Analysis of the Humoral Immune Response to Chlamydia Outer Membrane Protein 2

The humoral immune response to Chlamydia outer membrane protein 2 (Omp2) was studied. Omp2 is a highly genus-conserved structural protein of all Chlamydia species, containing a variable N-terminal fragment. To analyze where the immunogenic parts were localized, seven highly purified truncated fusion proteins constituting different regions of the protein were produced (Chlamydia pneumoniae-Omp2_aa23-aa93, Chlamydia psittaci-Omp2_aa23-aa94, and Chlamydia trachomatis-Omp2_{aa23-aa84, aa87-aa547, aa23-aa182, aa167-aa434, aa420-aa547}). By an enzyme-linked immunosorbent assay with serologically defined patient sera, Omp2 was found to be a major immunogen of both C. pneumoniae and C. trachomatis infections (P 0.0001). The humoral immune responses were not confined to any particular region of the Omp2 protein, and no species-specific anti-Omp2 immunoglobulins were detected.     

Toll-Like Receptor 2-Dependent Activity of Native Major Outer Membrane Protein Proteosomes of Chlamydia trachomatis

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen and the etiologic agent of blinding trachoma. Intracellular signaling pathways leading to host cell inflammation and innate immunity to Chlamydia include those mediated by Toll-like receptors (TLRs) and nucleotide binding oligomerization domain 1 (Nod1) protein. In epithelial cells, TLR-dependent signaling contributes to local immune responses via induction of inflammatory mediators. There is evidence that TLR3, TLR4, and, particularly, TLR2 are critical for Chlamydia-mediated host cell activation and pathology. Despite the importance of TLR2, major chlamydial TLR2 antigens have not been identified so far. Numerous bacterial porins are known TLR2 agonists, i.e., porins from Neisseriae, Shigella, Salmonella, Haemophilus influenzae, and Fusobacterium nucleatum, which share structural and functional similarities with the chlamydial major outer membrane protein (MOMP), a strong antigen candidate for a potential vaccine against C. trachomatis. We describe the ability of purified, detergent-free MOMP to signal via TLR2 in vitro in TLR-overexpressing cells and TLR2-competent human reproductive tract epithelial cell lines. Using MOMP formed in pure protein micelles (proteosomes), we show the induction of TLR2-dependent interleukin-8 (IL-8) and IL-6 secretion in vitro, the involvement of TLR1 as a TLR2 coreceptor, and the activation of both NF-κB and mitogen-activated protein (MAP) kinase intracellular pathways. Interestingly, MOMP proteosomes induce cytokine secretion in endocervical epithelial cells (End/E6E7) but not in urethral epithelial cells (THUECs). A detailed understanding of the TLR2-dependent molecular mechanisms that characterize the effect of MOMP proteosomes on host cells may provide new insights for its successful development as an immunotherapeutic target against Chlamydia.     

The Bioinformatics Analyses Reveal Novel Antigen Epitopes in Major Outer Membrane Protein of Chlamydia trachomatis

Purpose: The aim of this study was to predict the T-cell and B-cell epitopes in major outer membrane protein (MOMP) of Chlamydia trachomatis (CT) by using online software and also to analyse the secondary structure of MOMP through bioinformatics tools. Materials and Methods: The predictions of secondary structure of MOMP protein were carried out using SOPMA software, and the prediction of B-cell epitopes in MOMP protein was carried out using IEDB and LEPS software, while the T-cell epitopes were predicted by the software of IEBD and SYFPEITHI. The predictions from the software were combined with MOMP protein characteristics, including surface features, hydrophilicity, flexibility, accessibility and plasticity, to analyse the common epitope areas’ response by T-cells and B-cells. Results: In the secondary structure of CT MOMP, the alpha-helices accounted for 41.62% of total amino acid, while the beta sheets and random coil accounted for 19.80% and 32.49%, respectively. Predictions combined with MOMP protein surface features, hydrophilicity, flexibility, accessibility and plasticity were further characterised, and three high-score B-cell epitope areas were found as located in 24–31, 307–311 and 318–327 amino acids of MOMP protein, respectively; in the meanwhile, three high-score T-cell epitope areas were found in 234–236, 323–329 and 338–343 amino acids of MOMP using major histocompatibility complex (MHC) class I HLA-A 0201 restrictive T-cell epitope analyser. Conclusion: We established the methods by using the biological information network technologies for looking the T-cell antigen epitopes and B-cell antigen epitopes in MOMP of CT, and three novel T-cell epitopes as well as three novel B-cell epitopes were identified in the current study. It provides important information for further studying the antigenicity of CT MOMP protein and also provides useful information for developing highly efficient subunit vaccines for CT.     

Phylogenetic Analysis of the Chlamydia trachomatis Major Outer Membrane Protein and Examination of Potential Pathogenic Determinants

Phylogenetic analysis was utilized to investigate biological relationships (tissue tropism, disease presentation, and epidemiologic success), as evidenced by coevolution, among human strains of Chlamydia trachomatis. Nucleotide sequences of omp1, the gene encoding the major outer membrane protein (MOMP) of C. trachomatis, were determined for 40 strains representing 11 serovars. These data were combined with available omp1 sequences from GenBank for an analysis encompassing a total of 69 strains representing 17 serovars infecting humans. Phylogenetic analysis of the nucleotide and inferred amino acid sequences showed no evolutionary relationships among serovars that corresponded to biological or pathological phenotypes (tissue tropism, disease presentation, and epidemiologic success). In addition, no specific residues that may have evolved to play a role in determining biologically relevant characteristics of chlamydia, such as tissue specificity, disease presentation, and epidemiologic success, were apparent in the MOMP. These results suggest that variation in MOMP may have arisen from a need to be diverse in the presence of immune pressure rather than as a function of pathogenicity. Therefore, the role of MOMP in disease pathogenesis and infection may be passive, and it may not be the major ligand responsible for directing infection of various human cell types.     

Characterization of the Humoral Immune Response to Chlamydia Outer Membrane Protein 2 in Chlamydial Infection

Detection of antibodies to an outer membrane protein 2 (OMP2) by enzyme-linked immunosorbent assay (ELISA) by using either the Chlamydia trachomatis- or the Chlamydia pneumoniae-specific protein was investigated. OMP2 is an immunodominant antigen giving rise to antibody responses in humans infected with different C. trachomatis serovars (A to C and D to K) or with C. pneumoniae, which could be detected by OMP2 ELISA. OMP2 ELISA is not species specific, but antibody titers were usually higher on the homologous protein. The sensitivity of this assay was high but varied according to the “gold standard” applied. Levels of antibody to C. pneumoniae OMP2 as detected by ELISA seem to return to background or near-background values within a shorter period of time compared to antibodies to C. pneumoniae detected by microimmunofluorescence (MIF), making it more likely that positive results in ELISA reflect recent infection. Thus, OMP2 ELISA has distinct advantages over MIF and commercially available ELISAs and might be a useful tool for the serodiagnosis of chlamydial infection.     

Antigenic Analysis of the Major Outer Membrane Protein of Chlamydia spp

The major outer membrane proteins (MOMPs) of several Chlamydia trachomatis serotypes (B, D, G, H, and L2) and of the C. psittaci meningopneumonitis strain were purified by preparatory sodium dodecyl sulfate-(SDS)-polyacrylamide gel electrophoresis. The isolated SDS-polypeptide complexes, which varied in their apparent subunit molecular weights, were used as immunogens to raise hyperimmune rabbit antisera. The specificities of these antisera were determined both by rocket immunoelectrophoresis with the soluble SDS-polypeptide complex as antigen and by micro-immunofluorescence with whole organisms. By rocket immunoelectrophoresis, each of the soluble C. trachomatis MOMPs was immunologically related; however, no immunological cross-reactions occurred with the C. psittaci meningopneumonitis polypeptide, indicating that the MOMPs are antigenically distinct among members of these two chlamydial species. The same antisera were highly reactive with intact organisms by micro-immunfluorescence, demonstrating that at least some of the antibodies raised with SDS-polypeptide complexes reacted with native antigenic sites of these surface proteins. By micro-immunofluorescence, anti-MOMP sera remained species specific; but, unlike the results observed by rocket immunoelectrophoresis, distinct differences in the reactivity and specificity of these antisera were observed among C. trachomatis serotypes. C. trachomatis isolates were separated into two distinct serogroups on the basis of their reactivity with anti-MOMP sera. B complex organisms (B, Ba, D, E, F, G, K, L1, L2, and L3) all reacted strongly with anti-MOMP sera of the B, D, G, and L2 serotypes. In contrast, these same antisera were poorly reactive with the C complex serotypes A, C, H, I, and J. Anti-H MOMP serum was the most serospecific, since high-antibody titers were found only against the homologous H serotype organism. These findings indicate that MOMPs of different strains of C. trachomatis are antigenically complex and that antigenic heterogeneity exists among the surface-exposed portions of the protein.     

The Major Outer Membrane Protein of Chlamydia psittaci Functions as a Porin-Like Ion Channel

The major outer membrane protein (MOMP) of Chlamydia species shares several biochemical properties with classical porin proteins. Secondary structure analysis by circular dichroism now reveals that MOMP purified from Chlamydia psittaci has a predominantly β-sheet content (62%), which is also typical of bacterial porins. Can MOMP form functional ion channels? To directly test the “porin channel” hypothesis at the molecular level, the MOMP was reconstituted into planar lipid bilayers, where it gave rise to multibarreled channels, probably trimers, which were modified by an anti-MOMP monoclonal antibody. These observations are consistent with the well-characterized homo-oligomeric nature of MOMP previously revealed by biochemical analysis and with the triple-barreled behavior of other porins. MOMP channels were weakly anion selective (P_Cl/P_K ∼ 2) and permeable to ATP. They may therefore be a route by which Chlamydia can take advantage of host nucleoside triphosphates and explain why some anti-MOMP antibodies neutralize infection. These findings have broad implications on the search for an effective chlamydial vaccine to control the significant human and animal diseases caused by these organisms.Members of the order Chlamydiales are distinguishable from other bacteria by their obligate intracellular mode of growth and their distinctive biphasic life cycle in which the small spore-like extracellular and infectious form, the elementary body (EB), alternates with the intracellular vegetative form, the reticulate body (RB). The four main species currently recognized, Chlamydia trachomatis, C. psittaci, C. pneumoniae, and C. pecorum, are diverse pathogens that cause a range of disease in both humans and animals. A common component of all these species is the 40-kDa major outer membrane protein (MOMP), present in both the EB and RB forms. The MOMP is a multifunctional protein which is thought to have a role both in the infectious process (3, 34–36) and in the maintenance of structural rigidity via disulfide bond cross-linking within the EB outer membrane (13, 15, 26).The antigenic properties of MOMP have been studied in detail since the landmark discovery that MOMP purified from sodium dodecyl sulfate (SDS)-gels was capable of raising antibodies which could neutralize the infectivity of C. trachomatis in vitro (6). Protein sequence comparisons of MOMPs both within (33) and between (18) species, combined with epitope mapping studies (8, 43), have shown that the epitopes responsible for neutralization lie within four variable segments. Vaccine preparations based on chlamydial outer membrane complexes, which are highly enriched for the MOMP in its native form, have been shown to be protective against chlamydial disease in sheep (37), guinea pigs (2), and mice (10, 28). However, experimental vaccines based on denatured or nonnative recombinant MOMP preparations have yielded, at best, only partial protection (28). Most recently, protection was demonstrated in mice administered a DNA vaccine comprising only the MOMP gene (42).These results clearly make MOMP the primary candidate for a subunit vaccine against chlamydial infection, but despite many years of intensive study, the paucity of structural information leaves unanswered many questions as to how MOMP fulfills its diverse functions. Structural studies are hampered first by the difficulty of growing chlamydiae in bulk and subsequently by problems with purifying and solubilizing a protein which both is highly cross-linked and normally resides in a hydrophobic environment. These factors have precluded attempts to crystallize the protein and have made it necessary to rely on analysis techniques that require relatively small quantities of protein.A recent report showed that MOMP solubilized with octyl glucoside (OG) in the presence of dithiothreitol (DTT) was oligomeric, with electrophoretic and sedimentation properties consistent with a trimeric structure (21). These oligomers resisted denaturation with SDS in a way similar to that for classical gram-negative porin molecules, which are also trimers (27). The result was consistent with an early observation by Bavoil et al. (3), who used liposome swelling to demonstrate that the chlamydial outer membrane contained pores and, due to its predominance in the outer membrane, that MOMP was the likely pore-forming protein. In this paper, we report direct evidence for porin function obtained by using native, oligomeric MOMP incorporated into planar lipid bilayers. Moreover, due to the traditional view that chlamydiae are required to scavenge ATP from the host cell, we have investigated the transport of ATP through the MOMP channel.     

Naturally Produced Outer Membrane Vesicles from Pseudomonas aeruginosa Elicit a Potent Innate Immune Response via Combined Sensing of Both Lipopolysaccharide and Protein Components

Pseudomonas aeruginosa is a prevalent opportunistic human pathogen that, like other Gram-negative pathogens, secretes outer membrane vesicles. Vesicles are complex entities composed of a subset of envelope lipid and protein components that have been observed to interact with and be internalized by host cells. This study characterized the inflammatory responses to naturally produced P. aeruginosa vesicles and determined the contribution of vesicle Toll-like receptor (TLR) ligands and vesicle proteins to that response. Analysis of macrophage responses to purified vesicles by real-time PCR and enzyme-linked immunosorbent assay identified proinflammatory cytokines upregulated by vesicles. Intact vesicles were shown to elicit a profoundly greater inflammatory response than the response to purified lipopolysaccharide (LPS). Both TLR ligands LPS and flagellin contributed to specific vesicle cytokine responses, whereas the CpG DNA content of vesicles did not. Neutralization of LPS sensing demonstrated that macrophage responses to the protein composition of vesicles required the adjuvantlike activity of LPS to elicit strain specific responses. Protease treatment to remove proteins from the vesicle surface resulted in decreased interleukin-6 and tumor necrosis factor alpha production, indicating that the production of these specific cytokines may be linked to macrophage recognition of vesicle proteins. Confocal microscopy of vesicle uptake by macrophages revealed that vesicle LPS allows for binding to macrophage surfaces, whereas vesicle protein content is required for internalization. These data demonstrate that macrophage sensing of both LPS and protein components of outer membrane vesicles combine to produce a bacterial strain-specific response that is distinct from those triggered by individual, purified vesicle components.The innate immune response to Gram-negative bacteria is dominated by recognition of lipopolysaccharide (LPS). LPS is sensed by the Toll-like receptor 4 (TLR4) complex, and numerous studies have focused on both how LPS sensitivity drives effective inflammatory responses, leading to the clearance of infection, as well as how uncontrolled TLR4 signaling can lead to LPS toxicity and play a role in septic shock (20, 39, 48). Much of this research has been performed using purified LPS in the experimental treatments, and yet it is unlikely that pure LPS is shed from bacteria in the context of an infection. Instead, LPS has been found to be shed from the bacteria in the form of outer membrane vesicles.Outer membrane vesicles are spherical, selective portions of outer membrane and periplasm that are naturally secreted by all Gram-negative bacteria (7, 31, 49). Vesicles are produced at all stages of bacterial growth and have been detected in infected human tissues (7, 23, 28). Vesicle production has been identified as an independent bacterial stress response pathway that is activated when bacteria are exposed to environmental stress, such as might be experienced during colonization of host tissues (35).Compared to preparations of pure LPS, natural outer membrane vesicles are heterogeneous proteoliposomes of a larger dimension (50 to 250 nm in diameter) than liposomes composed solely of LPS (4, 7). Natural outer membrane vesicles are heterogeneous complexes of pathogen-associated molecular patterns (PAMPs), such as LPS, flagellin, and CpG DNA, as well as other outer membrane proteins, virulence factors, and envelope lipids (7, 31). The molecular composition of vesicles varies with the bacterial strain of origin, while LPS structure remains relatively constant within a bacterial species. The combination of PAMPs, virulence factors, and other outer membrane components results in vesicles that are particularly laden with molecules that can be recognized by the immune system. In the present study we have focused on characterizing macrophage innate immune responses to the combined signals presented by the heterogeneous PAMP ligands presented in the native context of outer membrane vesicles.Studies of host immune responses to outer membrane vesicles have mainly addressed the generation of antibodies to vesicle components. Notably, a protective antibody response is elicited by outer membrane vesicles generated from Neisseria meningitidis and Vibrio cholerae (17, 18, 38, 40, 47). In contrast, there are relatively few studies characterizing how vesicles trigger the innate inflammatory response. Outer membrane vesicles from Helicobacter pylori and Pseudomonas aeruginosa have been shown to elicit interleukin-8 (IL-8) production by epithelial cells (3, 24), and Salmonella enterica serovar Typhimurium vesicles have been shown to activate dendritic cells to secrete IL-12 and tumor necrosis factor alpha (TNF-α) (1).This research has focused on outer membrane vesicles from the opportunistic pathogen P. aeruginosa. P. aeruginosa infects the respiratory tract of patients with nosocomial pneumonia, cystic fibrosis, or acute respiratory distress syndrome (14). Host responses to acute pulmonary infections with P. aeruginosa are characterized by an intense inflammatory response. Macrophage recognition of bacterial components initiates a cascade of proinflammatory cytokine secretion, resulting in large numbers of neutrophils infiltrating the lung to clear the bacteria (44).We hypothesize that naturally produced vesicles may play an important role in activating innate immune responses. Given their small size and high proportion of PAMPs, it is reasonable to expect that vesicles could easily infiltrate into infected tissues and stimulate widespread inflammation. In addition to TLR ligands, vesicles also contain a variety of protein virulence factors that may specialize vesicles for specific functions or types of host cell damage. P. aeruginosa vesicles have been shown to contain the adhesins OprF, OprG, and OprH, as well as virulence factors that include β-lactamase, hemolysin, phospholipase C, antimicrobial quinolones, and quorum-sensing molecules (3, 12, 25, 26, 31, 34). P. aeruginosa vesicle-associated proteins have also been shown to directly affect host cells. Vesicle-packaged CiF protein downregulates lung epithelial expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein (10, 33), while an aminopeptidase enriched in vesicles increases association of vesicles with cultured epithelial cells (2).The present study describes how vesicles, with their heterogeneous mixture of PAMPs, contribute to the innate immune response during P. aeruginosa infection. We have focused on vesicle-macrophage interactions, since macrophages are sentinel cells that initiate inflammatory defenses against colonizing bacteria. Our data demonstrate that macrophages are more sensitive to the potent stimulus of outer membrane vesicles from P. aeruginosa compared to equivalent levels of pure LPS. Neutralizing the LPS reactivity of the vesicles or altering the protein composition of the vesicles significantly impacted both the macrophage-vesicle interactions and the cytokine responses to the vesicles. Based on our findings, we conclude that a potent and distinct inflammatory response to vesicles is the cumulative effect of sensing vesicle-associated heterogeneous protein and LPS ligands by macrophages.     

The Immune Response against Hapten-Autologous Protein Conjugates in the Mouse

The carrier specificity of secondary anti-hapten responses using different haptenic densities on autologous or heterologous carrier proteins was studied. In addition, the T cell requirements for anti-hapten antibody synthesis were investigated. The results demonstrated a clearcut decrease in carrier specificity as hapten density goes up. Also, memory against autologous hapten carrier conjugates at the T cell level waned with time in contrast to the memory against heterologous hapten-carrier conjugates. Specific helper cells against autologous conjugates were demonstrated by filtration through anti-immunoglobulin coated columns.     

Chlamydia trachomatis Serology: Diagnostic Value of Outer Membrane Protein 2 Compared with That of Other Antigens

Different immunoassays using recombinant antigens or synthetic peptides were evaluated for the serodiagnosis of Chlamydia trachomatis infections. Antigens used included cysteine-rich outer membrane protein 2 (OMP2), heat shock protein 60, the polypeptide encoded by open reading frame 3 of the plasmid (pgp3), synthetic peptides derived from species-specific epitopes in variable domain IV of the major OMP (MOMP) (Labsystems, Helsinki, Finland), and a fragment of the total lipopolysaccharide (Medac, Hamburg, Germany). Because cross-reactions between chlamydial species have been reported, Chlamydia pneumoniae-specific antibodies were also determined by immunoassays (Labsystems). Responses obtained with serum samples from patients with well-defined diseases (i.e., urethral or endocervical samples from which C. trachomatis DNA was amplified) were compared to those obtained with samples from healthy blood donors. The best sensitivity (79%) associated with the best specificity (82%) was obtained when immunoglobulin G (IgG) responses to both MOMP and pgp3 were considered. The highest sensitivity (89%) was obtained with anti-OMP2 IgG, but the lowest specificity (57%) was obtained with this antibody, due to probable cross-reactivity with C. pneumoniae OMP2.     

Immunization with a Peptide Corresponding to Chlamydial Heat Shock Protein 60 Increases the Humoral Immune Response in C3H Mice to a Peptide Representing Variable Domain 4 of the Major Outer Membrane Protein of Chlamydia trachomatis

C3H (H-2^k) mice are susceptible to a vaginal challenge with human strains of Chlamydia trachomatis and thus are a useful strain for testing potential Chlamydia vaccine candidates. However, C3H mice are fairly poor responders in terms of the level of antibody resulting from immunization with potential protective peptides representing variable domains (VDs) of the major outer membrane protein (MOMP). C57BL/6 (H-2^b) mice, on the other hand, are moderately resistant to a vaginal challenge but are good responders to the chlamydial MOMP VDs. Peptides representing universal T-cell helper epitopes were employed to determine whether the antibody response to a peptide representing VD4 of the MOMP, which has been shown to contain neutralizing epitopes, could be enhanced in C3H and C57 mice. Universal T-cell helper peptides from tetanus toxin, the pre-S2 region of hepatitis B virus, and the mouse heat shock protein 60, as well as the corresponding segment of the Chlamydia heat shock protein 60 (hsp_ct), were coadministered with the VD4 peptide. Peptides were coencapsulated in liposomes containing the adjuvant monophosphoryl lipid A and administered by using a combination of mucosal and intramuscular injection. The only T-cell helper peptide that improved the immune response as judged by antibody level, in vitro neutralization assays, and T-cell proliferation was hsp_ct. The response in the C57BL/6 strain was not significantly enhanced with hsp_ct over levels achieved with VD4 alone; however, in C3H mice the levels of serum antibody to C. trachomatis increased to that seen in C57 mice. However, the molecular specificity and immunoglobulin subclass distribution differed from those of the C57 response, and the neutralizing titers and T-cell proliferation responses were lower. In both strains of mice, titers of vaginal antibody to C. trachomatis were low. In summary, of the T-helper peptides used, only hsp_ct significantly enhanced the immune response of C3H mice to the VD4 peptide, but it had only a modest effect on the immune response of C57 mice.Chlamydia trachomatis, being a leading cause of sexually transmitted diseases, has been the focus of efforts to develop a protective vaccine (4, 31, 37). Central to this effort has been the identification of host factors that may protect against infections caused by this pathogen as well as the definition of chlamydial components that confer pathogenic potential to this organism. To date the major outer membrane protein (MOMP) has been the most widely investigated vaccine candidate of the chlamydial proteins (17). Within the MOMP there are four variable domains (VDs), which differ among the serovars and are regions in which there are neutralizable epitopes (3, 17, 24, 30, 33, 40). In vitro experiments using monoclonal antibodies directed at the VDs have shown that attenuation of infection is narrow in terms of the number of serovars that are neutralized when any one epitope is targeted (24, 30, 40). Since neutralization of all serovars is not achieved by using antibodies directed at any one epitope, a subunit vaccine that incorporates an array of protective epitopes has been proposed (4, 31, 37). For the few attempts to utilize recombinant MOMP or peptides representing VD neutralizing epitopes to immunize mice, only modest attenuation of the infection has been reported (36, 38). There may be several reasons for this, including the requirement for T-cell help to boost and direct the immune response to critical regions within these peptides, the failure to elicit an adequate mucosal immune response, the requirement for a conformational epitope not provided by short synthetic peptides, and inherent differences in inbred mouse strains used in the vaccine trials.It has been shown that mouse strains of different H-2 haplotypes vary dramatically in their responses to a genital challenge with C. trachomatis (8, 9). As an example, C57BL/6 (H-2^b) mice are fairly resistant to a vaginal challenge with a human serovar, with vaginal cultures, depending on the challenge dose, being positive for only 1 to 2 weeks following inoculation. In contrast, C3H (H-2^k) mice are infected with lower numbers of organisms, and C. trachomatis can be cultured from the vagina for up to 4 to 6 weeks following challenge (9, 26). Therefore, because of the longer duration of infection, C3H mice are an attractive strain in which to test peptide vaccine candidates. However, it has been shown that a peptide representing VD4 of the MOMP was immunogenic in C57BL/6 (H-2^b) mice but was significantly less effective in eliciting a humoral response in C3H/HeJ and B10.BR/SgSnJ mice, both of which are of the H-2^k haplotype (29, 34).The purpose of this study was to determine whether universal T-cell helper peptides could enhance the immune response to a VD4 peptide in the otherwise nonresponsive C3H mouse or modify the response in C57 mice. Since the long-term goal is to protect against a genital mucosal challenge, the immunization strategy used was to coadminister, through a combination of systemic and mucosal routes, T-cell helper peptides and a VD4 peptide coentrapped in liposomes. Eliciting an immune response in the permissive but low- or nonresponsive C3H strain is essential for the future development and testing of subunit vaccines in this animal model.     

E血清型沙眼衣原体主要外膜蛋白B细胞表位的预测

基于E血清型沙眼衣原体(Chlamydia trachomatis,CT)主要外膜蛋白(Major outer membrane protein, MOMP)氨基酸序列,采用Hopp-Woods的亲水性方案、Emini表面可及性方案、Jameson-Wolf 抗原指数方案和Janin可及性方案等,辅以对MOMP蛋白的二级结构中的柔性区域及跨膜区域的分析,预测CT MOMP蛋白的B细胞表位.推测最有可能的B细胞表位位于MOMP蛋白N端第73～81区段、217～225区段、第377～386区段、第261～270区段和第161～175区段内或它们的附近.用多参数预测CT MOMP蛋白的B细胞表位,为进一步研究蛋白特性及表位疫苗研制奠定了基础.     

Surface Accessibility of the 70-Kilodalton Chlamydia trachomatis Heat Shock Protein following Reduction of Outer Membrane Protein Disulfide Bonds

Numerous investigations have shown that 70-kDa heat shock protein (Hsp70) homologs interact tightly with hydrophobic proteins and functionally assist proteins in membranous organelles and environments. One such protein is the Chlamydia trachomatis Hsp70 that is associated with isolated outer membrane complexes of infectious elementary bodies (EB). Previous observations have indicated that chlamydial Hsp70 plays a role in EB attachment to, or entry into, endometrial epithelial cells. In this study, immunofluorescence microscopy and transmission electron microscopy observations showed that chlamydial Hsp70 is not a surface-displayed ligand on purified EB. However, brief exposure of EB to the thiol reducing agent dithiothreitol (DTT) led to surface accessibility of the Hsp70 substrate-binding domain. Reduction of the highly disulfide-cross-linked EB outer membrane proteins with DTT resulted in a decrease in EB attachment and infectivity. Interestingly, exposure of EB to the membrane-impermeable thiol-alkylating reagent 5,5'-dithiobis(2-nitrobenzoic acid) enhanced attachment but compromised infectivity, suggesting that EB outer membrane proteins must be reduced for entry and productive infection. Together, our data suggest that (i) the structural integrity of the EB outer membrane, maintained by protein disulfide bonds, is important during the initial stages of attachment; (ii) reduction occurs within the localized microenvironment of host cell surfaces once intimate contact is established between EB and host cells; and (iii) subsequent conformational changes in EB ultrastructure allow productive infection in host cells. The accessibility of the Hsp70 substrate-binding domain may support the hypothesis that this protein plays a role in events following the initial stage of attachment instead of serving as a primary, surface-displayed adhesin.     

CD4+ T Cells and Antibody Are Required for Optimal Major Outer Membrane Protein Vaccine-Induced Immunity to Chlamydia muridarum Genital Infection

Despite effective antimicrobial chemotherapy, control of Chlamydia trachomatis urogenital infection will likely require a vaccine. We have assessed the protective effect of an outer membrane protein-based vaccine by using a murine model of chlamydial genital infection. Female mice were first vaccinated with Chlamydia muridarum major outer membrane protein (MOMP) plus the adjuvants CpG-1826 and Montanide ISA 720; then they were challenged with C. muridarum. Vaccinated mice shed 2 log₁₀ to 3 log₁₀ fewer inclusion-forming units (IFU) than ovalbumin-vaccinated or naïve animals, resolved infection sooner, and had a lower incidence of hydrosalpinx. To determine the relative contribution of T cells to vaccine-induced protection, mice were vaccinated, depleted of CD4⁺ or CD8⁺ T cells, and then challenged vaginally with C. muridarum. Depletion of CD4⁺ T cells, but not depletion of CD8⁺ T cells, diminished vaccine-induced protection, with CD4-depleted mice shedding 2 log₁₀ to 4 log₁₀ more IFU than CD8-depleted or nondepleted mice. The contribution of antibodies to vaccine-induced protection was demonstrated by the absence of protective immunity in vaccinated B-cell-deficient mice and by a 2 log₁₀ to 3 log₁₀ decrease in bacterial shedding by mice passively administered an anti-MOMP serum. Thus, optimal protective immunity in this model of vaccine-induced protection depends on contributions from both CD4⁺ T cells and antibody.New cases of sexually transmitted diseases number more than 340 million worldwide annually and pose a formidable health risk to infected individuals (67-69). It is estimated that Chlamydia trachomatis, the causative agent of chlamydia, is responsible for more than 92 million of these cases. In the United States, where C. trachomatis infections are the infections most commonly reported to the Centers for Disease Control and Prevention, there are more than 4 million new cases each year (14, 67). As a bacterial agent of infection, C. trachomatis can be eradicated efficiently with appropriate antibiotic treatment, but more than 50% of infected individuals are asymptomatic and therefore lack the impetus to seek treatment (14). When left untreated, infection in women can lead to pelvic inflammatory disease, ectopic pregnancy, and tubal factor infertility and can cause severe and sometimes irreparable damage to the reproductive organs (14, 67). To combat the high rate of infection and disease, the development of an efficacious vaccine is critical.Trachoma vaccine trials using whole organisms in the 1950s and 1960s had mixed results, with some studies inducing only partial, serovar-specific, short-lived immunity (4). In one study, a subset of vaccine trial participants experienced an increased incidence of disease and exacerbated pathology relative to that of their unvaccinated counterparts upon reexposure to chlamydiae, which led many researchers to abandon the use of whole organisms in immunizations (7, 8). Since then, no other human vaccine trials targeting ocular or urogenital C. trachomatis infections have been published. Instead, researchers have focused their efforts on animal models of ocular and genital infection.To this end, the murine model of chlamydial genital infection, which closely mimics acute genital infection in women, has been employed extensively for the study of the immunological parameters of infection and for vaccine development. Mice infected with C. muridarum naturally resolve infection in approximately 4 weeks and develop long-lived adaptive immunity that markedly protects against reinfection (3, 37). Infection elicits Chlamydia-specific CD4⁺ T cells, CD8⁺ T cells, and antibody, but only CD4⁺ T cells are necessary for resolution of the primary infection (42). In contrast, immunity to reinfection is dominated by both protective CD4⁺ T cells and antibody (38). Clearance and immunity are highly dependent on a Th1-type response, specifically gamma interferon (IFN-γ)-secreting CD4 cells (9). On the other hand, Th2 responses are associated with scarring and immunopathology (62). For example, antibody responses dominated by IgG1 are not protective and may be associated with an increase in pathology, whereas anti-chlamydial antibodies of the IgG2a and IgG2b isotypes are associated with a protective response (51).Using knowledge of the protective response gleaned from the murine model of infection-induced immunity, investigators have made modest strides toward the development of an efficacious vaccine. Studies utilizing whole elementary body (EB) immunization have induced significant protection, though most of these studies have limited real-world application. One notable example that induced almost sterilizing immunity involved the passive transfer of dendritic cells pulsed ex vivo with nonviable chlamydiae (65). Subunit antigen vaccines represent the bulk of vaccine studies, and vaccines based on combinations of a number of chlamydial antigens, adjuvants, and delivery systems have had various degrees of success in preventing infection (8, 22, 62). Chlamydial antigens, including secreted proteins, such as chlamydial protease-like activity factor (CPAF) (16, 33, 43-46), and membrane associated proteins, such as PorB (26, 30) and IncA (33), have also been used in subunit vaccines; however, the vast majority of studies have focused on the major outer membrane protein (MOMP), an immunodominant antigen in both human and animal studies (22, 62). Novel delivery systems, including Vibrio cholerae ghosts and cationic liposomes, have been introduced into chlamydial vaccine research, and while initial studies have shown incomplete protection, these systems may have the potential to elicit protective responses against chlamydial genital infection when used in conjunction with appropriate antigens (2, 19, 20, 23).Despite substantial effort, no vaccine licensed for human use is currently available. Recently, a MOMP-based vaccine utilizing the adjuvants CpG-1826 and Montanide ISA 720, which together drive a strong Th1-type response, has been shown to confer considerable protection when mice are challenged directly in the upper genital tract with C. muridarum (51). In our current study, we sought to determine if this vaccine protected against vaginal challenge (the natural route of infection) and to evaluate the contributions of T cells and antibody to the vaccine-induced protective response. We found that the MOMP vaccine conferred significant protection against vaginal challenge and protected against infection-induced pathology (hydrosalpinx). Furthermore, optimal protection was dependent on both CD4⁺ T cells and antibody.     

Expression and Purification of the Major Outer Membrane Protein of Chlamydia Trachomatis in Prokaryotic Cell

Chlamydiatrachomatis(C.trachomatis)isanobligate intracellularbacterialpathogen.Ocularinfectionwith C.trachomatisserovarsA,B,BaandCleadstotracho ma,aleadingcauseofpreventableblindnessinmanyde velopingcountries[1].Urogenital tractinfectionwithC. trachomat…     

Immunization with a Combination of Integral Chlamydial Antigens and a Defined Secreted Protein Induces Robust Immunity against Genital Chlamydial Challenge

We have previously demonstrated the efficacy of recombinant chlamydial protease-like activity factor (rCPAF; a secreted chlamydial protein) in inducing antigen-specific CD4⁺ T cell/gamma interferon (IFN-γ)-mediated but not antibody-mediated chlamydial clearance and reduction of upper genital tract (UGT) pathological sequelae. Since chlamydial integral antigens may induce neutralizing antibody protection, we further evaluated induction of protective immunity using a combination of rCPAF and UV-inactivated chlamydial elementary bodies (UV-EB) against vaginal chlamydial challenge in comparison to immunization with the individual components or live EB. The rCPAF-UV-EB immunization induced a significantly enhanced anti-UV-EB cellular and antibody response and a reduced anti-CPAF cellular and antibody response, compared to immunization with the respective individual components. Moreover, vaccination with UV-EB and rCPAF-UV-EB induced serum antibodies that neutralized chlamydial infectivity. The rCPAF-UV-EB immunization resulted in a significant reduction of vaginal chlamydial shedding and induced earlier bacterial clearance than vaccination of mice with the individual components. Importantly, the UGT sequelae were significantly reduced in mice immunized with rCPAF or rCPAF-UV-EB, but not in those immunized with UV-EB alone, and approached the levels of protection induced by live EB. These results collectively suggest that a combination of neutralizing antibodies induced by integral chlamydial antigens and cell-mediated responses induced by secreted proteins such as CPAF induces optimal protective immunity against genital chlamydial infections.There is currently no licensed vaccine against Chlamydia trachomatis, the leading cause of bacterial sexually transmitted disease worldwide (2, 16). We have previously demonstrated the efficacy of recombinant chlamydial protease-like activity factor (rCPAF) in inducing protective immunity against genital chlamydial challenge (23). Immunization using rCPAF with a T helper 1 (Th1)-type adjuvant induces significantly enhanced bacterial clearance and robust protection against upper genital tract (UGT) pathology following vaginal challenge with homologous or heterologous serovars/species of Chlamydia (5, 6, 23). The high degree of cross-serovar/species protection against UGT sequelae highlights the importance of further characterizing the potential of rCPAF as a component of an antichlamydial vaccine for humans (25). rCPAF-vaccinated mice display significant protection against UGT chlamydial sequelae and clear the bacteria with significantly accelerated kinetics, achieving complete clearance by day 18 (day 30 in mock-vaccinated mice) after challenge. However, vaginal bacterial shedding in rCPAF-vaccinated mice is comparable to the level for mock-vaccinated controls during the initial week after challenge (6, 23). Such enhanced clearance kinetics, in the absence of resistance to infection, may be attributed to the dependence of the protective response on gamma interferon (IFN-γ)-producing CPAF-specific CD4⁺ T cells (15), a limited role for anti-CPAF antibody (22), and the restriction of CPAF to replicating reticulate bodies.Chlamydia muridarum infection in mice induces a high level of protective immune responses, including a certain degree of resistance to reinfection, mediated by robust IFN-γ-producing CD4⁺ T cell responses (4, 11-13, 16, 17, 20, 28-31, 34) and antibodies (16,18-20). A single immunogenic subunit that induces protective immunity comparable to that induced by live, replicating chlamydial organisms has yet to be identified (2, 16, 25). The immunogenic proteins that serve as targets for antibody and T cell responses may be broadly categorized, albeit with some overlap, as proteins that are integral to the chlamydial organism and those that are secreted from the organism, respectively. Specifically, proteins integral to the chlamydial organism would likely serve as targets for neutralizing infectivity extracellularly but may not be candidates of choice for eliciting T cell-mediated killing, due to the sturdy inclusion membrane barrier between the organisms and antigen-presentation pathways during the intracellular developmental cycle (25). On the other hand, secreted proteins such as CPAF are not present on the infectious chlamydial elementary body (EB) and therefore would not be expected to serve as targets for neutralizing chlamydial infectivity (25). However, proteins secreted into the host cytosol, and thereafter into extracellular compartments, may serve as exogenous antigens and a suitable target for CD4⁺ T cell-mediated effector responses (25, 37). Thus, it would appear that both integral and secreted proteins of Chlamydia may serve as targets for complementary immune responses and that the greatest potential for successful vaccination could be derived by combining them in a multisubunit vaccine.In this study, we compared the protective immunities induced by intranasal (i.n.) immunization with rCPAF, UV-inactivated EBs (UV-EB), rCPAF-UV-EB, or live EB against genital C. muridarum challenge in female BALB/c mice. The combination of integral and secreted proteins enhanced protective immunity compared to the individual components and approached the high level of protection induced by live, replicating chlamydial organisms.     

Protective Immunization with a Novel Membrane Protein of Plasmodium yoelii-Infected Erythrocytes

Immunization with a particulate fraction of blood-stage antigens was shown previously to protect mice against Plasmodium yoelii malaria. To identify antigens inducing the protective response, sera from immunized mice were used to screen a P. yoelii cDNA expression library. Sequence analysis of one 2.6-kb cDNA clone indicated that the identified gene, pypag-1, encoded a novel plasmodial antigen. Two nonoverlapping regions of pypag-1 were expressed in Escherichia coli. The first recombinant antigen, pAg-1N, contained the N-terminal 337 residues, which included a putative transmembrane domain and a region relatively rich in tryptophan residues. The second recombinant antigen, pAg-1C, contained the remaining C-terminal 211 residues, which included 31 copies of a 5-amino-acid degenerative repeat. Immunoblot studies using rabbit antiserum raised against recombinant pAg-1N showed that the native pypAg-1 protein migrated at approximately 98 kDa, considerably slower than its predicted molecular mass of 66 kDa. Immunofluorescence studies localized the expression of the native pypAg-1 protein both to the cytoplasm and at the surface of P. yoelii-infected erythrocytes. Immunization with either pAg-1N or pAg-1C induced a four- to sevenfold reduction in P. yoelii blood-stage parasitemia. As such, pypAg-1 appears to contain at least two distinct protective epitopes. To our knowledge, this is the first characterization of a protective antigen of P. yoelii that is associated with the erythrocyte membrane.     

Chlamydia trachomatis Polymorphic Membrane Protein D Is an Oligomeric Autotransporter with a Higher-Order Structure

Chlamydia trachomatis is a globally important obligate intracellular bacterial pathogen that is a leading cause of sexually transmitted disease and blinding trachoma. Effective control of these diseases will likely require a preventative vaccine. C. trachomatis polymorphic membrane protein D (PmpD) is an attractive vaccine candidate as it is conserved among C. trachomatis strains and is a target of broadly cross-reactive neutralizing antibodies. We show here that immunoaffinity-purified native PmpD exists as an oligomer with a distinct 23-nm flower-like structure. Two-dimensional blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that the oligomers were composed of full-length PmpD (p155) and two proteolytically processed fragments, the p73 passenger domain (PD) and the p82 translocator domain. We also show that PmpD undergoes an infection-dependent proteolytic processing step late in the growth cycle that yields a soluble extended PD (p111) that was processed into a p73 PD and a novel p30 fragment. Interestingly, soluble PmpD peptides possess putative eukaryote-interacting functional motifs, implying potential secondary functions within or distal to infected cells. Collectively, our findings show that PmpD exists as two distinct forms, a surface-associated oligomer exhibiting a higher-order flower-like structure and a soluble form restricted to infected cells. We hypothesize that PmpD is a multifunctional virulence factor important in chlamydial pathogenesis and could represent novel vaccine or drug targets for the control of human chlamydial infections.Chlamydia trachomatis is a mucosotropic obligate intracellular gram-negative pathogen that is a leading cause of sexually transmitted and ocular infections. Infection can result in serious sequelae such as infertility and blindness (54, 56) and an increased risk of human immunodeficiency virus infection and transmission (38). The pathophysiology of chlamydial infection is associated with the pathogen''s propensities to cause persistent infection and to suppress host immunity (3). A vaccine is needed to control chlamydial diseases; however, progress toward this goal will not be forthcoming until more is known about the virulence factors that mediate persistence and immune evasion.Chlamydiae are characterized by a unique biphasic developmental cycle that modulates between an extracellular, metabolically inactive, infectious elementary body (EB) and an intracellular, metabolically active, noninfectious reticulate body (RB) (34). Their obligate intracellular niche and the lack of a tractable genetic system present unique challenges in the study of chlamydial biology and pathogenesis. To overcome these hurdles, chlamydial genomes from a diverse spectrum of host-specific strains have been sequenced. Comparative genomics have shown considerable homology among various chlamydial species and have provided important insights into shared and species-specific virulence factors (7, 24, 41, 42, 46, 49).The type V or autotransporter (AT) secretion pathway is the most widespread secretion mechanism employed by gram-negative bacteria to deliver virulence factors involved in initiating infection, disease progression, and immune evasion (reviewed in references 11 and 21). AT proteins are characterized by three domains, (i) a signal sequence (SS), (ii) a diverse N-terminal passenger domain (PD) that confers effector function, and (iii) a conserved C-terminal translocator domain (TD). The TD inserts into the outer membrane (OM) by assembling into a β-barrel pore that facilitates PD translocation to the bacterial surface. The PD remains tethered to the TD or is cleaved and either is released or remains noncovalently associated with the OM. Well-characterized examples of ATs found on the bacterial cell surface as monomers or oligomers are Neisseria meningitidis NalP (37) and Helicobacter pylori VacA (31), respectively.C. trachomatis has a nine-member AT family (20), termed polymorphic membrane proteins (Pmps), whose role(s) in chlamydial pathogenesis has yet to be defined. The pmp paralogs (pmpA to pmpI) constitute 3.2% of the ∼1-Mb genome and are found at three chromosomal loci composed of two gene clusters (pmpA to pmpC and pmpE to pmpI) and the genetically isolated gene pmpD (46). Notably, PmpD is the second most highly conserved Pmp, exhibiting 99.2% amino acid identity among C. trachomatis serovars (16). Despite relatively low abundance in the chlamydial OM, Pmps are major immunogens and may be important virulence factors (29). C. trachomatis PmpD is a target of broadly cross-reactive neutralizing antibodies (Abs), which makes it an attractive vaccine candidate for the prevention of human infections (10).Previous reports have described proteolytic processing of C. pneumoniae and C. trachomatis PmpD (25, 52). Furthermore, recombinant C. pneumoniae PmpD has been suggested to function as an adhesin capable of inducing proinflammatory cytokine production (35, 52). Nothing is known about the native structure of C. trachomatis PmpD or the potential significance of its structure to chlamydial pathogenesis. Here we show that C. trachomatis PmpD is present on the organism''s surface as an oligomer with a higher-order flower-like structure. Moreover, we describe novel infection-dependent proteolytic processing of PmpD that produces soluble fragments with predicted eukaryotic motifs, implying a multifunctional protein important to chlamydial pathogenesis.     

Susceptibility of Mice to Vaginal Infection with Chlamydia trachomatis Mouse Pneumonitis Is Dependent on the Age of the Animal

Mice from three strains, BALB/c (H-2(d)), C3H (H-2(k)), and C57BL/6 (H-2(b)), ranging from 5 to 14 weeks of age, were inoculated intravaginally with different doses of the Chlamydia trachomatis mouse pneumonitis serovar. Vaginal swabs taken at weekly intervals showed that the percentage of animals with positive cultures and the number of inclusion-forming units recovered per mouse were higher in the younger animals. Furthermore, vaginal shedding lasted longer in the young mice than in the older mice. In addition, following mating higher rates of infertility and a decrease in the number of embryos were observed in the infected young mice. In conclusion, susceptibility to a chlamydial vaginal infection is dependent on the age of the mice, with the older animals being more resistant.     

Mapping of a Protective Epitope of the CopB Outer Membrane Protein of Moraxella catarrhalis

A monoclonal antibody (MAb) (MAb 10F3) directed against the CopB outer membrane protein of Moraxella catarrhalis previously was found to enhance pulmonary clearance of M. catarrhalis in an animal model (M. Helminen, I. Maciver, J. L. Latimer, L. D. Cope, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 61:2003–2010, 1993). In the present study, this same MAb was shown to exert complement-dependent bactericidal activity against this pathogen in vitro. Nucleotide sequence analysis of the copB gene from two MAb 10F3-reactive and two MAb 10F3-unreactive strains of M. catarrhalis revealed that the deduced amino acid sequences of these four CopB proteins were at least 90% identical. Comparison of the amino acid sequences of these proteins allowed localization of possible MAb 10F3 binding sites to five relatively small regions of the CopB protein from M. catarrhalis O35E. When five synthetic peptides representing these regions were tested for their ability to bind MAb 10F3 in a direct enzyme-linked immunosorbent assay system, an oligopeptide containing 26 amino acids was shown to bind this MAb. The actual binding region for MAb 10F3 was localized further through the use of overlapping decapeptides that spanned this 26-mer. A fusion protein containing the same 26-mer readily bound MAb 10F3 and was used to immunize mice. The resultant antiserum contained antibodies that reacted with the CopB protein of the homologous M. catarrhalis strain in Western blot analysis and bound to the surface of both homologous and heterologous strains of M. catarrhalis.