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Revisión Inmunología Vol. 24 / Núm 3/ Julio-Septiembre 2005: 298-312

Chlamydia trachomatis genital infection: Immunity and prospects for vaccine development M.R. Caro Vergara1, A.J. Buendía Marín2, L. del Río Alonso1, F. Cuello Gijón1, N. Ortega Hernández1, M.C. Gallego Ruiz1, J. Salinas Lorente1 Departamento de Sanidad Animal. 2Departamento de Histología y Anatomía Patológica. Facultad de Veterinaria, Universidad de Murcia, Murcia, Spain.

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INFECCIÓN GENITAL POR CHLAMYDIA TRACHOMATIS: INMUNIDAD Y PERSPECTIVAS PARA EL DESARROLLO DE VACUNAS

Recibido: 3 Junio 2005 Aceptado: 25 Julio 2005

RESUMEN

ABSTRACT

Chlamydia trachomatis es una bacteria intracelular obligada con un ciclo de desarrollo bifásico. Actualmente este organismo es considerado mundialmente como la principal causa de enfermedad sexual transmisible en medicina humana. Paralelamente a este hecho, en los últimos años se ha incrementado notablemente la prevalencia de enfermedad pélvica inflamatoria, gestaciones ectópicas e infertilidad asociadas a C. trachomatis. El control definitivo de todos estos procesos solo será posible si es desarrollada una vacuna segura y eficaz. Un conocimiento más profundo de la inmunidad protectora y de la inmunopatología de C. trachomatis en los últimos años ha sido posible gracias a la utilización de un modelo murino de infección. Estos importantes avances en la investigación de la inmunobiología de C. trachomatis ha permitido establecer cuales son los parámetros inmunológicos esenciales para la selección de vacunas eficaces frente a la infección como el establecimiento de una respuesta inmune tipo Th1, necesaria para la eliminación del microorganismo. Los recientes avances en el estudio del genoma clamidial ha permitido la identificación de productos génicos clamidiales que podrían ser candidatos óptimos para ser incluidos en una vacuna subcelular. No obstante es necesario desarrollar nuevos y efectivos sistemas asociados al antígeno vacunal como adyuvantes, sistemas de liberación o vesículas transportadoras de antígenos (liposomas). En el presente artículo se revisa el estado actual de la investigación en el campo de la inmunobiología, patología y nuevos diseños de vacunas frente a C. trachomatis. Además, hemos creído conveniente incluir en la presente revisión datos aportados en otros estudios sobre infecciones genitales por clamidias de gran importancia en medicina veterinaria, como es el caso de Chlamydophila abortus, agente etiológico del aborto enzoótico ovino y causante de abortos en la mujer.

Chlamydia trachomatis is an obligate intracellular bacteria characterized by a biphasic development cycle of replication. The organism is now recognized the major cause of sexually transmissible human bacterial infections throughout the world. Paralleling this rise in chlamydial infection during recent decades, the prevalence of pelvic inflammatory disease (PID), ectopic pregnancy and tubal infertility has undergone a steady increase. Definitive control of C. trachomatis sexually transmitted diseases (STDs) is possible through the development of a safe and effective vaccine. A better understanding of the protective immunity and immunopathology of C. trachomatis has emerged in recent years from studies using a mouse model of chlamydial genital tract infection. The important progress in our knowledge of functional immunobiology of Chlamydia has established the essential immunologic parameters for vaccine selection and evaluation, including the obligatory requirement for a vaccine to induce a T-helper Type 1 immune response that controls chlamydiae. Recent advances in chlamydial genomics should facilitate identification of likely chlamydial gene products that fulfil the antigenic requirements of putative vaccine candidates. Further studies are however needed in the development of novel and effective delivery systems, vehicles and adjuvants. This review summarizes the status of contemporary C. trachomatis immunobiology, immunopathology and vaccine research. Also, in this article we review data generated from other studies on chlamydial genital infections in veterinary medicine such as those into Chlamydophila abortus, the most frequent cause of infectious abortion in sheep (ovine enzootic abortion, or OEA) and goat. This organism is also considered an important zoonotic agent cause of severe, life threatening disease in pregnant women.

PALABRAS CLAVE: Chlamydia trachomatis/ Chlamydophila abortus/ Respuesta inmune/ Inmunopatología/ Vacunas

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KEY WORDS: Chlamydia trachomatis/ Chlamydophila abortus/ Immunity/ Immunopathology/ Vaccines.

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CHLAMYDIA TRACHOMATIS GENITAL INFECTION: IMMUNITY AND PROSPECTS FOR VACCINE DEVELOPMENT

INTRODUCTION Chlamydiae are obligate intracellular Gram-negative bacteria that are responsible for a diverse range of diseases in humans, other mammals and birds. Following reclassification of the order Chlamydiales in 1999, the family Chlamydiaceae is now divided in two genera, Chlamydia and Chlamydophila(1).The genus Chlamydia comprises the species Chlamydia trachomatis (pathogen of man), Chlamydia suis (swine) and Chlamydia muridarum (hamsters and mice). The Chlamydophila genus contains the species Chlamydophila pneumoniae (pathogen of man), Chlamydophila abortus (ruminants and swine), Chlamydophila pecorum (ruminants, swine and marsupials), Chlamydophila felis (cats), Chlamydophila psittaci (birds and poultry) and Chlamydophila caviae (guinea pigs). Chlamydia trachomatis, one of three major species within the genus Chlamydia, is an etiological agent for a group of common genital tract syndromes, including urethritis and epididymitis in men and urethritis, cervicitis and pelvic inflammatory disease (PID) in women. C. trachomatis is now recognized as one the most common sexually transmissible bacterial infections among persons under than 25 years of age living in industrialized nations such as the United States, where the rate of prevalence runs at 4.2%(2). Similar prevalence rates have also been documented in a recent populationbased study in Britain. Sub-Saharan Africa and Southern and Southeast Asia have particularly high burdens of disease, with an estimated 15 million new cases occurring in Africa and 45 million new cases in Southern Asia every year. All these data highlight a universal feature of C. trachomatis: that infection is mainly observed in adolescents and young adults(2). Chamydial urogenital tract infections are readily cured with antibiotics, but measures based on antimicrobial chemotherapy alone are hampered by the frecuency of asymptomatic infections and delayed diagnosis. In addition, a number of studies have documented that within a year after treatment of a previous chlamydia infection, 13-26% of individuals show persistent or recurrent infection. In fact, Burstein and colleagues(3) have estimated that the mean time of reinfection is 6-7 months in a sexually active adolescent population. Therefore, considering the high rate of reinfection, screening programs must be extremely aggressive and frequent, in order to reduce the prevalence of chlamydial infection. The definitive control of C. trachomatis sexually transmitted diseases (STDs) will only be possible through the development of a safe and effective vaccine that induce an adequate immune response, avoiding immunopathological consequences. Progress toward the development of an effective vaccine has been disappointingly modest, as it has

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been for vaccines against other sexually transmitted pathogens that infect the genital tract mucosa. The strict tropism for mucosal epithelial cells, the complex biology and antigenic structure, and the predilection to cause persistent infection have presented formidable challenges to chlamydial vaccine development. A better understanding of protective immunity to C. trachomatis urogenital infection has emerged in the past decade from studies using a mouse model of chlamydial genital tract infections. The insights are of considerable interest because they offer promise for the development of an effective chlamydial vaccine. This review focuses on the progress made and summarizes the current understanding of the immunological and pathological basis of C. trachomatis genital tract infections. This will provide a rational foundation for the design of a vaccine against infection of this organism. We also review data from similar studies concerning animal chlamydial genital infections, including Ovine Enzootic Abortion (OEA) disease, which is associated with Chlamydophila abortus that causes abortion in both ruminants and humans.

THE INTRACELLULAR DEVELOPMENT CYCLE OF CHLAMYDIA The key to understanding the pathophysiology of genital tract disease caused by C. trachomatis, is the biphasic development cycle of these organisms (Fig. 1). The bacteria exist in two development forms: the infectious extracellular elementary bodies (EBs) which attach to the host-cell and are internalized in an entry vacuole that avoids fusion with host-cell lysosomes. Within 8-10 h, the small EB (0.2-0.3 μm in diameter) differentiate into the second form, the larger (0.5-1.6 μm) non-infectious metabolically active, reticulate bodies (RBs), which proliferate within the same membranebound vacuole. After several divisions by binary fission, the RB differentiate back into EB towards the end of the cycle (24-48 h, depending on species) and the EBs are released from the infected cell by lysis or exocytosis to begin a new cycle of infection(4). The EB, in contrast to the RB, is structurally rigid, as a result of extensive disulphide linkages between various cysteine-rich proteins in, or associated with, the outer membrane. This rigidity results in EBs being resistant to both chemical and physical factors and therefore they are adapted for prolonged extracellular survival, an important factor in terms of chlamydial pathogenesis and treatment of chlamydial infections. The host-cell death observed at the end of the infection cycle could thus be involved in the release of EB from the host cell and could partially contribute to the inflammatory response of the host, since macrophages undergoing apoptosis secrete inflammatory

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Expression of IDO INF-γ Replication

Tryptophan

Persistent form Immune cells

Reactivation

RB

No immune stress

↑ Tryptophan

Removal of immune stress

EB Inclusion

Indole

Microbes

Nucleus

Figure 1. The development cycle of Chlamydiae. Chlamydial infection is initiated by the attachment of the infectious EB to the host cell, followed by entry of the EB into a membrane-bound vesicle, called inclusion. The inclusion evades fusion with host lysosomes and the EB rapidly differentiates into a RB that replicate by binary fision within the inclusion. Following several rounds of replication, the RBs reorganize and form infectious EBs, which are released from the cell. Under certain conditions, such as an inflammatory environment where IFN-γ is produced, the intracellular growth of chlamydial strains may be altered. IFN-γ induces IDO, which results in a marked decrease in available tryptophan. Depletion of tryptophan either results in chlamydiae cell death or causes Chlamydia to adopt a non-infectious, nonreplicationg form (aberrant form) that retains viability (persistence). Persistent forms can redifferentiate into infectious EBs upon removal of IFN-γ and subsequent replenishing of intracellular strain. Alternatively, even in an IFN-γ-rich environment, strains of chlamydiae that posses a functional tryptophan synthase (i.e., genital strains) may use indole (perhaps produced by local microbial flora) as a substrate for triptophan synthesis to counter the growth inhibitory effects of IFN-γ.

cytokines and cells dying necrotically stimulate inflammation(4). Deviating from this typical development cycle, a third persistent form exits in vitro (i.c., in cell-culture systems), where enlarged aberrant RBs, have been experimentally induced by a variety of stimuli, including IFN-γ, antibiotics and nutrient deprivation (Fig. 1). Mention should be made of the ability of these stimuli, particularly IFN-γ, to alter chlamydial growth and to facilitate persistent or chronic infection. C. trachomatis has been reported to cause chronic infections detectable by nucleic acid amplification tests but not in i.c. systems, suggesting that this persistent form may also occur in vivo(5). However, it remains unclear whether some antimicrobials induce these persistent forms in vivo or which therapy might be optimal for eradicating such a persistent form. While in vitro induction of persistent chlamydial forms is an important area of research, extensive discussion on this topic is beyond the scope of this review.

GENITAL TRACT CHLAMYDIAL INFECTION C. trachomatis is the most common cause of STD(6) with approximately 90 million new cases estimated to occur

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worldwide each year. In young women, the majority of genital tract infections are asymptomatic and so remain undetected and untreated. This leads to persistent infections, which in a large number of cases can result in PID, leading to chronic pelvic pain, ectopic pregnancy or infertility. C. trachomatis EBs normally infect the single-cell columnar layer of the epithelium in the endocervix of women and the urethra of men. At the site of mucosal infection, intense inflammation characterized by redness, oedema and discharge can occur, resulting in the syndrome of mucopurulent cervicitis in women and non-gonococcal urethritis in men. Asymptomatically infected women can show signs of disease as mucopurulent endocervical discharge, hypertrophic cervix, and friability. Clinical symptoms include dysuria, abnormal vaginal discharge, abnormal menstrual bleeding, postcoital bleeding and lower abdominal pain. In some untreated women (20-40%), infection ascends the endometrial epithelium to the fallopian tubes, where C. trachomatis can establish persistent infection and cause PID. Overall, 11% of women with PID develop tubal factor infertility and 9% develop ectopic pregnancies. Moreover, this risk seems to be higher for those suffering PID caused by infection with

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TABLE I. Chlamydia trachomatis serovars and their associated human diseases Serovars

Human disease

Method of spread

A, B, Ba and C

Ocular tracoma

D, Da and E, F, G, H, I, Ia, J, Ja, K

Oculogenital disease

Hand to eye, fomites and eye-seeking files Sexual and perinatal

L1, L2 and L3

Lymphogranuloma venereum

Sexual

C. trachomatis than PID caused by others factors, such as infection with Neisseria gonorrhoeae(2).

THE IMMUNOBIOLOGY OF C. TRACHOMATIS Currently, there are 18 serovars of C. trachomatis (Table I) based on immunoepitope analysis using monoclonal antibodies directed against the major outer membrane protein (MOMP) of chlamydiae (7). The MOMP comprises approximately 60% of the EB envelope. Among the serovars, the DNA sequence homology of omp1, the gene for MOMP, is greater than 80%, with most of the variance found within the variable domains(8). The four variable domains are flanked and interspersed by five constant domains. Many of the C. trachomatis serovars can cause genital infection in humans, in particularly serovars D though K. The most commonly isolated serovars from infected individuals are D, E and F. Knowledge of the immune response against C. trachomatis is essential for developing a vaccine. A vaccine needs to induce a protective immune response and avoid responses associated with persistence of infection or immunopathology. A mouse model of vaginal infection, using C. muridarum as well as a guinea pig inclusion conjunctivitis (GPIC) model, have been used to analyse the innate and adaptive responses to infection with C. trachomatis, since both seem to closely mimic acute infection of the genital tract in women. The Innate Immune Response in Chlamydial Infection Generally, female genital tract (FGT) contains few leukocytes and must recruit immune cells capable of eradicating infections from the central circulatory system(9). One early mechanism that appears to reduce the number of organisms early after infection is an influx of neutrophils, which have the capacity to destroy chlamydiae, as demonstrated in vitro(10). Likewise, in mice that were depleted of neutrophils by antibody treatment, the number of organisms isolated from the FGT were approximately 10-fold greater the day after infection. However, neutrophils are not critical for the

Pathology Conjunctivitis, and conjunctival and corneal scarring Cervicitis, urethritis, endometritis, pelvis inflammatory disease, tubal infertility, ectopic pregnancy, neonatal conjunctivitis and infant pneumonia Submucosa and lymph-node invasión, with necrotizing granulomas and fibrosis

eradication of C. trachomatis genital infection since all the mice were able to resolve the infection within the same time frame(11). Thus, neutrophils appear to play a role in reducing the initial amplification of C. trachomatis and possibly in limiting the spread locally within the FGT. Natural killer (NK) cells, as well γδ T lymphocytes have also been implicated in the initial control of clamydial infections. Tseng and Rank(12) reported that mononuclear cells isolated from the FGT of infected mice showed YAC1 cell cytotoxicity in vitro, which is a measure of NK cell function. Although the depletion of NK cells did not reduce the number of organisms isolated from the FGT during the first week after infection, continued depletion throughout the course of infection resulted in the delayed clearance of Chlamydia. Interestingly, NK cells appear to be necessary for the development of a Th1 protective response against Chlamydia. The authors indicated that NK cells are responsible for the early production of IFN-γ, which would suggest that the primary role of early IFN-γ production by NK cells is to down-regulate the Th2 response, thereby allowing expression of a strong Th1 response which has been shown to be essential for resolution of the infection. In contrast, studies on the role of γδ T cells have shown that these cells play a modest positive role in host defense early in C. trachomatis infection(13). Role of DTH in Protective Immunity against and in the Pathology of Chlamydial Infection The Delayed Type Hypersensitivity (DTH) response has long been considered as both potentially protective and pathological, and this is represented the functional differences in Th1 and Th2- type DTH responses in chlamydial infection. Chlamydia is able to induce both Th1 and Th2 DTH responses each being associated with its respective cytokines which are related with either the clearance of infection or immunopathology. Many groups have shown that DTH and Th1 responses (IFN-γ production) are the major protective mechanisms against chlamydial infection(14-18). Paradoxically, immunopathological responses (mucosal scarring) to

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chlamydia have also been found to be mediated by DTH(1921) .Yang and colleagues (22) studied the protective and immunopathological immune response to C. trachomatis using IL-10 gene knockout (KO) and IFN-γ KO mice. The results indicated that, in the absence of IFN-γ, mice are still capable of mounting a significant CD4 T cell-mediated DTH to chlamydial infection, which is associated with Th2 cytokine production and characterized by eosinophil infiltration. The Th2 type DTH response in these mice induced by chlamydial infection was similar in cellular pattern and kinetics to the type 2 cytokine-associated inflammatory reactions in other intracellular models systems(23). Therefore, this Th2 type DTH in IFN-γ KO mice was not protective in controlling local chlamydial infection and preventing its dissemination. The study by Yang and colleagues(22) suggested that DTH is a double-edged sword in Chlamydia-induced immune responses. Although DTH type 1 is critical for host defense against chlamydial infection thus providing protection, DTH type 2 is not protective and may even promote pathology. The existence of the distinct types of DTH response may be one of the reasons for the dual role of DTH in chlamydial protective immunity and immunopathology. Cytokines Recent progress in elucidating the development basis for Th1/Th2 differentiation resulted from the finding that microenviromental cytokines are key factors that influence the behaviour of Th cell precursors to become Th1 or Th2 cells. In particular, the early presence of IFN-γ and IL-12 favors Th1 polarization, whereas the early presence of IL4 and IL-10 is a potent stimulus for Th2 commitment. In general, the source of these cytokines during the early phases of the immune response often depends on the innate defense mechanisms mobilized by the pathogen. Given the intracellular development cycle of Chlamydia, there is strong evidence for the involvement of cell-mediated immunity (CMI) (Th1-pathway) and its associated cytokines as IFN-γ, IL-2, and IL-12, in resolving a chlamydial infection. After infection with Chlamydia spp., epithelial cells produce various pro-inflammatory mediators, including CXCchemokine ligand 1 (CXCL1), CXCL8 (also known as IL-8), CXCL16, granulocyte/monocyte-colony stimulating factor (GM-CSF), IL-1γ, IL-6 and tumor necrosis factor α (TNFα). Also, an upregulate expression of the chemokines CCchemokine ligand 5 (CCL5) and CXCL10 is produced, and the epithelial infected cells secrete cytokines that promote the production of IFN-γ, including IFN-α, IFN-β and IL-12. Infected fibroblasts secrete IFN-γ, IFN-β and nitric oxide, whereas infected macrophages produce TNF-α and IL-6(24). Previous studies showed that Th1 CD4+ T cell responses

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play the dominant role in protective immunity against chlamydial infection, whereas Th2 cytokine responses, especially IL-10, may be associated with immunopathological responses. The Th2 cells appear to accelerate tissue fibrosis and granuloma reactions, fail to reach areas of chlamydial infection and by definition do not secrete cytokines such IFN-γ that inhibit the growth of chlamydiae(22). Recent studies have provided data that confirming the extreme importance of IL-10 in mediating host susceptibility to chlamydial infection and the development of pathological changes. However, besides IL-10, other cytokines may be also involved in granuloma formation and fibrosis in the course of chlamydial infection. For example, transforming growth factors-β (TGFβ), a cytokine produced predominantly by Th3 cells(25,26), and other cell types can promote the growth of fibroblasts and induce the synthesis of extracellular matrix proteins(27). Moreover, TGF-β1 is able to alter the antigen presenting cells (APC) thus polarizing T cell responses towards a Th2 type immune response(28). Carefully regulated cytokine production is crucial to successful immune responses to intracellular pathogens. Deficiencies or overstimulation in the production or activity of these pro-inflammatory cytokines may be associated with the failure of protective immunity or harmful inflammation. The pathways trough which acute chlamydial infection is resolved or, alternatively, progresses to chronic infection with severe pathology, appears to be varied and dependent on a closely entwined interplay between host and pathogen. Are still to be resolved the means by which a particular immune pathway (humoral or cell-mediated immunity) is adopted, strengthened, or shifted away from balance in favour of one, as demonstrated in other infections(29), all under the influence of changing cytokine patterns, and the degree to which each of these effect the outcome of infection. One obstacle to understanding the significance of cytokines in infection is the difficulty of extrapolating from animal to human models(30). The contribution of IFN-γ to chlamydial resistance has become less clear due to variations in cytokine susceptibility across different C. trachomatis strains(31) and genetic differences between mouse strains(32). Role of Dendritic Cells in the Immune Response to Chlamydial Infection The Th cells are activated by recognition of antigenic peptides presented with histocompatibility complex (MHC) class II molecules by APCs, including dendritic cells (DCs), macrophages and B cells. A recent hypothesis states that differential expression and engagement of Toll Like Receptor (TLR) family members at the surface of DCs influences

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the type of immune response that is induced by a microbial pathogen (33). Given the high level of expression of TLRs by DCs and the ability of DCs to polarize immune responses, identification of the role of DCs in Chlamydia-specific immune response is crucial for understanding the type of immune response that is elicited and therefore also for designing a vaccine against infection by C. trachomatis. The capacity of DCs to present chlamydial antigens has been demonstrated by in vitro and in vivo studies with controversial results. Waalen and colleagues(28) found that DCs were much more potent than monocytes in presenting C. trachomatis antigens. They also found that the DCs from different sources all expressed high levels of MHC class II molecules and that the DC-induced T cell responses to chlamydial antigens could be inhibited by antibodies against HLA-DQ and HLA-DR molecules. The data from this study indicate that DCs may play an important role in presenting chlamydial antigens to T cells in rheumatoid inflammation. Ojcius and colleagues(34) found that Chlamydiae were internalized by DCs in a non-specific manner through macropinocytosis followed by the fusion of the macropinosomas with DC lysosomes which express MHC class II molecules. DCs which have internalized chlamydial organisms can present chlamydial antigens and activate chlamydia-specific CD4+ T cells. Detailed data on the uptake and processing of C. trachomatis antigens by human DCs(35) showed that the entry of C. trachomatis was mediated by the attachment of the organism to heparin sulfates, which could be blocked by heparin. Infection of DCs with C. trachomatis led to activation of these APCs, which produce IL-12 and TNF-α but not IL10. Besides their ability to present chlamydial antigen to CD4+ T cells, infected DCs were also found to be able to activate chlamydia-specific CD8+ T cells(34). Several studies have been performed to study the role of DCs in inducing an anti-chlamydial immune response in vitro using bone marrow (BM)-derived DCs. Lu and Zhong(36) showed that vaccination with murine BM-derived DCs pulsed with heat-killed Chlamydia could induce protective immune responses in a genital infection mouse model. The protective immune response induced by Chlamydia-pulsed DCs was correlated with a Chlamydia-specific Th1 type immune response, similar to that immunized by live chlamydial strains. They also found that BM-derived DCs could efficiently phagocytose Chlamydia, secrete IL-12 and present chlamydial antigen to infection-sensitized CD4+ T cells in vitro. A very interesting study reported by Shaw and colleagues(37) showed that DCs pulsed with a recombinant chlamydial MOMP antigen had a different role on activating CD4+ T cells in vitro and in vivo experiments. The authors found that, although DCs pulsed with recombinant MOMP secreted

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IL-12 and stimulated infection-sensitized CD4+ T cells to proliferate and secrete IFN-γ in vitro, the adoptive transfer of the pulsed DCs to naive mice unexpectedly generated a Th2 rather than Th1 anti-MOMP immune response. These findings suggest that the immunological properties of ex vivo pulsed DCs are not necessarily predictive of the immune response generated in vivo following adoptive transfer. Although there have been substantial studies on in vitro DCs cultures, the recruitment and characteristics of DCs in a natural infection circumstance has yet to be well documented. Studies(38) reported that immunity to C. trachomatis lung infection induced by vaccination with live organisms was correlated with early production of GM-CSF and IL-12 and the maturation of DCs. The immune response in mice vaccinated with viable organisms included high levels of organism-specific DTH reaction, IFN-γ production and IgA responses. In contrast, vaccination with inactivated organisms mainly induced IL-10 production and IgG1 antibody response without IgA or DTH reaction. The results suggest that early production of pro-inflammatory cytokines and the recruitment of DCs may be the key mechanism by which live-organism vaccination induces active immunity to C. trachomatis infection. Recently, Rey-Ladino and colleagues(39), in a interesting study using murine BM-derived DCs to examine DC maturation and immune effector function induced by live and UV-irradiated C. trachomatis EBs, confirmed that the level of protection induced by UV-EB-pulsed DC in mice was significantly less than achieved using DC pulsed ex vivo with viable EBs. Thus, exposure of DC to live EBs of C. trachomatis resulted in a mature DC phenotype which was able to promote protective immunity, while exposure to UV-Ebs generates a semimature phenotype with less protective potential. This result may explain in part the differences in protective immunity induced by natural infection and immunization with whole inactivated organisms. This study indicates that future directions for rational vaccine design require the use of strategies that cause full DC activation and that the ideal antigen-adjuvant combination would mimic the DC maturation effects induced by live EBs. The CD4+ and CD8+ T Cells in the Immune Response to Chlamydial Infection In animal models, the transfer of T lymphocytes from infected or immunized mice can facilitate the clearance of infection in T cell-deficient mice, and this effect has been demonstrated for both CD4+ and CD8+ T lymphocytes(40-44). In humans, both T cell subsets can be detected at the site of C. trachomatis infection, but most work on defining the immune response to C. trachomatis in humans has concerned

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CD4+ T cells. A number of C. trachomatis antigens which can be recognized by human CD4+ T cells have been identified, including MOMP(45), the 60-kD cysteine-rich outer membrane protein 2 (Omp2)(46), polymorphic outer membrane protein D (POMP-D(47), heat shock protein 60 (hsp 60)(48), the histonelike protein Hcl and enolase(47); several epitopes have been mapped within these antigens. In contrast, much less is known about the antigenic specificity and roles of CD8+ T lymphocytes during C. trachomatis infection. In recent years human CD8+ T cells able to recognize MOMP(49, 50) or Hsp60(49) have been isolated from infected humans, but the approach in each case was to identify peptides in C. trachomatis proteins which could be predicted to bind to common class I HLA alleles and to determine whether infected subjects had CD8+ T cells able to recognize these peptides. Cellular toxicity and cytokine-mediated functions are two possible mechanisms that may participate in the CD4+ Th1 immune response that is essential for host resistance to chlamydial genital tract infection. The Th1 cytokine IFN-γ and TNF-α are essential for optimal clearance of infection from genital tract tissue(31, 39, 51-56) in humans and in experimental animals. The effector role for IFN-γ in mediating chlamydial clearance could include both immunoregulatory and nonregulatory functions. An immunoregulatory function for IFNγ production CD4+ Th1 cells would be in the activation of antigen-specific cytotoxic CD8+ T cells, although such a mechanism seems unlikely because CD8+ T cells are not required for immunity(57-60). Although the effector mechanisms of IFN-γ mediated in vivo infection with C. trachomatis are not completely understood, it is well established that IFN-γ limits the in vitro growth of C. trachomatis through inducing production of tryptophan-decyclizing enzyme indoleamine 2, 3-dioxygenase (IDO). Activation of IDO by IFN-γ leads to the degradation of thryptophan, and lack of this essential aminoacid causes the death of C. trachomatis through tryptophan starvation(61) (Fig. 1). Recently, it has been showed that genital, but not ocular, serovars of C. trachomatis can use indole as a substrate to synthesize tryptophan in the presence of IFN-γ. This finding suggest that genital strains of C. trachomatis might escape IFN-γ mediated eradication in the genital tract by using indole provided by the local microbial flora of the FGT(62). Another IFN-γ inducible host cell function in chlamydial immunity is the induction of inducible nitric oxide synthase, although the production of bactericidal nitric oxide free radicals is not a plausible mechanism because mice genetically deficient in inducible nitric oxide synthase resolve both primary and secondary chlamydial infections with kinetics similar to those of wild type mice(63). In passive transfer experiments, CD4+ T cells which produce IFN-γ have been shown to provide protection against

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C. trachomatis in rodents but clearance of the pathogen was slower than in animals to which both CD4+ and CD8+ T cells were transferred, suggesting that additional IFN-γ might be produced by the CD8+ T subset(40, 44). The C. trachomatisspecific CD8+ T cells also play a role in the activation of DC. Matyszak and Gaston(64) showed that clones of CD8+ when co-cultured with infected DCs, induced a rapid increase in IL-12 production by DC. Since IL-12 is critical for stimulating the Th1 response and, therefore the expansion of IFN-γproducing CD4+ T cells, the effects of CD8+, in addition to their own ability to produce IFN-γ, may be to obtain an appropiate Th1-polarized CD4+ T-cell response to C. trachomatis, although the CD8 T cells might have a contributory role in limiting infection with Chlamydia spp., mainly via cytokines, they are apparently not essential for the clearance of chlamydial infection(57,59). Some observations suggest that antigen-specific cytotoxic T cells may contribute more to the pathogenesis of chlamydial infection than to protective immunity. For example, chlamydiae-specific T cell-mediated cytolysis requires high lymphocyte-to-target cell ratios and the lysis of infected targets occurs late in the chlamydial development cycle, at a time when the majority of organisms have differentiated into EBs. This finding does not support a mechanism that would favour inhibition of intracellular growth and argues that cytolytic T cells could potentially contribute more to the pathology or spread of infection than to its eradication(65). Role of Humoral Immune Response in Chlamydial Immunity A definitive role for anti-chlamydial antibodies has been more difficult to demonstrate than for a cellular immune response. However it is increasingly clear that a specific humoral immune response, including secretory and systemic antibodies, appears to play a role in protective chlamydial immunity, facilitating memory response and augmenting the primary CMI during reinfection (58,65,66). Several immunobiological studies in a murine model of chlamydial genital and pulmonary infection revealed that efficient clearance of reinfections and the development of a protective memory response were dependent upon a competent humoral immune response with an intact B-cell function and antibody production(59, 65). Interestingly, mice that lack Fc receptors suffer more severe secondary infection with C. muridarum than wild-type mice, owing in part to impaired cellular immune responses, which indicates that B cells and antibodies might also be important for enhancing protective effector T-cell responses(67). Possible mechanisms for how B cells contribute to immunity to re-infection include antibodymediated neutralization and opsonization, as well as enhanced

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antigen presentation to T cells, possibly following Fc-receptormediated uptake of antigen-antibody complexes (59, 68). However, these mechanisms may be more important in preventing the dissemination of chlamydiae to distant sites rather than in resolving infection of the mucosal epithelium. Antibodies might also contribute to the resolution of intracellular infection by an antibody-dependant cellular cytotoxicity (ADCC) mechanism. The potential role for an ADCC mechanism in immunity to chlamydial infection comes from studies demonstrating than an immunoglobulin A (IgA)-dependent CD4+ T cell ADCC mechanism functions in immunity to other intracellular bacterial pathogens such Salmonella and Shigella(69-71). In addition, B cells are important antigen presenting cells in the activation of memory Th cells and function by promoting the clonal expansion of high frequencies of antigen-specific memory Th cells(72). Thus, current findings support the operational paradigm that a potentially efficacious chlamydial vaccine should elicit high levels of both mucosal and systemic Th1 responses, as well as a humoral response that rapidly foments Th1 activation following reinfection.

VACCINE DEVELOPMENT From the above discussion, based on current knowledge, an ideal vaccine against C. trachomatis genital infection would need to induce both a systemic CMI response, to deal with C. trachomatis as an intracellular pathogen, plus a local mucosal IgA response to reduce bacterial shedding and the resulting spread of infection. At this moment, this represents a daunting challenge for several reasons: little knowledge exists on regulation of the immune response to C. trachomatis in the FGT which seems influenced by sex hormones(73, 74), the lack of adjuvants that target vaccines to the genital mucosa and our limited knowledge concerning which C. trachomatis antigens induce protective immune response. The observation that the immune response is directly or indirectly involved in the pathogenesis of disease caused by Chlamydia spp. further complicates the vaccine development process. The successful design and delivery of a future chlamydial vaccine will depend on a better understanding of these factors and how they can be manipulated to achieve optimal vaccine efficacy. Antigens The form, structural and immunobiochemical properties of an antigen selected as potential vaccine determine its capability to induce the required immune effectors that provide protective immunity against chlamydiae. Experimental vaccine selection efforts using outer membrane fractions, recombinant

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proteins, naked DNA and ex vivo antigen-pulsed DCs in models of chlamydial genital, respiratory and ocular infections have revealed promising vaccine candidates(75,76). Because immune protection against C. trachomatis infection is probably mediated by immunization with targets protein of CD4+ and possible CD8+ T cells, identification of such proteins is particularly important. In fact, recent years have seen the characterization of eight C. trachomatis proteins that are target for T-cell recognition(43,46,48,50,77,78). However, the most studied and most promising vaccine candidate is C. trachomatis MOMP, which contains serovar-specific epitopes, and five constant domains which are highly conserved among the different serovars and which contain several conserved CD4+ and CD8+ T-cell epitopes. Another vaccine candidate is C. trachomatis Omp2 which is also an immunodominant antigen that contains CD4+ and CD8+ Tcell epitopes, this could provide protection against the different C. trachomatis serovars because it has more highly conserved in amino-acid sequence than MOMP(79). The entire genome of C. trachomatis strain D has suggested several new candidate chlamydial antigens that contain known T-cell epitopes, including Hsp60, YopD homologue (homologue of Yersinia pseudotuberculosis), enolase and POMP(78,46). Among the many exciting findings was the identification of genes encoding the components for a complete type III secretion system (TTSS), the principal virulence mechanism of the organism(80). As for the outer membrane proteins, the effector proteins secreted by TTSS are possible immunotherapeutic targets. Possible candidates for secretion via the TTSS include the Inc proteins which lack classical amino terminal signal sequences but which have been shown to be localised in the inclusion membrane(81). Indeed, one such protein known as CrpA, has been shown to be targeted by CD8+ T cells and to confer partial protection to mice infected with C. trachomatis(43). It is also important to consider that some C. trachomatis antigens contain epitopes that might be associated with pathogenic responses. For example, C. trachomatis Omp2 or Hsp60 have been found in patients with reactive arthritis triggered by previous infection with C. trachomatis(47). Preference for Subunit Vaccines Following immunization with the whole organism, poorly protected individuals who were re-exposed to chlamydiae developed a more severe disease than individuals who were not immunized(82) (Table II). Mounting evidence suggests that the Hsp60 may account for the negative effects observed during vaccination trials and for some of the longterm sequelae that may result from chlamydial infection(83). In recent years attention has turned to DNA vaccination as a way of inducing a protective response against chlamydial

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TABLE II. Recent results of vaccination trials using Chlamydia muridarum major outer membrane protein as antigen Immunogen and adjuvant*

Route of chlallenge

Protection level

Immune response

Ref

MOMP DNA

Intranasal

~103 less IFUs in lungs

84

MOMP DNA MOMP DNA (ISCOMs)

Vaginal Intranasal

No effect ~106 less IFUs in lugs

Conformational MOMP (Freud’s adjuvant) MOMP (rVCGs)

Upper genital tract

~70% reduction in IFUs in vagina ND

Th1-like, enhanced DTH and IgG2a and IFN-γ production Weak DTH and antibody production Th1-like, enhanced DTH and IgG2a and IFN-γ production Th1-like, increased IgG2a in serum and IFN-γ production by splenocites CD4+ T cells protected naïve mice on adoptive transfer Mixed Th1/Th2 Mixed Th1/Th2

98

Th1, IgG2a in serum and IFN-γ production by splenic T cells

24

MOMP (CT and CpGcontaining ODNs) MOMP (OspA of Borrelia burgdorferi) MOMP and OMP2 (rVCGs)

ND Vaginal Vaginal Vaginal

~50% reduction in IFUs in vagina ~50% reduction in IFUs in vagina 80% of animal protected

85 84 24 99 24

*Adjuvant(s) shown in parentheses. CT, cholera toxin; DTH, delayed-type hypersensitivity; IFN-γ, interferon-γ; IFU, inclusion-forming unit; ISCOM, immunostimulating complex; MOMP, major outer membrane protein; ND, not determined; ODN, oligodeoxynucleotide; OMP2, outer membrane protein 2; OspA, outer surface protein A; rVCG, recombinant Vibrio cholerae ghost; Th, T helper.

genital infection(84). However in the search for a vaccine against human chlamydial infection this method has generally been more successful at eliciting a protective response in the murine respiratory model than in the equivalent genital tract model(85). In general, DNA vaccination has been shown to be more effective in mice than in humans or large animals(86). As a consequence, the focus of C. trachomatis vaccine research has now turned to the production of subunit vaccines that are based on individual C. trachomatis protein antigens, which are administered with adjuvant or other delivery vehicles. In this respect, recent studies in chlamydial genomics have predicted several immunogenic proteins that may serve as potential chlamydial vaccines(87-90). In addition, the formely serologically defined and molecularly characterized chlamydial antigens of the MOMP are identifying additional vaccine candidates(89-92). However, vaccine effectiveness based upon MOMP has been limited, due in part, to poor immunogenicity and consequently producing only partial immunity, as measured by a reduction in the infectious burden or pathology. The lack of satisfactory protective immunity with MOMP-based vaccine regimens would suggest that either MOMP alone is inadequate as a vaccine (calling for multisubunit vaccines), or that more effective delivery systems are needed to optimize the effect of MOMP and potentially other emerging single subunit vaccine candidates. Interestingly, some of the POMPs have been shown to induce antigen-specific T cell response against chlamydia antigens(47).

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Comparative structural and immunological analysis of these antigens could well lead to the judicious selection of a combination of immunogens for a multisubunit vaccine. A major advantage of the multiple subunit approach is the potential synergistic immunological benefit of a combination of epitopes from multiple antigens, which could likely induce a higher frequency of immune effectors that ensures an effective long-lasting immunity. In addition, the associated epitopes required for optimal Th1 activation may depend on antigen conformation. Additional studies are therefore needed to clarify the role of antigen conformation in the induction of protective T-cell immunity against chlamydiae. Vaccine Delivery Systems The focus on a chlamydial subunit vaccine against C. trachomatis requires the development of safe and effective delivery vehicles, such as adjuvants and vectors, or biological manipulations techniques capable of boosting the Th1 response and targeted to the genital mucosa, which is the major goal in chlamydial research. There have been a limited number of vaccine studies that have investigated the role of adjuvants in the protective immune response against chlamydial infection in humans. To date, only aluminium salts, liposomes and MF59 have been approved for use in humans(93). Aluminium adjuvants alone are probably not suitable for controlling primary chlamydial infections as they induce a Th2 humoral response, whereas MF59 and ISCOM matrix are perhaps more appropiate

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as they can induce a Th1 cellular response(94). In a murine model of C. trachomatis genital tract infection following intramuscular immunization MOMP incorporated into ISCOMs has been shown to induce a local genital mucosal Th1 response and protection(95). Vector-mediated immunization with naked DNA has received much attention, with delivery of MOMP and Hsp60 genes showing some promising results(96,97). However, this approach has been successful in the murine lung model not in the genital tract(85). Other approaches have investigated the use of bacteria and bacterial antigens as delivery vehicles and adjuvants. Co-administration of the outer surface protein, OspA, of Borrelia burgdorferi with C. muridarum MOMP offered significant protection in mice against chlamydial genital challenge (98). Recent efforts in designing multisubunit experimental ghost vaccines, by expressing select chlamydial proteins on the epitheliotropic Vibrio cholera ghosts (VCG) have also provided attractive results. Intramuscular immunisation with recombinant VCG-MOMP has been found to induce elevated local genital mucosal and systemic Th1 responses in a murine model of C. trachomatis genital tract infection(99). This option has the advantage that the VCGs are non-toxic, possess intrinsic adjuvant properties, maintain the structural functional and immunological integrity of expressed antigens, adequately target the primary APCs and are likely to induce mucosal immune responses since Vibrio is an epitheliotropic pathogen. In addition it has been demonstrated that the cell targeting and adjuvant properties of recombinant VCG are superior to classical adjuvants such as complete Freund’s or aluminium salts, while their safety and relatively cheap production cost offer a technological and manufacturing advantage for a vaccine needed on a global scale(100). Other promising studies, such as using of DCs to deliver and present chlamydial antigens in vivo, have shown some of the most interesting experimental results(101,102). It has been suggested that this phenomenal efficacy of the DC-based cellular vaccine makes them «natural adjuvants or preeminent delivery vehicles», useful as tools in the design of effective delivery systems that mimic the action of DCs, for immunizing against chlamydial infections and for unravelling the necessary vaccine machinery in terms of antigens and immunostimulatory and homing requirements(66).

CHLAMYDIAL GENITAL INFECTIONS IN VETERINARY MEDICINE: OVINE ENZOOTIC ABORTION Following reclassification of the Order Chlamydiales in 1999(1) the genus Chlamydophila contains the species C. abortus. This species is the most common infectious cause of abortion in sheep (ovine enzootic abortion, or OEA) and goats in

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Europe, and results in significant losses to agricultural industries worldwide(103). It is also recognized as a cause of reproductive failure in cattle, horses and pigs, although the economic impact of such infections is unknown because of the lack of epidemiological data. C. abortus is also a zoonotic pathogen and can cause severe, life threatening diseases in pregnant women. Infection in women during pregnancy can result in spontaneous abortion or stillbirths, which are typically preceded by several days of acute influenza-like illness, as well as renal failure, hepatic dysfunction and disseminated intravascular coagulation(104,105). As regards OEA, adult ewes are infected as a result of the contamination of lambing pens or pasture by fetal membranes and discharges. Few or no clinical signs are observed in non-pregnant animals until, during a subsequent pregnancy, ewes abort. Abortion always appears in the last weeks of gestation, regardless of the moment of infection(106). Immune response against Chlamydophila abortus As in the case of C. trachomatis, mouse models have been widely used to study the pathogenesis and the immune response in C. abortus infections(107-110). In experimental murine infections the systemic spread of C. abortus is followed by the establishment of an effective immune response capable of eliminating the infection from every organ except placenta where the bacteria multiply, inducing abortion. Studies from our laboratory have described the kinetic of C. abortus colonization in placenta using a mouse model(108) and natural host(111). The important role of innate immunity, especially associated with neutrophils, has been shown(112) in the early stages of a primary infection, when it contributes to establish a specific immunity through the secretion of different cytokines. Neutrophils also influence the recruitment of other leukocyte populations, especially CD8+ T cells(113) in a primary response, although these cells have been shown to be of limited relevance in a secondary infection(114). Another component of innate immunity, the natural killer (NK) cells, has been studied in C. abortus infection by Buendía and colleagues(115). The authors demonstrated the relationship between the NK cells and early IFN-γ production in the control of infection of C. abortus, as well as the complex and close relationship with neutrophils, which produce cytokines that are chemotactic and activators for NK cells. Although innate immunity plays an important role, C. abortus infection is mainly controlled by a specific Th1 immune response which is, at least partly, IL-12-independent and characterized by the early production of high concentrations of IFN-γ (116) and the presence of T cells, particularly CD8+(109,113) in contrast with of C. trachomatis

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immune response(57-60). In fact, unpublished findings(117) suggest an additional regulatory role for CD8+ T cells in the primary response to C. abortus infection. The balance of the specific immune response is a complex feature. Some reports have shown that the exacerbated production of cytokines in response to C. abortus infection can induce pathological changes(116,118) and abortion has been associated with the detrimental effect of inflammatory cytokines (IFN-γ, TNF-α) induced by the infection in the placenta(119). The role of humoral immunity against C. abortus has been poorly studied, although we have demonstrated that the passive transfer of anti-MOMP monoclonal antibodies protect pregnant mice against chlamydial abortion(107). We also studied the role for B cells during C. abortus infection(118) and found that B cells are not essential for controlling the multiplication of C. abortus in a secondary infection but could have take some part in controlling the exacerbated inflammatory response induced by C. abortus primary infection. Vaccination against Chlamydophila abortus infection The goal for the prevention of OEA is to obtain an effective vaccine against C. abortus infection. Inactivated vaccines prepared from egg-grown or cell cultures have formed the basis for the prevention of the infection since the early 1950s(120). However, efficiency varies, since outbreaks of the disease have been reported in vaccinated flocks(121,122). Killed vaccines can reduce the incidence of abortion and also the shedding of C. abortus at lambing, although they may not stop shedding completely in all cases, which leads to endemic cycles of infection that have serious consequences regarding the epidemiology of OEA. An alternative approach to solve this problem has been to develop a live temperature-sensitive attenuated vaccine, which is commercially available and which has been shown to offer good protection against C. abortus-induced abortion(123,124). However, the potential dangers of this kind of vaccine make them a less advisable choice, particularly since C. abortus can also cause abortion and severe illness in pregnant women (104). Therefore, the identification of new strains of C. abortus with a different antigenic structure from the vaccinal strain may affect the very protection for which this vaccine was designed(125). To date, efforts directed at obtaining a suitable subcellular vaccine have largely focused on the MOMP, which is reportedly highly immunogenic if it used in its native oligomer form(107) in contrast to C. trachomatis. However, since cell culture yields of C. abortus are poor, the purification of MOMP oligomer from the bacteria is very difficult and prohibitively expensive for an ovine vaccine. Furthermore,

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vaccine studies performed in ewes to examine the efficacy of different forms of recombinant MOMP against experimental infection have been disappointing (126). Finally, all the vaccination attempts in mouse models for C. abortus with different DNA vaccines including the genes of Dnak (Hsp70) and MOMP have failed in the induction of a protective immune response(127). The experimental mouse model is a useful tool for validating commercial or experimental vaccines against C. abortus(107,127-131). Our group demonstrated that none of the inactivated vaccines commercially available in Spain provides an acceptable degree of protection against the bacteria(130). Therefore, in the same murine model, our lab tested different vaccine-production procedures in order to design new inactivated vaccines against C. abortus(131). The results showed that two experimental vaccines (QS-21 and Montanide ISA 773 adjuvated vaccines) induced good protection and elicited an adequate degree of the cellular immune response required for the clearance of infection. Finally, the selected vaccines were seen to prevent abortion and C. abortus shedding at delivery in pregnant mice. One important aspect of vaccination against OEA is to choose specific adjuvants that help activate the effector cells or cytokines, polarizing the immune response towards a Th1 type. Related with this fact, in a study on the influence of a Nippostrongylus brasiliensis parasite infection before or after C. abortus vaccination(132), using QS-21 and aluminium hydroxide as adjuvants, we determined that the best protection was offered by QS-21 vaccine. However, the effectivity offered by the vaccine adjuvated by aluminium hydroxide, an adjuvant usually included in vaccines for veterinary medicine, was reduced when the Th2 response induced by N. brasiliensis was established just prior to infection with C. abortus. In field conditions it is usually not possible to prevent parasitic infections before C. abortus infection, so the authors concluded that care should be taken in choosing the most effective adjuvant or type of vaccine being used to avoid the deleterious immune effects of a high parasitic burden. Recently(133), our group tested these vaccines in sheep and compared the results with those obtained with two representative commercially available vaccines in the natural host. The results showed that the new inactivated vaccines notably increased the protection conferred and minimized C. abortus shedding at delivery. Finally, the recent publication of the complete genome sequence of C. abortus(134), as it is already available for C. trachomatis, will undoubtedly contribute to the identification of other potential protective antigens that could serve as candidates in experimental vaccines against ovine enzootic abortion.

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CLOSING REMARKS In human and veterinary medicine, contemporary immunological, antigenic and immunomodulatory paradigms guiding vaccine design against Chlamydiae are and well grounded on reproducible and experimental evidence. Experimental findings in animal models are being extended and substantiated by more recent results from clinical studies in humans, which have confirmed the crucial role of the Th1 response in anti-chlamydial immunity. The complementary role of antibodies is appealing in that they contribute to chlamydial immunity by facilitating an enhanced Th1 response. Vaccine capable of eliciting high levels of Th1 response and the complementary CMI- associated IgG2a and IgA antibodies are the current focus of anti-chlamydial vaccine research. More recent approaches involving the use of recombinant antigens and peptides have been relatively unsuccessful, and so current research aims to improve the presentation of the chosen antigens, and thus the protective efficacy of the vaccine, through the careful selection of appropriate adjuvants, delivery vehicles and routes of inoculation. Chlamydiae vaccine research will continue to focus on the identification of additional antigens that induce protective T cell responses, more feasible now that the complete sequence of the chlamydial genome is available, and on the mechanisms that promote protective immunity in FGT, including the role of DCs in antigen uptake and presentation and the role of pro-inflammatory cytokines in influencing the Th1/Th2 response bias. Further data are required to understand the mechanisms that downregulate the immune response in the FGT, including the effects of sex hormones and the menstrual cycle. Ideally, suitable vaccines should also limit the shedding of infectious organisms and the spread of infection, but also offer heterotypic cross-protection against the various serovars and subtypes of each species of Chlamydia.

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ACKNOWLEDGMENTS Some investigations reviewed in this work were financed by the following grants: AGF97-0459, 1FD97-1242-CO2-01, AGL2001-0627 and AGL2004-06571, all from MEC, MCyT and FEDER.

CORRESPONDENCE TO: J. Salinas Phone 968 364729 Fax: 968 364147 E-mail: [email protected]

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