Division of Infectious Diseases, School of Public Health, 235 Earl Warren Hall, University of California, Berkeley, CA 94720-7360, USA1
Departments of Anesthesia and Pharmaceutical Chemistry2 and the Francis I. Proctor Foundation3, University of California, San Francisco, CA 94143, USA
Author for correspondence: Richard S. Stephens. Tel: +1 510 643 9900. Fax: +1 510 643 1537. e-mail: rss{at}uclink4.berkeley.edu
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ABSTRACT |
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Keywords: intracellular bacteria, outer-membrane proteins, recombinant antibody
Abbreviations: EB, elementary body; HRP, horesradish peroxidase; scFv, single-chain variable fragment
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INTRODUCTION |
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Chlamydia spp. are obligate intracellular bacteria that cause disease in humans and other animals. C. trachomatis causes a spectrum of diseases in humans, including trachoma (a leading cause of blindness) and sexually transmitted infections that often result in severe sequelae in women such as infertility, ectopic pregnancy and chronic pelvic pain (Schachter, 1999 ). Chlamydia spp. have a developmental cycle that begins with the EB, the infectious, metabolically inactive, osmotically stable extracellular form that enters the host cell and remains within a specialized host-cell vacuole, referred to as an inclusion. The EB outer membrane is extremely stable and contains multiple disulfide-bonded outer-membrane proteins (Hatch et al., 1984
). Because Chlamydia spp. are obligate intracellular pathogens, the macromolecular surface components of EBs are of particular importance for their unique biology. However, there is a narrow understanding of the macromolecular composition of chlamydial outer membranes, and few surface-exposed components have been defined at a molecular level. Even though extensive research has been conducted, all of the potential mechanisms for chlamydial attachment and infectivity have yet to be elucidated (Stephens, 1999
). Adhesion or invasion ligand candidates include heparan-sulfate-like molecules (Zhang & Stephens, 1992
), the major outer-membrane protein (MOMP) (Su et al., 1988
), glycosylated MOMP (Swanson & Kuo, 1994
), 18 kDa and 32 kDa glycoproteins (Swanson & Kuo, 1990
) and exoglycolipid (Stuart et al., 1991
). Of these, only a few have been demonstrated as surface exposed and studied at a molecular level.
Investigations to define the composition of the outer membrane have been biased by the procedures available to extract the outer membrane as well as limited by availability of probing reagents, primarily monoclonal antibodies. Monoclonal antibodies are typically dependent upon selection to dominant immunogens. The information inferred by electron microscopy of chlamydial EBs shows complex surface structures (Matsumoto, 1981 ), and proteins identified from the genome sequence of C. trachomatis predicted to be localized to the outer membrane (Stephens et al., 1998
) suggest a more diverse composition than has been elucidated thus far.
In this study, a panel of scFv phage antibodies to EB surface molecules was generated by panning the phage antibody library on C. trachomatis EBs adsorbed to polystyrene tubes. The scFv antibodies were characterized for reactivity by ELISA, dot-blot, immunoblot and immunocytochemistry. Several chlamydial surface proteins probably not previously identified by other methods were identified by specific scFv antibodies. In addition, scFv antibodies specific for host-cell antigens associated with the surface of EBs were selected that would not otherwise have been identified from the C. trachomatis genome sequence (Stephens et al., 1998 ).
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METHODS |
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Selection of scFv antibodies specific for EBs.
The selection for scFv antibodies that bound chlamydial EBs included three discrete steps: selection of bound phage particles, recovery of phagemids encoding scFv antibody, and production of phage for additional rounds of selection. For the selection procedure, the method of Marks et al. (1991) was used with the exceptions noted below. Briefly, immunotubes (Nunc Maxisorp) were coated overnight with a 1 ml suspension of C. trachomatis (serovar L2) EBs (0·1 mg ml-1 in PBS) at 4 °C. The tubes were washed, blocked and 1012 phage particles from a phage library containing 6·7x109 members (Sheets et al., 1998
) were added and allowed to bind for 1 h at room temperature. The nonadherent phage particles were removed by washing. Bound phage were eluted using three separate methods from three EB-coated immunotubes in an effort to obtain a variety of scFv antibodies with different specificities. First, 1 ml 100 mM triethylamine was added for 10 min then neutralized with 0·5 ml 1 M Tris/HCl pH 7·4. Second, phage were eluted by shifting the temperature to 43 °C for 15 min. Third, 0·2 units heparitinase (Seikagaku America) in 250 µl PBS was used to elute phage for 15 min at 37 °C.
Eluted phage were used to infect E. coli TG-1 which were plated on 2x TYE plates (Miller, 1972 ) containing 100 µg ampicillin ml-1 and 1% glucose (2x TYE/amp/glu). Following overnight incubation, the bacteria were scraped from the plates into 2x TYE medium containing 2x TYE/amp/glu and phage antibodies were prepared by rescue with VCSM13 helper phage (Stratagene) as previously described (Marks et al., 1991
). After overnight growth, the sample was centifuged and phage were concentrated from the supernatant by precipitation with polyethylene glycol and filtered through a 0·45 µm filter. The concentrated phage (1·0x1011 particles ml-1) were used for the next round of selection on EB. A total of three rounds of selection was performed.
After the third round of selection, soluble scFv antibodies were expressed from individual E. coli colonies as described by Marks et al. (1991) . Briefly, scFv phage-displayed antibodies were screened for binding to EBs by ELISA (see below). The number of unique ELISA-positive clones was determined by BstNI fingerprinting (Clackson et al., 1991
). scFv genes were amplified from single E. coli colonies with the primers LMB3 (5'-CAGGAAACAGCTATGAC-3') and fd-SEQ1 (5'-GAATTTTCTGTATGAGG-3') followed by digestion of the PCR product with BstN1 (Marks et al., 1991
). Clones were classified according to the fingerprint pattern and one clone of each fingerprint was selected for further characterization.
Isolation of soluble scFv antibodies.
Soluble scFv antibodies were prepared for the antibody-binding studies. scFv genes were initially contained in the vector pHEN1 (Schier et al., 1995 ) for secretion as scFv antibodies with a C-terminal c-Myc- epitope tag (Evan et al., 1985
). Native scFv antibodies were expressed from E. coli TG-1 as previously described by induction with IPTG (0·5 mM) and growth at 25 °C for 5 h (Marks et al., 1991
). scFv antibodies were harvested from the bacterial periplasm by osmotic shock (Breitling et al., 1991
). Because of cross-reactivity of the anti-c-Myc antibody 9E10 (Santa Cruz Biotechnology) with several chlamydial proteins, scFv genes were also subcloned into the vector pCANTAB 5E (Amersham Pharmacia Biotech). This vector fused a C-terminal E-epitope tag to the scFv antibodies which could be detected with anti-E monoclonal antibody (Amersham Pharmacia Biotech). Conservation of the scFv gene was confirmed by repetition of the fingerprinting assay using the primers LMB3 and LMB2 (5'-GTAAAACGACGGCCAGT-3').
Binding assays.
scFv antibodies were normalized by examination of relative amounts of scFv antibody in each individual periplasmic preparation. The relative amount of scFv antibody was quantified by nitrocellulose spot assay as observed by intensity of the dots. Briefly, 50 µl of each scFv periplasmic preparation was spotted onto nitrocelluose membranes. Membranes were blocked with 3% BSA in PBS for 1 h at room temperature. scFv antibodies were detected using either 9E10 or anti-E-tag antibody followed by the addition of horseradish peroxidase (HRP)-conjugated anti-mouse Fc antibody (Sigma) and 4-chloro-1-naphthol substrate (Fisher Biotech).
ELISA.
scFv antibodies were screened for binding to chlamydial EBs by ELISA. Briefly, 96-well microtitre plates (Immulon 2, Dynex Technologies) were coated overnight with 50 µl 0·1 mg ml-1 purified EBs in PBS. Plates were blocked with 5% skim milk in PBS (MPBS) and 50 µl of E. coli supernatant containing scFv antibodies was added. scFv antibody binding was detected using either 9E10 or anti-E antibody, HRP-conjugated anti-mouse Fc antibody and OPD substrate (Dako Corporation).
Immunoblot.
Immunoblot assays were used to determine if scFv antibodies bound identifiable bands in lysates of EBs and/or host cells. Fifty micrograms of purified EBs, HeLa229 or L929 cells were tested per lane and immune reactions were determined using either c-Myc-tag or E-tag scFv antibodies, their corresponding anti-tag antibodies and anti-Fc peroxidase conjugate. Ultimately the anti-E antibody was found to react to EBs by immunoblot; therefore the anti-E antibody was preabsorbed with a final concentration of 10 µg ml-1 of lysed (20 mM DTT, boiled) purified EBs. Reactivity was detected using enhanced chemoluminescence (ECL, Amersham Pharmacia Biotech). EBs were run on 10% SDS-PAGE gels for all immunoblots and transferred to nitrocellulose.
Immunocytochemistry.
Immunocytochemistry of Chlamydia-infected and uninfected HeLa229 cells and L929 cells was performed using confluent host cell monolayers on 12 mm glass coverslips in 24-well tissue culture plates. Samples of 200 µl containing 8x106 inclusion-forming units ml-1 were used to infect monolayers (multiplicity of infection of 4). The infected cells were incubated for 48 h at 37 °C, washed with PBS and fixed with 100% methanol for 10 min; subsequent washes were performed with PBST (0·05% Tween 20). Each scFv antibody was incubated on the fixed cells for 1 h at room temperature and probed with anti-E antibody in 2% MPBS. Immune reactions were detected using FITC-conjugated anti-mouse Fc sera (Zymed) diluted in 2% MPBS for 1 h at room temperature. The cells were counterstained with Evans Blue and visualized using a fluorescent microscope. For immunofluorescence assays involving unfixed cells, scFv antibodies were applied directly to the live cells for 1 h at 37 °C, after which the cells were methanol fixed. Subsequent detection incubations were as stated above.
EB dot-blot assay for surface-accessible antigens.
The dot-blot assay developed by Zhang et al. (1987) permits the specific detection of surface-localized antigens. The assay was performed as previously described (Zhang et al., 1987
) with modifications noted. Briefly, 50 µl samples of purified serovar L2 EBs (5 µg ml-1) were applied to nitrocellulose in a dot-blot apparatus (Gibco-BRL) by gravity filtration for 10 min then vacuum was applied for 10 s. The nitrocellulose was then blocked with 3% BSA in PBS and washed. Fifty microlitres of normalized amounts of scFv antibody were added to each well and left for 1 h at room temperature. Anti-E antibody and anti-mouse Fcperoxidase conjugate were used to probe for bound scFv antibodies. Reactivity was detected by ECL. For some dot-blot assessments EBs were digested using 50, 100 or 500 µg proteinase K ml-1 for 20 min at 37 °C prior to conducting the assay. To inhibit activity of proteinase K, PMSF at a concentration of 1 mM was added simultaneously with the proteinase K to the solution containing EBs. To ensure that intact EBs were predominant in the dot-blot assay, EBs were subjected to 2% SDS to lyse the organisms and expose intracellular antigens.
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RESULTS |
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A total of 368 scFv clones from the enrichment/selection methods was individually assessed for binding to purified EBs by ELISA. ELISA-positive reactions were considered arbitrarily to be twice the absorbance of the background conjugate control. This limit was selected so scFv antibodies that might be binding molecules of low abundance on EB would not be eliminated. By this criterion 132 scFv clones were considered positive for binding EBs. The nitrocellulose spot assay was used to determine which of the clones were efficiently expressing scFv antibodies. Forty-seven of the scFv clones produced weak reactions in the nitrocellulose spot assay and were eliminated. Next, restriction endonuclease fingerprint analysis was used to evaluate the diversity of scFv clones. By this assay 61 scFv clones were likely duplicates and not evaluated further. Nine clones were lost during these steps due to a variety of causes such as loss of clone viability or scFv antibody expression. A total of 15 scFv antibodies that met the screening criterion was used in the subsequent analyses.
Species specificity of scFv antibodies
Although the scFv phage antibody library was selected using C. trachomatis serovar L2 EBs, it could be expected that some scFv antibodies may not only recognize serovar-specific antigens, but species- and genus-specific antigens as well. This spectrum of reactivity has been observed previously using monoclonal antibodies (Stephens et al., 1982 ). Soluble scFv antibodies were prepared and relative quantities normalized using the nitrocellulose spot assay. scFv antibody specificity was assayed by ELISA, immunoblot, and immunofluorescence testing the reactivity to EBs prepared from C. trachomatis LGV biovar (serovar L2) and a trachoma biovar (serovar D) and C. psittaci (Cp) (AP-1, avian) (Table 1
). Ten individual scFv antibodies bound all three strains of Chlamydia by at least one of the assays (ELISA, immunoblot or immunofluorescence), suggesting that the scFv antibodies recognize a genus-specific antigen. Four scFv antibodies bound only to EBs from C. trachomatis serovars L2 and D, suggesting an antigen shared at the species level. One scFv antibody (3H1-F2) was shown ultimately not to bind EBs by ELISA, immunoblot and immunofluorescence staining, although by the nitrocellulose spot assay large amounts of scFv antibodies were produced.
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Because many of the scFv antibodies reacted to specific antigens in purified serovar L2 EB lysates and had genus-specific or species-specific reactivity as determined by ELISA, each scFv was used in immunoblot assays using lysates from C. trachomatis serovar D and C. psittaci. For the scFv antibodies that recognized serovar D and/or C. psittaci antigens, the estimated molecular masses were the same as those observed in serovar L2, except for E-A11, E-B11 and H-E11 (data not shown). Each of these latter three scFv antibodies bound an antigen of 165 kDa in serovar L2 and one of
110 kDa in C. psittaci, but in serovar D they were reactive to a
75 kDa antigen and a
110 kDa antigen (Table 1
). scFv antibodies 2H-G2, H-B6 and H-B11 bound only a
69 kDa doublet antigen in both of the C. trachomatis biovars. 3H1-B1 recognized a
55 kDa component only in serovar L2 and C. psittaci but not in serovar D. An example of each antigen-specific reactivity is shown in Fig. 1
.
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Despite the observation that 3H1-A5, 3H1-E10 and 3H1-D2 stained uninfected host cells by immunofluorescence, consistent with the host cell immunoblot data, the scFv antibodies nevertheless bound to purified EBs by ELISA, immunoblot and dot-blot (see below). These data suggest that host-cell antigens not associated with intracellular chlamydiae become associated with EBs following lysis and exit from the host cell.
Surface localization of scFv-antibody-recognized antigens
Due to the nature of the selection procedures, elution of phage-displayed antibodies from EBs would be expected to generate probes for chlamydial surface components. Surface localization of the scFv-antibody-recognized antigens was tested using the dot-blot assay to detect chlamydial surface antigens as described by Zhang et al. (1987) . scFv antibody preparations were adjusted for approximately equal amounts of scFv antibody and used in the dot-blot assay probing purified C. trachomatis EBs. Although some of the scFv antibody reactivities were relatively weak, each of the scFv antibodies bound EBs by dot-blot (Fig. 3
). To ensure that EBs were intact during the dot-blot assay, EBs were subjected to lysis by SDS treatment then probed with the anti-E-tag antibody that is cross-reactive to an internal chlamydial protein as well as all scFv antibodies (data not shown). Following lysis of EBs, anti-E-tag antibodies showed increases in dot intensity, indicating that EB lysis occurred without overall loss of antigen quantity and that internal antigens became accessible to antibody binding by EB lysis (Fig. 3
). Additionally, scFv antibodies 3H1-B1, 3H2-D6 and E-A11 also showed an increased binding upon EB lysis, whereas the remaining scFv antibodies revealed dots of equal intensity regardless of whether the EBs were lysed or not (data not shown).
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Characterization of host-cell-reactive antigens recognized by scFv antibodies
scFv antibodies 3H1-D2 and 3H1-E10 recognized a 230 kDa antigen in Chlamydia purified from L929 cells and uninfected L929 cell lysates but this antigen was not readily detected in uninfected HeLa229 cell lysates nor in Chlamydia purified from HeLa229 cells (Fig. 4a
), suggesting that the molecule is from the host cell. While 3H1-E10 and 3H1-D2 had similar reactivity patterns, 3H1-D2 recognized an additional
42 kDa antigen (Fig. 1
). The
230 kDa antigen was visibly abundant in both C. trachomatis serovar L2 and C. psittaci EB lysates, yet was reproducibly less abundant in C. trachomatis serovar D lysates (Fig. 4a
). The
42 kDa antigen recognized by 3H1-D2 was evident in all strains of chlamydiae purified from both HeLa229 and L929 cells and was not observed in uninfected host cell lysates (data not shown). This suggests that the
42 kDa molecule is chlamydial in origin.
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Localization and identification of a host-cell-reactive antigen recognized by scFv antibodies
scFv antibodies 3H1-E10 and 3H1-F2 were used to probe uninfected host cells to localize the recognized antigen within the host. Both fixed and unfixed live cells were probed. 3H1-F2 detected an antigen in fixed L929 cells but not in unfixed cells (Fig. 5c, d
), suggesting that fixation was required to allow access of the scFv antibodies to an internal antigen following permeabilization of the host cell. In contrast, scFv antibody 3H1-E10, which did not recognize an antigen in HeLa229 cells, recognized an antigen in both fixed and unfixed L929 cells (Fig. 5a
, b
). It is likely that the antigen recognized by 3H1-E10 is surface localized, because scFv antibody binding to this antigen was not dependent on permeabilization of the cells by fixation. Similar results to those for 3H1-E10 were observed for 3H1-D2 (data not shown).
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DISCUSSION |
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Gough et al. (1999) attempted to select phage antibodies to Phytophthora infestans. It remained a question whether phage-display would be an effective tool to select a diverse population of phage antibodies to a complex antigen because they reported only selecting phage antibodies to a few different antigens despite panning and subpanning on two distinct antigen groups. Gough et al. (1999)
speculated that recovery of so few unique phage antibodies was due to a high concentration of a single antigen. It is possible that the diversity of selected phage antibodies obtained from Chlamydia is due to multiple factors. Some important differences could be the variation in the starting phage-display libraries, and differences in the complexity and/or stability of the surfaces of the complex antigens.
Of the fourteen scFv antibodies that bound Chlamydia-associated antigens, most reacted to proteins by immunoblot. Proteins of approximately 42, 55, 69, 71, 73, 75, 110, 165 and 230 kDa were identified. By the dot-blot assay each of these proteins was found to be surface-accessible on EBs to antibody binding. Almost all the proteins were sensitive to proteinase K. Four scFv antibodies (2H-G2, H-B6, H-B11 and 2H-B9) recognized a species-specific antigen by ELISA, immunoblot (69 kDa) and immunofluorescence. Because all four of these scFv antibodies have unique fingerprint patterns, their corresponding binding sites are different and each scFv antibody probably recognizes either a different epitope on the same antigen or, more unlikely, each scFv antibody recognizes a different antigen of similar molecular mass. scFv antibodies E-A11, E-B11 and H-E11 were genus specific by ELISA and immunoblot and recognized bands of dissimilar size in different strains of Chlamydia. This suggests that although epitopes are conserved across chlamydial species, there is at least size polymorphism for the analogues in C. psittaci. Given the high molecular mass, size polymorphism and surface localization of these antigens one could speculate that they may be members of the polymorphic membrane protein (Pmp) antigen family (Tanzer & Hatch, 2001
; Tanzer et al., 2001
; Grimwood & Stephens, 1999
; Grimwood et al., 2001
; Longbottom et al., 1998
). Likewise, scFv reactivity to a 42 kDa protein may represent recognition of a novel surface protein (protein 4) recently described by Tanzer & Hatch (2001)
. It was surprising that among the scFv antibodies that were reactive by immunoblot that these did not recognize the quantitatively predominant major outer-membrane protein (MOMP). This may be due to the dominance of conformation-dependent antigenicity of native MOMP (Wolf et al., 2001
; Fan & Stephens, 1997
) and the fact that we selected antibodies with a wide spectrum of reactive intensities in the initial screening process, not merely antibodies with the strongest reactivity.
Ten scFv antibodies were reactive to a genus-specific antigen by at least one of the immunoassays. One genus-specific chlamydial antigen, previously characterized using monoclonal antibodies, is lipopolysaccharide (LPS). LPS can typically be observed in immunblots and in Chlamydia-infected cells, where immunofluorescence staining shows a markedly diffuse pattern (Stephens et al., 1982 ). However, all but E-B11 and 3H1-D2 recognized antigens that were sensitive to proteinase K digestion, suggesting that these antibodies were not recognizing LPS but some other shared, perhaps protein molecule. Although E-B11 and 3H1-D2 recognized antigens that were resistant to proteinase K digestion their immunofluorescence staining pattern did not appear to be similar to the staining pattern observed for monoclonal antibodies to LPS, nor did the antigens resemble the immunoblot staining observed for LPS (Stephens et al., 1982
). Thus, phage-display technology allowed the identification of several previously undescribed surface molecules in C. trachomatis.
scFv antibodies 3H1-D2, 3H1-E10 and 3H1-A5, although selected from EBs and bound EBs by ELISA and dot-blot, did not react by immunofluorescence staining to chlamydial antigens within inclusions, and each reacted by immunoblot to murine L929 cell antigens in uninfected cells. Of these three scFv antibodies, only 3H1-A5 also strongly reacted to an antigen in HeLa229 cells, suggesting that its respective antigen is conserved among mammalian cell types. 3H1-D2 and 3H1-E10 both recognized a molecule of 230 kDa detected in L929 cells but not in HeLa229 cells. Additionally, the
230 kDa protein was apparently less abundant by immunoblots of serovar D EB lysates as compared to serovar L2 and C. psittaci EB lysates. Physical differences among chlamydial strains and species have been described previously (Söderlund & Kihlström, 1982
); thus, it is not surprising that there are physical differences between these strains detected by scFv antibody probes.
scFv antibodies with high specificity and affinity were selected to various molecular components associated with Chlamydia and proved useful in a variety of analytical methods including ELISA, immunofluorescence staining and immunoblotting. While the relatively limited numbers of antibodies characterized in this study provided unexpected diversity of reactivities, many more antibodies could be selected or tested using other criteria. Unequivocal identification of protein antigens recognized by scFv antibodies requires sequence identification. Biochemical approaches such as amino-terminal microsequencing are challenging for chlamydiae because of the limited quantities of organisms that can be grown in tissue culture. However, testing scFv antibody reactivity to expressed proteins for all open reading frames identified in the chlamydial genome should permit the determination of immune-specific reactivity and the identity of the reactive antigen. This approach using phage-display antibodies has expanded the repertoire of techniques useful in the identification of antigens associated with micro-organisms, especially including host antigens that would not be identified in studies based upon genome sequences or standard monoclonal antibody techniques.
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ACKNOWLEDGEMENTS |
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Received 19 July 2001;
revised 28 September 2001;
accepted 4 October 2001.