Interaction of chlamydiae and host cells in vitro.

Persistent chlamydiae: from cell culture to a paradigm for chlamydial pathogenesis.

Chlamydia trachomatis sexually transmitted infections cause considerable morbidity and socioeconomic burden worldwide, despite significant advances in our understanding of the biology (29, 31, 57), pathogenesis (11, 83, 117), genomics (94), and epidemiology (91) of this parasite. Chlamydial urogenital tract infections are readily cured with antibiotics, but control measures based on antimicrobial chemotherapy alone are hampered by the frequency of asymptomatic infections and delayed diagnosis (9). Definitive control of C. trachomatis sexually transmitted diseases (STDs) is possible through the development of a safe and efficacious vaccine (24). Progress toward the development of an effective vaccine has been disappointingly modest, as it has been for vaccines to other sexually transmitted pathogens that infect the genital tract mucosae. 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 heightened 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 infection. The insights are of considerable interest because they offer promise for the development of an efficacious chlamydial vaccine. This review focuses on that progress and summarizes the current understanding of protective immune mechanisms that function against murine chlamydial urogenital infection. We also discuss specific requirements for a vaccine to protect against chlamydial STDs and the challenges presently confronting us in achieving that goal.

https://iai.asm.org/content/iai/70/6/2741.full.pdf

Immunity to Murine Chlamydial Genital Infection

Previous studies have shown that the immune-regulated cytokine gamma interferon (IFN-gamma) activates host cells to restrict intracellular growth of the bacterial pathogen Chlamydia trachomatis by induction of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). Recently, subinhibitory levels of IFN-gamma were used to generate an in vitro persistent chlamydial infection characterized by large aberrant, noninfectious reticulate bodies from which infectious progeny could be recovered following the removal of IFN-gamma. Studies were done to determine if the mechanism functioning to induce chlamydiae to enter a persistent state in the presence of low levels of IFN-gamma was similar to that reported to inhibit chlamydial growth. Host cells treated with levels of IFN-gamma required to induce persistence were assessed for IDO activity by high-performance liquid chromatography analysis of tryptophan and its catabolic products. Substantial tryptophan catabolism was detected in acid-soluble cellular pools, indicating that the intracellular availability of this essential amino acid was limited under these conditions. In addition, a mutant cell line responsive to IFN-gamma but deficient in IDO activity was shown to support C. trachomatis growth, but aberrant organisms were not induced in response to IFN-gamma treatment. Analyses of infected cells cultured in medium with incremental levels of exogenous tryptophan indicated that persistent growth was induced by reducing the amount of this essential amino acid. These studies confirmed that nutrient deprivation by IDO-mediated tryptophan catabolism was the mechanism by which IFN-gamma mediates persistent growth of C. trachomatis.

https://iai.asm.org/content/iai/62/9/3705.full.pdf

Tryptophan depletion as a mechanism of gamma interferon-mediated chlamydial persistence.

Chlamydia trachomatis infection of humans is commonly a localized inflammation that can result in infertility, blindness, and perhaps arthritis. The pathogenic process(es) that cause these sequelae are thought to be immunological. A 57-kD protein that is common among Chlamydia elicits ocular inflammation when introduced onto the conjunctivae of guinea pigs or nonhuman primates previously sensitized by chlamydial infection. This protein is thought to mediate the immunopathology that follows chlamydial infection. To more thoroughly characterize this chlamydial component, we cloned its gene from a C. psittaci strain and identified a particular recombinant that produced the 57-kD polypeptide. The recombinant gene product was immunoreactive with a monospecific anti-57-kD serum, and elicited an ocular inflammation similar to that produced by the 57-kD antigen isolated from chlamydiae. Sequencing identified two ORFs that encode polypeptides of 11.2 and 58.1 kD and are co-transcribed. These two polypeptides show homology with Escherichia coli groE and Coxiella burnetii htp heat-shock proteins. Striking homology (greater than 50%) was found between the 57-kD protein and the HtpB, GroEL, 65-k Mycobacterium tuberculosis and Hsp60 proteins. Thus, the 57-kD chlamydial protein, previously implicated as mediating a deleterious immunologic response to chlamydial infections, is a stress-induced protein similar to those that occur universally in both prokaryotic and eukaryotic organisms.

https://rupress.org/jem/article-pdf/170/4/1271/1099774/1271.pdf

Chlamydial disease pathogenesis. The 57-kD chlamydial hypersensitivity antigen is a stress response protein.

A panel of monoclonal antibodies (MAb) was generated against Chlamydia trachomatis serovar B, an etiologic agent of blinding trachoma. The specificities of MAb were determined by dot blot assay by using viable elementary bodies of 13 C. trachomatis serovars and two C. psittaci strains. The dot blot assay was used to identify those antigens that were unique and immunoaccessible on the chlamydial surface. MAb were identified that recognized bi-specific (serovars B and Ba) or subspecies-specific (various B complex serovars) surface-exposed antigenic determinants that were either resistant or sensitive to heat denaturation (56 degrees C, 30 min). All of the MAb recognized the major outer membrane protein as determined by either immunoblotting or radioimmunoprecipitation. MAb specific for immunoaccessible major outer membrane protein epitopes protected mice from toxic death after i.v. injection of B serovar elementary bodies and neutralized the infectivity of the organism for monkey eyes. In contrast, MAb reactive against non-immunoaccessible subspecies- or species-specific major outer membrane protein epitopes or against an immunoaccessible genus-specific epitope located on chlamydial lipopolysaccharide did not protect mice from toxic death or neutralize infectivity of the parasite for monkey eyes. These data suggest that those major outer membrane protein antigenic determinants that are serovar or serogroup specific and are accessible to antibody on the chlamydial cell surface may be useful as a recombinant subunit vaccine for trachoma.

Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis.

Monoclonal antibodies exhibiting Chlamydia trachomatis serovar specificity (serovar A, B-Ba, or C) and serogroup specificity (B, intermediate, or C serogroup) were produced and characterized. These antibodies reacted with the major outer membrane protein, recognized epitopes located at the chlamydial cell surface, and passively neutralized chlamydial toxicity for mice. The antibodies should be useful reagents for defining the molecular structure of these protective epitopes, a necessary step toward the development of a subunit or recombinant C. trachomatis vaccine.

Protective monoclonal antibodies to Chlamydia trachomatis serovar- and serogroup-specific major outer membrane protein determinants.

In this study, we examined the temporal antibody response by immunoblotting analysis in tears and sera of three cynomolgus monkeys (Macaca fasicularis) with primary acute Chlamydia trachomatis serovar B conjunctivitis. The objective was to identify chlamydial antigens stimulating antibody during the host responses in the course of this self-limiting infection with the rationale that they may be protective antigens. The major outer membrane protein (MOMP), lipopolysaccharide (LPS), and polypeptides of 60 and 68 kilodaltons (kDa) were the predominant antigens recognized by immunoglobulin A (IgA) in monkey tears. Tear IgA antibody specific for the MOMP was first detected 14 days postinfection, whereas tear IgA reactive with LPS or the 68- and 60-kDa polypeptides was first detectable on day 21. Tear IgA antibodies specific for each of these antigens persisted in tears through day 56, 4 weeks after both peak clinical disease and recovery of the organism from the conjunctivae. In contrast, tear IgG antibodies peaked at approximately 28 days postinfection, the time of maximal inflammatory response. The IgG response in monkey sera was similar to that observed for tear antibodies, in that the MOMP, 60-, and 68-kDa polypeptides were the primary immunogens. The exception was that IgG antibody against these antigens was detected 1 week later than that observed for tear IgA antibodies. Of three monkeys that responded with tear IgA antibody against LPS, one did not have detectable serum IgG LPS antibody. The specificity of the tear IgA antibody response of monkeys was determined by immunoblotting nine other C. trachomatis serovars in addition to the homologous B serovar. The tear IgA response to the MOMP was predominantly B complex subspecies-specific (serovars B, Ba, D, and E), whereas the response to chlamydial LPS was found to be species-specific. The significance of these observations in relation to previous vaccine studies in nonhuman primates is discussed. Images

Tear and serum antibody response to Chlamydia trachomatis antigens during acute chlamydial conjunctivitis in monkeys as determined by immunoblotting.

We isolated, by hydroxylapatite high-performance liquid chromatography, 14- and 15-kilodalton (kDa), cysteine-rich outer membrane proteins from Chlamydia trachomatis TW-5/OT (serovar B) and LGV-434 (serovar L2), respectively. Monoclonal antibodies (MAbs) were generated against the purified proteins, and their specificities were determined by immunoblotting. MAb B-14k recognized an epitope located on the 14-kDa cysteine-rich protein of the TW-5/OT strain and was immunoreactive with a comigrating 14-kDa protein that was common to all trachoma biovar strains, but it did not react with the 15-kDa, cysteine-rich protein of LGV biovar strains. In contrast, MAb L2-15k, which recognized an epitope located on the 15-kDa protein of the LGV-434 strain, reacted with the 15- and 14-kDa, cysteine-rich proteins of both LGV and trachoma biovar strains, but did not react with related proteins of two Chlamydia psittaci strains. Thus, the low-molecular-mass, cysteine-rich outer membrane proteins of C. trachomatis possess antigenic determinants that are both biovar and species specific. Neither MAbB-14k nor MAb L2-15k was reactive by dot-blot assay when viable chlamydiae were used as test antigens, indicating that the cysteine-rich proteins are not accessible to antibody on the native chlamydial cell surface.

The low-molecular-mass, cysteine-rich outer membrane protein of Chlamydia trachomatis possesses both biovar- and species-specific epitopes.

The major outer membrane protein (MOMP) of Chlamydia trachomatis was expressed in Escherichia coli. To assess whether it assembled into a conformationally correct structure at the cell surface, we characterized the recombinant MOMP (rMOMP) by Western immunoblot analysis, indirect immunofluorescence, and immunoprecipitation with monoclonal antibodies (MAbs) that recognize contiguous and conformational MOMP epitopes. Western blot analysis showed that most of the rMOMP comigrated with authentic monomer MOMP, indicating that its signal peptide was recognized and cleaved by E. coli. The rMOMP could not be detected on the cell surface of viable or formalin-killed E. coli organisms by indirect immunofluorescence staining with a MAb specific for a MOMP contiguous epitope. In contrast, the same MAb readily stained rMOMP-expressing E. coli cells that had been permeabilized by methanol fixation. A MAb that recognizes a conformational MOMP epitope and reacted strongly with formalin- or methanol-fixed elementary bodies failed to stain formalin- or methanol-fixed E. coli expressing rMOMP. Moreover, this MAb did not immunoprecipitate rMOMP from expressing E. coli cells even though it precipitated the authentic protein from lysates of C. trachomatis elementary bodies. Therefore we concluded that rMOMP was not localized to the E. coli cell surface and was not recognizable by a conformation-dependent antibody. These results indicate that rMOMP expressed by E. coli is unlikely to serve as an accurate model of MOMP structure and function. They also question the utility of rMOMP as a source of immunogen for eliciting neutralizing antibodies against conformational antigenic sites of the protein.

https://iai.asm.org/content/iai/61/10/4093.full.pdf

S. Stewart

Papers

Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis.

Protective monoclonal antibodies to Chlamydia trachomatis serovar- and serogroup-specific major outer membrane protein determinants.

Tear and serum antibody response to Chlamydia trachomatis antigens during acute chlamydial conjunctivitis in monkeys as determined by immunoblotting.

The low-molecular-mass, cysteine-rich outer membrane protein of Chlamydia trachomatis possesses both biovar- and species-specific epitopes.

Expression of the major outer membrane protein of Chlamydia trachomatis in Escherichia coli.