Characterization of a Neutralizing Monoclonal Antibody Directed at the Lipopolysaccharide of Chlamydia pneumoniae

Abstract

Identification of protective epitopes is one of the first steps in the development of a subunit vaccine. One approach to accomplishing this is to identify structures or epitopes by using monoclonal antibodies (MAb) that can attenuate infectivity in vitro and in vivo. To date attempts to use this approach with Chlamydia pneumoniae have failed. This report is the first description of a MAb directed to the lipopolysaccharide (LPS) of Chlamydia that neutralizes both in vitro and in vivo the infectivity of C. pneumoniae. MAb CP-33, an immunoglobulin G2b (IgG2b), was identified from a fusion using splenocytes from mice immunized with C. pneumoniae TW-183. By Western blot analysis, MAb CP-33 exhibited genus-specific reactivity in that it recognized the LPSs of C. pneumoniae, Chlamydia trachomatis, and Chlamydia psittaci. MAb CP-33 did not react with 15 genera of gram-negative and gram-positive bacteria and Candida albicans. By using isolated LPS of Re mutants of Escherichia coli, Salmonella enterica serovar Minnesota, and recombinants expressing the 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) transferase gene kdtA of C. trachomatis, MAb CP-33 was shown to require for binding the presence of the genus-specific trisaccharide epitope αKdo(2→8)αKdo(2→4)αKdo. By employing synthetic oligosaccharides and neoglycoconjugates in an enzyme immunoassay (EIA) and EIA inhibition, it was further shown that MAb CP-33 differed from the extensively investigated prototype chlamydial LPS MAb S25-23. Most likely, MAb CP-33 recognizes a conformational epitope in which the αKdo(2→8)αKdo(2→4)αKdo trisaccharide is an essential structural component. When tested in an in vitro neutralization assay, MAb CP-33 gave a 50% neutralization titer of 8 ng/ml against C. pneumoniae TW-183. However, this MAb did not neutralize other C. pneumoniae strains, C. trachomatis, or C. psittaci. C. pneumoniae TW-183 was treated with either MAb CP-33 or a control IgG and then used to inoculate mice by the respiratory route. Five days after inoculation, there was a difference between the mice inoculated with the control IgG-treated inoculum and those inoculated with the MAb CP-33-treated organisms as to the number of mice infected as well as the number of inclusion-forming units recovered from lung cultures (P < 0.05). In summary, a Chlamydia-specific LPS MAb was able to neutralize in vitro the infectivity of C. pneumoniae TW-183.

Chlamydia pneumoniae has been shown to be a common cause of human respiratory infections which range from pharyngitis to fatal pneumonia (19, 21, 37). Epidemics of pneumonia caused by C. pneumoniae in several geographical locations have been documented (13, 14, 19, 31). The prevalence of antibodies to C. pneumoniae rises from late childhood to early adolescence and throughout life. Serological surveys from the United States, Japan, and Europe have documented a prevalence of C. pneumoniae antibodies of over 50% in adults (24). This organism has also been implicated as a factor in adult onset asthma as well as in reactive airway disease in children (23). Furthermore, a number of investigators have presented evidence which suggests a role of C. pneumoniae in atherosclerosis (36, 54). In an effort to reduce the morbidity and mortality due to this pathogen, consideration needs to be given to the long-term goal of developing a vaccine. However, the key factors of the host immune response that are essential in protecting the host from infection or severe disease, as well as key structures or functions of the pathogen that contribute to its pathogenicity, have not been established.

C. pneumoniae shares many characteristics of other members of the genus Chlamydia, including its growth cycle and overall outer membrane composition (5, 20, 37, 42, 43, 45). The Chlamydia lipopolysaccharide (LPS) has been characterized as having a rough phenotype that has a genus-specific epitope(s) (5, 9). Therefore, it is similar to the LPS in the Re mutant of Salmonella enterica serovar Minnesota, since it has the core lipid A moiety and 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) core but lacks the distal O-polysaccharide region. With the exception of one report by Girjes et al. (18), in which borderline neutralization at high concentrations of a genus-specific anti-LPS monoclonal antibody (MAb) was shown, there are no reports of the involvement of LPS in neutralization of this pathogen. As with the other species of Chlamydia, the most abundant protein by weight in the outer membrane is the 40-kDa major outer membrane protein (MOMP) (11, 43). In Chlamydia trachomatis the MOMP is immunodominant, the target of neutralizing antibodies, and thus a candidate for acellular vaccines (11, 51, 62). In contrast, however, the MOMP of C. pneumoniae, while antigenic, is not immunodominant; no MAbs have been mapped to it, and antisera to peptides representing the variable domains (VDs) do not neutralize C. pneumoniae (12, 52). Also, in contrast to C. trachomatis, in which the variation in the VDs of the MOMP appears to be the basis for the serovariants of this species, there does not appear to be variation in VD 4 of the C. pneumoniae strains so far examined (17, 30, 58). However, the existence of different strains or serovariants of C. pneumoniae is still controversial, and if they exist, they may be due to surface structures other than the MOMP (2, 29, 30). Therefore, the basic architectures of the outer membrane components, while they may be similar among the species, exhibit differences in antigenicity and function.

Puolakkainen et al. (55) were the first to describe MAbs that neutralized the infectivity of C. pneumoniae, but despite several attempts, they were not successful in defining structures recognized by these MAbs. The reason for this remains unclear but may be due to the strict conformational nature of the epitope(s) recognized. In this study an attempt was made to define a surface structure of C. pneumoniae that was the target of a neutralizing antibody. We describe a MAb that recognizes a genus-specific LPS epitope that specifically neutralizes the infectivity of C. pneumoniae TW-183.

MATERIALS AND METHODS

Organisms.

The Chlamydia strains used in this study were C. pneumoniae TW-183, obtained from the Washington Research Foundation (Seattle, Wash.); 1497, an isolate obtained from a throat culture from a patient at the University of California, Irvine; and 2043, CM-1, and CWL-029, obtained from the American Type Culture Collection (Rockville, Md.). C. trachomatis serovars L1 (440), L3 (404), A (G-17), B (HAR-36), C (TW-3), D (IC-Cal), E (Boor), I (UW-12), J (UW-36), K (UW-31), and mouse pneumonitis (Nigg II), as well as Chlamydia psittaci (Texas turkey), were obtained from the American Type Culture Collection. All Chlamydia isolates were raised for 48 to 72 h in HeLa 229 cells, and C. pneumoniae was also propagated in HEp-2 cells. Chlamydiae were harvested by sonication of infected monolayers in 0.2 M sucrose–0.02 M sodium phosphate (pH 7.2)–5 mM glutamic acid (SPG). Organisms were stored at −70°C. Where indicated, elementary bodies (EBs) of Chlamydia were further enriched by centrifugation through 35% Renografin-76 (E. R. Squibb & Sons, Princeton, N.J.) (10).

Bacterial and fungal isolates were obtained from the Medical Microbiology Laboratory at the University of California, Irvine Medical Center. All isolates were subcultured twice to 5% sheep blood agar before being used.

Bacterial LPS, synthetic oligosaccharides, and neoglycoconjugate antigens.

The Re mutant strains of Escherichia coli F515 and S. enterica serovar Minnesota R595 (25, 26) were transformed with plasmid pFEN207 (46), containing the Kdo transferase gene kdtA of C. trachomatis L₂ (1, 41). Recombinant bacteria and the parent bacteria were grown in a fermentor, killed with phenol (0.5%), washed successively with ethanol, acetone, and ether, and then dried. LPS was extracted from dry bacteria by the phenol-chloroform-petroleum ether method, purified by repeated ultracentrifugation, and converted into the uniform triethyl-ammonium salt (16). Throughout this report, the resulting LPS are abbreviated as F515, F515-207, R595, and R595-207. De-O-acetylated LPS (LPS_de-O-ac) was prepared by hydrazinolysis (37°C, 30 min), and dephosphorylated LPS (LPS_de-P) was prepared by treatment with 48% aqueous hydrogen fluoride (4°C, 48 h), as reported elsewhere (27).

The synthetic oligosaccharides αKdo(2→allyl, [where Kdo is 3-deoxy-d-manno-oct-2-ulopyransylonic acid], αKdo(2→4)αKdo(2→allyl, αKdo(2→8)αKdo(2→allyl, αKdo(2→4)αKdo(2→4)αKdo(2→allyl, αKdo(2→8)αKdo(2→4)αKdo(2→allyl, αKdo(2→8)αKdo(2→4)αKdo(2→6)βGlcNAc(1→allyl, and αKdo(2→8)αKdo(2→4)αKdo(2→6)βGlcNAc(1→6)αGlcNAc(1→allyl were synthesized as described previously (32–35). The allyl glycosides R-CH₂-CH=CH₂ were conjugated with cysteamine (38), yielding R-(CH₂)₃-S-(CH₂)₂-NH³⁺, which was activated with thiophosgen into the isothiocyanate derivatives R-(CH₂)₃-S-(CH₂)₂-N=C=S and then conjugated to bovine serum albumin (BSA), yielding R-(CH₂)₃-S-(CH₂)₂-NH-CS-NH-BSA, where R represents the glycosyl residue. The last compounds are abbreviated R-BSA. The amount of ligand present in the conjugates was determined by measuring the amounts of protein (Bradford assay; Bio-Rad, Richmond, Calif.) and Kdo (thiobarbiturate assay) (4). The isolation and characterization of the pentasaccharide bisphosphate αKdo(2→8)αKdo(2→4)αKdo(2→6)βGlcNAc(1→6)αGlcNAc1,4′P₂ has been reported earlier (25).

Hybridoma production.

Six- to 8-week-old female BALB/c mice (Simonsen Laboratories, Gilroy, Calif.) were injected intraperitoneally (i.p.) with 10⁷ inclusion-forming units (IFU) of C. pneumoniae TW-183 in complete Freund’s adjuvant on day 0 and with the same number of IFU i.p. in incomplete Freund’s adjuvant on day 14. This was followed by an intravenous injection of the same infectious dose in phosphate-buffered saline, (PBS) (10 mM, pH 7.2) on day 21. Three days later, the animals were sacrificed and the spleen was removed for cell fusion as previously described (49). Hybridoma supernatants were screened by indirect inclusion immunofluorescence assay (IFA) and enzyme immunoassay (EIA) with EBs of C. pneumoniae. Ascitic fluid was produced by injecting 10⁶ hybridoma cells i.p. into 4- to 6-week-old BALB/c mice that had been injected i.p. with pristane 10 days previously. Immunoglobulin was purified from mouse ascitic fluid by using the Affi-Gel protein A MAPS II system (Bio-Rad). Upon elution from the column, fractions containing MAb were pooled and dialyzed against three 1-liter changes of PBS. Protein contents of the purified MAbs were determined by the method of Lowry et al. (39), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to examine the purity of the preparations (59). Purified MAbs were also tested for reactivity by an IFA and an in vitro neutralization assay (49, 50). Isotyping was performed as previously described with microtiter plates coated with EBs of C. pneumoniae and a mouse typer kit (Bio-Rad) (51).

Immunoassays.

The IFA was performed as previously described, using HEp-2 cells for preparing slides of cells infected with C. pneumoniae (50). Briefly, cells were infected, incubated for 24 to 48 h, trypsinized, washed in Eagle’s minimal essential medium containing Earle’s salts (E-MEM), resuspended in E-MEM supplemented with 10% fetal bovine serum and gentamicin (50 μg/ml) (E-MEM-S), and applied to 5-mm-diameter wells on a glass slide. The slides were incubated in a humid chamber at 37°C for 4 h or until a confluent monolayer coated the slide well surface. Slides were washed with PBS, fixed with acetone for 10 min, and stored dry at −70°C. For performance of the IFA, slides were allowed to reach room temperature before the addition of the antisera. Primary- and secondary-antibody incubations were at 37°C for 30 min, followed by several washes in PBS.

Dot blotting, EIAs, and immunoblotting were performed as previously described (49, 50). For the immunoblots, low-range prestained SDS-PAGE protein standards (Bio-Rad) were used for determination of molecular weights. For some EIAs, EBs (10 μg/ml) that had been treated with 50 mM sodium metaperiodate in 50 mM sodium acetate buffer (pH 5.5) or buffer alone for 6 h were used to coat plates. For selected immunoblots, prior to separation by PAGE, EBs (1 mg/ml) were boiled for 5 min in SDS-PAGE sample buffer and then treated with proteinase K (2.5 mg/ml) for 1 h at 60°C.

The epitope specificity of MAb CP-33 was determined by EIA and EIA inhibition with synthetic neoglycoconjugates or LPS as the solid-phase antigen; details of both assays have been previously described (57). Briefly, neoglycoconjugates or LPS was applied to MaxiSorp (U-bottom; Nunc, Roskilde, Denmark) or polyvinyl (U-bottom; Becton Dickinson) microtiter plates, respectively. Neoglycoconjugate solutions were adjusted to equimolar concentrations based on the amount of ligand present in the respective conjugate. For comparison, the murine MAb S25-23 (immunoglobulin G1 [IgG₁]), which has been described in detail (15) was used. For EIA inhibition, serial twofold dilutions of inhibitor (30 μl) were mixed in V-shaped microtiter plates (Nunc) with an equal volume of antibody diluted in the same buffer to give an optical density at 405 nm (OD₄₀₅) of 1.0 without the addition of inhibitor. After incubation (15 min, 37°C), 50 μl of the mixture was added to antigen-coated EIA plates. All measurements were done in triplicate; confidence values never exceeded 10%.

In vitro neutralization assay.

The in vitro neutralization assay used has been previously described (49). Briefly, antisera were diluted in PBS containing 5% guinea pig serum (GPS) (BioWhittaker, Walkersville, Md.). Chlamydiae (10⁴ IFU) were added to the antiserum dilutions. The antigen-antibody mixtures were incubated at 37°C for 45 min and inoculated into duplicate confluent HEp-2 or HeLa cell monolayers contained in glass vials (15 by 45 mm), which had been washed twice with PBS immediately before inoculation. Cells were infected by centrifugation at 1,000 × g for 1 h followed by stationary incubation at 37°C for 1 h, after which cultures were fed with 1 ml of E-MEM-S containing cycloheximide (1 μg/ml). Infected monolayers were incubated for 48 or 72 h, fixed, and stained either with MAb E4, which recognizes the MOMP of C. trachomatis, or with MAb CP-31, which recognizes the 57-kDa heat shock protein (hsp) of Chlamydia. IFU were counted in 10 fields at a magnification of ×200, and the neutralization titer was that dilution that gave 50% inhibition of control IFU. Each test was repeated a minimum of two times.

Animal model.

The mouse model described by Yang et al. (61) was used to assess the ability of MAb CP-33 to attenuate an in vivo infection. Briefly 5 × 10⁸ to 5 × 10⁵ IFU of C. pneumoniae per ml was incubated in vitro with either 100 μg of purified IgG (Sigma Chemical Co., St. Louis, Mo.) per ml, which served as a control, or 100 μg of MAb CP-33 per ml. These mixtures were made in PBS containing a final concentration of 5% GPS. Chlamydiae were incubated with antibody for 45 min at 37°C. Male Swiss Webster mice (Simonsen Laboratories), 4 to 6 weeks of age, were lightly anesthetized with metophane prior to intranasal installation of 2 × 10⁷ to 2 × 10⁴ IFU in 40 μl. Mice were administered 20 μl of inoculum in each nostril. Once inoculated, mice were housed in a laminar flow facility for 5 days, at which time they were euthanized and the lungs and tracheae were removed and weighed. The lung tissue was placed in 5 ml of SPG and homogenized in a Stomacher Lab Blender 80 (Dynatech Laboratories, Inc., Alexander, Va.), and 0.2 ml of undiluted or diluted lung homogenate was inoculated into monolayers of HEp-2 cells contained in 1-dram glass vials. Cultures were centrifuged for 1 h at 30°C, fed with E-MEM-S containing cycloheximide (1 μg/ml), fixed with methanol at 72 h, and stained with a fluorescence-labeled MAb to the LPS of Chlamydia (Meridian Diagnostics, Inc., Cincinnati, Ohio). To compare the groups in terms of yield of IFU from lungs, a Mann-Whitney U test with the Statview IV software program (Abacus, Berkeley, Calif.) was employed.

RESULTS

Antigenic specificity of MAb CP-33.

MAb CP-33, an IgG2b antibody, when tested by Western blotting recognized the LPS of five strains of C. pneumoniae, all 11 serovars of C. trachomatis tested, and C. psittaci (Fig. 1). Similar results were obtained when these organisms and MAb CP-33 were tested by dot blotting and IFA (Fig. 2). Reactivity appeared to be specific to the genus Chlamydia, because there was no reactivity by Western blotting when MAb CP-33 was tested against species within the genera Escherichia, Klebsiella, Enterobacter, Proteus, Morganella, Acinetobacter, Neisseria, Gardnerella, Pseudomonas, Haemophilus, Enterococcus, Streptococcus, Staphylococcus, Lactobacillus, and Candida.

FIG. 1.

Immunoblot of C. pneumoniae TW-183 (Cpn), C. trachomatis serovar E (Ct), C. psittaci Texas turkey (Cps), and S. minnesota Re LPS probed with a 1/200 dilution of ascitic fluid containing MAb CP-33. MW, molecular mass.

FIG. 2.

Dot blot of native EB preparations (1 μg/dot) probed with a 1/200 dilution of ascitic fluid containing MAb CP-33. Dots: A1, C. pneumoniae TW-183; A2, C. pneumoniae 2043; A3, C. pneumoniae 1497; A4, C. pneumoniae CM-1; A5, C. pneumoniae CWL-029; A6, C. trachomatis serovar E; A7, C. trachomatis serovar C; B1, C. trachomatis serovar L3; B2, C. trachomatis serovar F; B3, C. trachomatis mouse pneumonitis biovar; B4, C. psittaci Texas turkey; B5, Re LPS; B6, HeLa cells; B7, HEp-2 cells.

Periodate treatment of EB-coated EIA plates essentially abolished the EIA reactivity of MAb CP-33 (Fig. 3). In contrast, there was only a slight decrease in the reactivity of MAb CP-31, which recognizes a 57-kDa hsp of C. pneumoniae. When EBs of C. pneumoniae were treated with proteinase K, there was no significant decrease in the Western blot reactivity of MAb CP-33 toward LPS (data not shown). However, when polyclonal serum raised to C. pneumoniae was used to probe proteinase K-treated and nontreated C. pneumoniae EBs, the reactivities of the majority of the bands diminished in the proteinase K-treated sample, with the LPS band remaining.

FIG. 3.

EIA showing the effect of periodate treatment on the binding of MAb CP-33 to EBs of C. pneumoniae TW-183. Microtiter plates coated with 11 μg of EBs per well were treated with 50 mM sodium metaperiodate in 50 mM sodium acetate buffer (pH 5.5) or with buffer alone for 6 h. These plates were then used in an EIA with dilutions of MAb CP-33 or MAb CP-31 (control), which recognizes a 57-kDa hsp of C. pneumoniae.

Having established that MAb CP-33 recognized LPS, the binding specificity of MAb CP-33 was determined by EIA and EIA inhibition with chemically defined antigens and inhibitors. For comparison, MAb S25-23 (IgG1), another MAb to chlamydial LPS which has been previously characterized in detail (15), was included. The data obtained with LPS from the Re mutants of either E. coli or S. enterica serovar Minnesota indicated that MAb CP-33 did not react with Re-type LPS, thus corroborating the Western blot results showing this MAb to be Chlamydia specific. However, when LPS of recombinant bacteria expressing the Kdo transferase of C. trachomatis were used, MAb CP-33 bound to LPS derived from E. coli F515-207 but not to that from S. enterica serovar Minnesota R595-207 (Table 1). With the LPS of the latter strain after dephosphorylation or after de-O-acylation, binding was observed at a concentration comparable to that obtained with MAb S25-23.

TABLE 1.

Binding specificity of MAb CP-33 in EIA with LPS or chemically defined partial structures

Antigena	Concn of MAb (ng/ml) yielding an OD405 of >0.2
	S25-23	CP-33
Synthetic neoglycoconjugates
αKdo	>1,000	>1,000
αKdo(2→4)-αKdo	>1,000	>1,000
αKdo(2→8)αKdo	>1,000	>1,000
αKdo(2→4)-αKdo(2→4)αKdo	>1,000	>1,000
αKdo(2→8)αKdo(2→4)αKdo	1	125
αKdo(2→8)αKdo(2→4)αKdo(2→6) βGlcNAc	1	>1,000
αKdo(2→8)αKdo(2→4)αKdo(2→6)βGlcNAc(1→6)αGlcNAc	1	>1,000
LPSs
E. coli F515	>1,000	>1,000
S. enterica serovar Minnesota R595	>1,000	>1,000
E. coli F515-207	2	40
S. enterica serovar Minnesota R595-207	8	>1,000
S. enterica serovar Minnesota R595-207de-O-ac	4	10
S. enterica serovar Minnesota R595-207de-P	2	10
S. enterica serovar Minnesota  R595-207de-O-ac/de-P	4	625

^aThe indicated carbohydrate ligands were synthesized and covalently linked to BSA as described in Materials and Methods. LPS were obtained from Re mutant strains of E. coli or S. enterica serovar Minnesota transformed with plasmid pFEN207 carrying the Kdo transferase gene kdtA of C. trachomatis. 

With synthetic neoglycoconjugates containing as ligands the carbohydrate backbone or partial structures of chlamydial LPS as solid-phase antigens in EIA, no reactivity against Kdo, Kdo disaccharides, or the Kdo trisaccharide αKdo(2→4)αKdo(2→4)αKdo was observed with either antibody. Binding was observed with the Kdo trisaccharide αKdo(2→8)αKdo(2→4)αKdo, however, at concentrations 100 times higher than those required for MAb S25-23. Whereas MAb S25-23 bound equally well to higher oligosaccharides, the reactivity of MAb CP-33 toward αKdo(2→8)αKdo(2→4)αKdo(2→6)βGlcNAc and αKdo(2→8)αKdo(2→4)αKdo(2→6)βGlcNAc(1→6)αGlcNAc decreased significantly.

By using synthetic haptenic oligosaccharides in EIA inhibition (Table 2), MAb S25-23 could be inhibited with all oligosaccharides containing the αKdo(2→8)αKdo(2→4)αKdo trisaccharide, whereas none of these compounds was able to inhibit MAb CP-33, even at concentrations higher than 100 μM.

TABLE 2.

Binding specificity of MAb CP-33 in EIA inhibition with LPS or chemically defined partial structures

Inhibitora	Concn (μM) yielding 50% inhibition with MAbb:
	S25-23	CP-33
αKdo	>360	>360
αKdo(2→4)-αKdo	>200	>200
αKdo(2→8)αKdo	>200	>200
αKdo(2→4)αKdo(2→4)αKdo	>140	>140
αKdo(2→8)αKdo(2→4)αKdo	0.28	>140
αKdo(2→8)αKdo(2→4)αKdo(2→6)βGlcNAc	0.11	>100
αKdo(2→8)αKdo(2→4)αKdo(2→6)βGlcNAc (1→6)αGlcNAc	0.36	>100
αKdo(2→8)αKdo(2→4)αKdo(2→6)βGlcNAc (1→6)αGlcNAcP2	0.31	>100

^aAll structures were used as synthetic allyl glycosides except αKdo(2→8)αKdo(2→4)αKdo(2→6)βGlcNAc(1→6)αGlcNAcP₂, which was isolated from F515-207 LPS (26). 

^bEIA plates were coated with LPS F515-207 at a concentration of 8 μg/ml (400 ng/well). Antibody concentrations were adjusted to give an OD₄₀₅ of 1.0 without the addition of inhibitor. 

Neutralization of infectivity by MAb CP-33.

The first series of neutralization experiments was performed with C. pneumoniae raised in Hep-2 cells. In vitro neutralization results for MAb CP-33 against C. pneumoniae TW-183 gave a 50% neutralization titer of 8 ng/ml (Fig. 4). However, when MAb CP-33 was tested against three other C. pneumoniae strains, CM-1, 1497, and CWL-029, also raised in HEp-2 cells, there was no attenuation of infection. This was also true for C. trachomatis serovars E, C, D, and L3 and C. psittaci Texas turkey which had been raised in HeLa cells. All of these neutralization assays were originally performed with Hep-2 cells. The same neutralization assays were also repeated with HeLa 229 cells and the same neutralization stocks, with similar results. Therefore, from these data it appeared that neutralization was specific for the TW-183 strain of C. pneumoniae. This was also the strain used to immunize the mice from which the hybridoma producing MAb CP-33 was identified.

FIG. 4.

In vitro neutralization results for three C. pneumoniae isolates and C. trachomatis serovar E tested with MAb CP-33 on HEp-2 cells. Neutralization was defined as ≥50% inhibition compared to the control value. Bars represent standard deviations.

To ensure that the same result would be obtained with different batches of EBs, we regrew both C. pneumoniae TW-183 and CM-1 in HEp-2 cells. Here again MAb CP-33 was unable to neutralize CM-1 but neutralized TW-183. The 50% reduction from the control value for this preparation of TW-183 was achieved with 250 ng of MAb CP-33 per ml, which was considerably more than that required for the 50% neutralization point for the previous preparation of TW-183 (data not shown). Seeing this variability, we wanted to establish whether the time of harvest had an effect on the amount of MAb CP-33 that was necessary to achieve the 50% end point. In addition, we wanted to determine whether harvesting at different times would affect the neutralization result with CM-1. Both C. pneumoniae strains were raised in HeLa and HEp-2 cells and harvested at 48, 72, and 96 h after infection. The harvesting conditions were strictly standardized, and aliquots of each harvest were frozen at −70°C. An aliquot of each harvest was used to determine the IFU present. The peak infectious yield was achieved at 72 h after infection. Neutralizations were performed with the stock raised in HEp-2 cells, using both HEp-2 and HeLa cells in the neutralization assay. The time at which the TW-183 stock to be used in the neutralization assay was harvested had a significant effect on the overall neutralization result. When harvested at 48 h, the organisms were more susceptible to neutralization by MAb CP-33. Interestingly, this is in contrast to the case for organisms harvested at 72 h, which yielded the largest amount of infectious organisms. CM-1, however, regardless of the harvest time or whether it was raised in HEp-2 or HeLa cells or tested for neutralization in either of these two cell lines, did not show neutralization with MAb CP-33.

With the mouse model described by Yang et al. (61), male Swiss Webster mice were inoculated intranasally with C. pneumoniae TW-183 that had been incubated for 45 min with MAb CP-33 or, in the case of the controls, with IgG and 5% GPS. The lungs and tracheae were removed 5 days after inoculation, homogenized, and cultured on HEp-2 cells. When the IFU present in the control (IgG-treated) and test (MAb CP-33-treated) mixtures immediately before inoculation were compared by in vitro methods, there was approximately a 10-fold decrease, or 14% of control IFU, in all test mixtures (Table 3). This decrease paralled the recovery of IFU from the control and test mice for the different doses used to infect the mice (Fig. 5). The control doses used to inoculate mice ranged from 2 × 10⁷ to 2 × 10⁴ IFU per animal. When the animals receiving the highest inocula, 2 × 10⁷ to 2 × 10⁶ IFU, were compared, there was no significant difference between the two groups. However, in the group inoculated with 2 × 10⁵ IFU, there was a significant difference between the control and MAb CP-33-treated mice as to the recovery of IFU from the infected animals (P < 0.05). In the MAb CP-33-treated group, viable organisms were recovered in only two of the five mice, in contrast to 100% (five of five) of the control mice. In order to verify that the in vitro neutralization results with MAb CP-33 correlated with the in vivo results, two strains of C. pneumoniae, CM-1 and 2043, that were not neutralized in vitro by MAb CP-33 were also used to infect mice. After pretreatment of doses equivalent to 2 × 10⁵ IFU per mouse with either control IgG or MAb CP-33, there was no significant difference (P > 0.1) between control and MAb CP-33-treated CM-1 and 2043 EBs in the number of mice infected or the recovery of IFU per lung at 5 days after infection (Fig. 6). Thus, both the in vitro and in vivo neutralizations seen with MAb CP-33 appeared to be specific for C. pneumoniae TW-183.

TABLE 3.

Culture results for lungs 5 days after infection with different doses of MAb CP-33-treated EBs^a

Treatment	Inoculum after incubation	No. culture positive/total no. of mice
Control IgG	2.5 × 106	5/5
	2.2 × 105	5/5
	1.5 × 104	2/5
MAb CP-33	4 × 105	5/5
	3.3 × 104	2/5b
	2.1 × 103	0/5

^aThree 10-fold dilutions of EBs were incubated in vitro with 100 μg/ml of either purified IgG (control) or MAb CP-33 per ml in 5% GPS–PBS for 45 min at 37°C. The treated EBs were cultured prior to being used to infect mice intranasally. Five days after infection, the mice were sacrificed and the lungs and tracheae were excised, homogenized, and cultured. 

^bP = 0.016 by the Mann-Whitney U test. 

FIG. 5.

Scattergram of the yield of C. pneumoniae TW-183 IFU from the lungs of Swiss Webster mice harvested 5 days after intranasal inoculation with three different doses of C. pneumoniae TW-183 treated with 100 μg of either MAb CP-33 or control IgG per ml immediately before inoculation into mice.

FIG. 6.

Scattergram of the yields of three C. pneumoniae strains, TW-183, 2043, and CM-1, from the lungs of Swiss Webster mice harvested 5 days after intranasal inoculation. The three strains (5 × 10⁶ IFU/ml) were pretreated with 100 μg of either MAb CP-33 or control IgG per ml immediately before inoculation of 2 × 10⁵ IFU into mice.

DISCUSSION

MAb CP-33 is the first MAb described that both neutralizes the infectivity of C. pneumoniae and has been mapped to an antigenic structure of this pathogen. Although polyclonal serum raised to this organism exhibits the ability to neutralize the infectivity of this organism in vitro (reference 44 and unpublished results), until this study, efforts to identify a structure recognized by neutralizing MAbs have been unsuccessful (55). Polyclonal serum raised to a recombinant 76-kDa protein of this species has been reported to show neutralization of infectivity in vitro; however, this attenuation of infectivity was borderline (44). Puolakkainen et al. (55) have described two MAbs that neutralize the infectivity of C. pneumoniae, MAbs RR-402 and TT-205. MAb RR-402 was identified from a fusion using splenocytes from a mouse immunized with C. pneumoniae AR-39 and neutralized the homologous C. pneumoniae strain. Likewise, MAb TT-205 was selected from a fusion using TW-183-immunized splenocytes and was able to neutralize the homologous strain. Despite several attempts, the structure recognized by these MAbs was not determined. Identification of key structures that may be the target of future vaccine or treatment efforts is an important area of research with C. pneumoniae.

While LPS predominates on the surface of Chlamydia, it has not been ascribed a specific function in terms of host-Chlamydia interactions. This alone is intriguing, since in gram-negative bacteria, LPS plays a central role in bacterium-host cell interactions. LPS in several gram-negative species has been considered a target of the host immune response in terms of protection. It is on the basis of this concept that MAbs to LPS have been used in humans to reduce the sequelae of a gram-negative sepsis (53). Chlamydia spp., like with other gram-negative bacteria, such as Neisseria, Haemophilus, and Bordetella spp., that inhabit mucosal surfaces that are not rich in bile, do not possess the long carbohydrate O side chains that are so distinctive of enteric gram-negative rods (22). Instead, Chlamydia LPS has been described to be of the rough phenotype, resembling that of the rough Re LPS mutant of Salmonella enterica serovar Minnesota (5, 7). By using MAbs to Chlamydia, at least two distinct LPS epitopes have been described, a Chlamydia genus-specific epitope that binds to the core region of LPS and one that cross-reacts with Re LPS of other enterobacteria (6, 9).

Our data show that MAb CP-33 belongs to the former group. The lack of reactivity with two different Re-type LPSs and the reactivity with LPS or synthetic structures containing the trisaccharide αKdo(2→8)αKdo(2→4)αKdo clearly indicate Chlamydia specificity. Nevertheless, MAb CP-33 is not a typical Kdo trisaccharide antibody such as MAb S25-23, which has been most intensively investigated (8, 15). Also, for MAb CP-33 the Kdo trisaccharide is a structural prerequisite but is not sufficient for optimal binding. Whereas MAb S25-23 bound to neoglycoconjugates and various LPS from recombinant Re mutant bacteria in similar concentrations (1 to 8 ng/ml), MAb CP-33 gave the best results with dephosphorylated or de-O-acylated LPS of S. enterica serovar Minnesota R595-207. The native LPS of the same strain was not at all recognized by MAb CP-33, although MAb S25-23 gave similar results. Obviously, the epitopes recognized by these two antibodies are different; however, they both have the requirement for the Kdo trisaccharide. We hesitate to speculate at present on the fine specificity of MAb CP-33, but the following facts should be taken into consideration. The difference between the LPS of E. coli F515-207 and of S. enterica serovar Minnesota R595-207 concerns the presence in the latter of an additional palmitic acid residue at the reducing glucosamine unit of lipid A (bound to the hydroxyl group of the amide-linked 3-hydroxytetradecanoic acid) and of 4-aminoarabinose and ethanolaminephosphate replacing the phosphate groups of lipid A. It is presently not known whether such substituents also occur in chlamydial LPS and, if so, whether they are equally present in all species or serovars. All of these substituents could influence considerably the conformation of the carbohydrate backbone, particularly the conformation of the 2→8 bond, which, due to its additional degree of freedom, exhibits much more flexibility than the 2→4 linkage. Thus, the epitope recognized by MAb CP-33 could well be the Kdo trisaccharide in a specific conformation (3). This hypothesis is supported by the result that MAb CP-33 bound less to the tetrasaccharide αKdo(2→8)αKdo(2→4)αKdo(2→6)βGlcNAc or the pentasaccharide αKdo(2→8)αKdo(2→4)αKdo(2→6)βGlcNAc(1→6)αGlcNAc than to the Kdo trisaccharide and by the results obtained with LPS from which the ester-linked acyl residues or the phosphate groups or both have been removed. Since removal of either phosphate or fatty acids did not change the binding, it is unlikely that these groups per se are relevant parts of the epitope.

Subtle conformational differences for C. pneumoniae TWAR-183 compared to all of the other species and strains tested may explain why MAb CP-33 can recognize, as determined by immunologic assays, the LPS of the three species of Chlamydia yet neutralize only the homologous strain to which it was produced. Work by Brade et al. (7) with the LPS of C. psittaci and C. trachomatis supports this concept. In performing passive hemolysis inhibition tests, they found that while 1 ng of LPS from C. trachomatis or C. psittaci inhibited the respective homologous systems, significant larger amounts (16 and 32 ng, respectively) were needed for the same inhibition in the heterologous systems. This led these investigators to conclude that while C. psittaci has a genus-specific LPS epitope, it also has a second, species-specific determinant in the carbohydrate structure of the LPS. This hypothesis was verified recently when structural analysis performed on LPS from recombinant bacteria expressing the Kdo transferase of C. psittaci 6BC identified a branched Kdo tetrasaccharide with the sequence αKdo(2→8)[αKdo(2→4)]αKdo(2→4)αKdo (28). It may also be possible that the epitope recognized by MAb CP-33 is identical in all three species but that in C. pneumoniae TW-183 the surrounding outer membrane is different in that the binding of this MAb may interfere with the chlamydia-host interaction. Only by further investigation into the structure and conformation of the LPS and other outer membrane components of C. pneumoniae strains will the reasons for our findings be determined.

The question of whether there are differences among C. pneumoniae strains remains controversial. Our work with MAb CP-33 suggests that there are strain differences and that some of the differences reside in the LPS structure. When examined by sequencing either the VD 4 of the MOMP or 580 bases of the 60-kDa OMP-2 protein, from all strains of C. pneumoniae examined appear to be identical (17, 60). However, when several C. pneumoniae strains were studied with immune sera, strain differences within C. pneumoniae were observed. Black et al. (2), in examining immunoblot profiles by using human immune sera to probe different C. pneumoniae strains, concluded that there are strain variations within this species. Likewise, Wagels et al. (60), using immunoblot profiles of five C. pneumoniae isolates, found distinct antigenic differences between TW-183 and the four other C. pneumoniae isolates tested. These four other C. pneumoniae isolates, one of which was CM-1, while similar to one another, produced profiles which were not identical but were quite distinct from that of TW-183. These data, coupled with similarities of strains in the MOMP and OMP-2 DNA sequences, led these investigators to conclude that strain differences, while they exist in C. pneumoniae, do not reside in the these two outer membrane proteins. We too found C. pneumoniae CM-1 and TW-183 to have distinct differences as measured by neutralization by MAb CP-33.

In performing neutralization assays with MAb CP-33 and C. pneumoniae TW-183, we found that some batches of EBs were more susceptible to neutralization than others. Peeling and Brunham (48), in examining the parameters of a neutralization assay for C. trachomatis, reported that as the ratio of noninfective to infective chlamydiae increased, the efficiency of neutralization decreased exponentially. However, the parameters for the neutralization assay for C. pneumoniae have not been studied in detail. Therefore, we wanted to determine whether the time of harvest of C. pneumoniae TW-183 could affect the neutralization results. In addition, we wanted to determine whether the lack of neutralization of other C. pneumoniae strains was due to the time of harvest. We found that the maximum yield of infectious C. pneumoniae TW-183 and CM-1 was achieved at 72 h. This presumably would also correspond to the highest ratio of IFU to particles. Therefore, we expected that the stock harvested at 72 h would be more efficiently neutralized by MAb CP-33. However, we found that the TW-183 stock harvested at 48 h was clearly more susceptible to neutralization by MAb CP-33. Possible explanations for this finding could be that in the development of infectious EBs the LPS itself might be in a different conformation or that the surrounding outer membrane structure differs throughout the maturation stages within the EB. This would suggest that while organisms may be infectious and thus technically EBs, even within the EB there are different stages of maturation. It has been shown with C. trachomatis and C. psittaci that during the maturation of the reticulate body to the EB, there are different amounts of disulfide cross-linking of the outer membrane proteins. Newhall (47) has reported that with C. trachomatis serovar L2, while OMP-2 is extensively cross-linked throughout most of the growth cycle, the MOMP and OMP-3 gradually become cross-linked over the last 24 h of the growth cycle. Therefore, whether degrees of maturation within the EB of C. pneumoniae accounted for our findings remains to be determined.

Another explanation for the variation in sensitivity with stocks of TW-183 is the possibility of smooth and rough LPS variants of C. pneumoniae within a given population. Findings of distinct subpopulations with regard to LPS structure that can be modulated by bacterial growth phase and growth conditions have been reported for a variety of gram-negative bacteria. Lukacova et al. (40) have presented evidence for smooth-rough variation within C. psittaci and C. trachomatis. Although in general Chlamydia spp. have been characterized as having a rough phenotype, evidence of a smooth variant was detected in chlamydiae grown in the yolk sacs of embryonated eggs. These investigators also found that within a population of Chlamydia in tissue culture, a small percentage of organisms stained with a MAb to the smooth phenotype. Therefore, during the development of the mature EB, there could be different degrees of saccharide O side chains within TW-183, which could make MAb CP-33 more or less efficient at blocking entrance into host cells.

For the genus Chlamydia, most research defining protective epitopes has been with the MOMP of C. trachomatis, for which neutralizing MAbs have been mapped to the VDs, and thus these regions have been suggested to be candidates for subunit vaccines (49, 51, 56, 62). However, it has been shown with C. pneumoniae that these regions behave quite differently, in that they are less antigenic and peptides representing these regions fail to elicit neutralizing antibodies (52). Prior to this report, no purified MAbs recognizing the Chlamydia genus-specific LPS epitope have been shown to neutralize the infectivity of Chlamydia. Here we have described a MAb that recognizes the LPSs of the three species of Chlamydia but neutralizes only one. These findings suggest that key components in the outer membranes of C. pneumoniae and C. trachomatis, namely, LPS and the MOMP, may be quite different in structure. Although the epitope responsible for the neutralization of the strain reported here will not suffice as a vaccine candidate on its own due to the narrow protection afforded only to TW-183, it serves to point out that there may be other sites in the LPS molecule that may have a broader scope in terms of strain protection. Therefore, in light of the findings presented here, studies to investigate the role of LPS in the pathogenesis of C. pneumoniae and possible strategies that can be developed to block the critical epitopes of LPS that contribute to infectivity of this pathogen are warranted.