable variants that, in the case of C. trachomatis, arise from variation in MOMP gene sequences. T-helper 1 (Th1) cells – A CD4+ T-helper cell population characterized by the cytokines that they produce (interferon g, tumour necrosis factors a and b and interleukin 2). They tend to be involved in cell-mediated immunity.
mechanisms and the incorporation of these ideas into vaccine candidates, and we can expect our current knowledge of immune responses to chlamydiae to influence the design of future vaccines. To date, most MOMP-based vaccine candidates are designed to elicit protective antibody responses, although recent studies (see above) suggest that this approach is unlikely to be successful. MOMP is immunodominant for serological responses. Protective, serovar-specific antibodies recognize epitopes within VSI and VSII; species-specific and subspecies-specific epitopes are located in VSIV; and VSIII does not appear to contain important antibody recognition sites. This means that a vaccine protecting against all strains would have to contain variable regions from several different serovars. Regions of MOMP that stimulate T cells, in humans as well as in mice, have now been identified33–36. Although its dominance in cellmediated responses has not been established, many regions of 170
MOMP contain T-cell epitopes and many different MHC molecules can be used for recognition of MOMP epitopes. Furthermore, regions containing T-cell epitopes tend to be located in conserved rather than variable regions. These observations suggest that it should be possible to construct subunit vaccines containing relatively small, defined regions of MOMP capable of stimulating T-cell responses against all serovars in most or all of a population with different human leukocyte antigen (HLA) haplotypes. With the rationale that protection would be mediated by T-celldependent antibody production, two groups have engineered chimeric peptides in which variable segments containing B-cell epitopes are linked to regions containing one or more T-cell epitopes. The peptide produced by Su and Caldwell37 contained VSI of serovar A and induced antibodies to this sequence in six different mouse strains with disparate MHC haplotypes, indicating that the T-cell epitope(s) could be recognized in the context of multiple MHC alleles. However, the fine specificity of the antibody response differed between mouse strains: although sera from all strains recognized VSI, only two strains developed antibodies that recognized whole bacteria, and only these two sera had significant neutralization titres. Congenic mice revealed that the fine specificity of the antibodies raised to the vaccine was under the control of genes outside the MHC. Zhong and colleagues38 incorporated several variable segments into a single peptide with the intention of protecting against the multiple serovars. They increased antibody titres by incorporating polymers of the peptides into a lipid-based hydrophobic core. However, the antibody titres to whole organisms were again several orders of magnitude lower than titres to the peptide; neutralizing titres were lower still. These studies with chimeric peptides illustrate the importance of generating antibodies to conformation-dependent epitopes if neutralization is to be achieved. They also highlight the confounding factor of genetically determined variation in host response. These are challenges to designing peptide-based vaccines that elicit antibody production. Future vaccines might need to include structural features of the native B-cell epitope, as well as the correct linear sequence. Another potential problem arises from MOMP variants within the same serovar; these are small deviations from the prototype sequence but can be sufficient to allow escape from neutralization by antibodies39. MOMP and a number of these immunogenic subunits have been evaluated in animal models for their ability to confer protection against infection or disease but to date they have been at best partially effective (Table 2). This is despite the stimulation of both mucosal and serum antibody responses40–43. Some protection was also obtained by immunization with MOMP-derived peptide in the absence of antibody production44. There are several possible explanations for the poor protection conferred by these candidate vaccines: (1) Although immunogenic, the vaccine candidates might fail to stimulate protective immune mechanisms. Perhaps the antibody responses generated at mucosal surfaces are of insufficient titre or an inappropriate subclass, or perhaps they are not directed against protective (possibly conformation-dependent) epitopes. Alternatively, they might not stimulate the protective cell-mediated mechanisms revealed by recent knockout experiments (see above). Although systemic T-cell responses were observed in some studies, nothing is known about the responses stimulated at the site of infection. The chimeric peptide containing VSIV developed by Caldwell and colleagues42 can induce high titres of neutralizing antibody in
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Table 2. Protection conferred by MOMP-based vaccines in animal models of chlamydial infectiona Immunogenicity
Protection
Vaccine
Host and site of infection
Challenge organism (route)
Route of immunization (adjuvant)
Specific antibody
CMI
Colonization
Disease
Ref.
Partially purified (OGP) MOMP C. psittaci GPIC
Guinea pig GT
C. psittaci GPIC (ivag)
Subcutaneous (Freunds)
IgG: serum; mucosal IgA: mucosal
+
Reduced
ND
40
Recombinant 1/2–3/4 MOMP (serovar L1)
Mouse GT
Serovar F (iu)
Systemic (aluminium hydroxide gel) Mucosal (via Peyer’s patches)
IgG: serum; mucosal IgA: mucosal IgG: mucosal IgA: mucosal
ND
No effect
Reduced
41
ND
Reduced
No effect
41
P11 synthetic peptide (Conserved 12mer)
Mouse GT
Serovar F (iu)
Intradermal (aqueous solution)
None
+
Shedding delayed
Slightly delayed
44
Chimeric peptide (Th epitope + VSIV serovar A)
Mouse GT
Serovar D (ivag/iu)
Subcutaneous (aluminium hydroxide gel)
IgG: serum; mucosal No IgA
ND
No effect
ND
42
Partially purified (OGP) MOMP (serovar C)
Monkey eye
Serovar C (ocular)
Oral (cholera toxin) or systemic (Freunds)
IgG: serum; mucosal IgA: mucosal
+
Reduced
No effect
43
a
Abbreviations: CMI, cell-mediated immunity (assessed by in vitro proliferative responses of spleen or lymph node cells); GPIC, guinea pig inclusion conjunctivitis agent; GT, genital tract; iu, intrauterine; ivag, intravaginal; ND, not determined; OGP, octyl-b-D glucopyranoside.
the mouse strain used for the protection studies, but parenteral immunization failed to induce IgA responses in either serum or genital tract secretions. Mucosal immunization may be required. The importance of route of delivery is also illustrated by the finding that different routes have differing effects upon colonization and disease41. In mice, intra-nasal immunization appears particularly effective at inducing long-term memory responses at mucosal sites, and might be the route of choice in future studies. (2) MOMP might not be the best antigen to use in a subunit vaccine. MOMP was the antigen of choice for first-generation subunit vaccines because it is immunodominant for antibody responses, but perhaps other components of the bacterium will prove to be more potent stimulators of protective cell-mediated mechanisms. Much remains to be learnt about the specificity of Chlamydia-reactive T cells, particularly in humans. A comparison of responses of T cells from infected individuals with those from exposed but uninfected individuals, for instance in trachoma-endemic regions, might be informative. Similarly, the fine specificity of response in mice with different susceptibilities to chlamydial disease might be informative. The DNA vaccine approach (see below) would permit rapid screening of chlamydial libraries for antigens that are protective in such models.
(3) The models used might not have been optimized for the detection of protective immunity. For instance, it might be difficult to demonstrate protective effects with a vaccine against genital infection when subsequent challenge involves direct inoculation of relatively large numbers of organisms into the upper genital tract41,44. Intrauterine challenge maximizes pathology and could overwhelm a weakly protective response, or bypass mechanisms that would operate in the cervix or vagina. In this context, the partial protection against intrauterine challenge, obtained by intradermal injection of a MOMP peptide in simple solution44, might be more impressive than it appears. Models of ascending infection employing small challenge doses to mimic the insidious nature of natural infection might be a more reasonable test of protective effects.
Anti-idiotypic vaccines Another approach to chlamydia vaccines is anti-idiotypic (anti-Id) antibodies. A monoclonal anti-Id that mimics the chlamydial exoglycolipid antigen GLXA – a poorly characterized antigen secreted by infected cells – has been produced by immunization of mice with a mAb to GLXA (Ref. 45). Administered subcutaneously in alumina adjuvant, the anti-Id conferred 50–90% protection against colonization 171
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of mouse conjunctivas and reduced disease severity. Unfortunately, it did not protect against genital tract infection (M. Tuffrey, pers. commun.).
Improved delivery of chlamydial vaccines There is considerable scope for improved delivery of MOMP-based vaccines. Microbial delivery vectors are being explored: hybrid polio viruses expressing MOMP VSI and VSIV have been produced and shown to be highly immunogenic in rabbits46; and MOMP epitopes have been expressed in attenuated strains of Salmonella47. More recently, a number of groups have begun to develop ‘naked’ DNA vaccines: intramuscular injection of mice with a plasmid containing the MOMP gene confers significant protection against infection of the respiratory tract by the MoPn biovar of C. trachomatis48. Recovery of the bacteria was reduced >100-fold and morbidity reduced. These results are at least as good as those obtained with MOMP protein and synthetic peptides (Table 2), and early experience suggests that intranasal administration of the DNA construct is even more effective (B. Brunham, pers. commun.). Intramuscular injection induced both cell-mediated immunity and serum antibodies recognizing native EBs. The protective component of the response remains unidentified but, if it proves to be neutralizing antibody, it might be necessary to incorporate several MOMP genes into a DNA vaccine to confer protection against multiple C. trachomatis serovars; T-cell mechanisms might be predicted to be more crossreactive. Attempts to protect mice against genital infection using DNA vaccines have so far proved unsuccessful (L.M. de la Maza, pers. commun.). This might be due to technical reasons, such as the construct used or the vaccination protocol, but could also reflect a real difference in protective mechanisms at different mucosal sites. In the future it might be possible to modulate mucosal immunity by administrating cytokines, chemokines or hormones; genes for such immunomodulators could be incorporated into DNA vaccines.
Summary Major advances have been made in our understanding of the complex immune response that develops in response to chlamydial infection. Although there are still areas of debate, this progress will inform fu-
The outstanding questions • Can protective immune mechanisms identified in mouse models be applied to human infection? • Can similar vaccine approaches be applied to genital and ocular infection? • Will carefully chosen vaccine components avoid immunopathology or is the type of response invoked of central importance? • Is MOMP the best candidate for a subunit vaccine and are conformational epitopes essential for protection? • Can alternative delivery systems (e.g. DNA vaccines) improve the efficacy of chlamydial vaccines and can the local mucosal immune response be modulated to favour protective mechanisms? • Would vaccines that protect against severe disease rather than infection be a more achievable goal? • Can systems for genetic manipulation of Chlamydia be developed and what will sequencing of the genome reveal?
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ture attempts at rational vaccine design, leading to an improvement upon the protection obtained with early subunit vaccines. If the optimism that an effective vaccine can be developed is to be fulfilled, delivery systems capable of stimulating effective mucosal responses, particularly T-cell responses, are likely to be required. Acknowledgements. I thank Stella Knight and Maureen Tuffrey for critical reading of the manuscript. The work at Imperial College was supported by The Arthritis and Rheumatism Council, the Medical Research Council and the European Union. I apologize to those chlamydiologists whose work is not fully cited owing to space constraints.
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New features on the international ImMunoGeneTics database… Protein displays of human immunoglobulin and T-cellreceptor variable regions, descriptions of mutations and allele alignments. 3-D representations of immunoglobulin and T-cell-receptor variable regions. DNA plots for the analysis of human immunoglobulin and T-cell-receptor rearranged sequences. All freely available at http://imgt.cnusc.fr:8104
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