Analysis of the Humoral Immune Response toChlamydia Outer Membrane Protein 2

Chlamydial strains. C. pneumoniae VR-1310 was obtained from the American Type Culture Collection, whereas C. trachomatis L2/434/Bu and C. psittaci cal10 were received from The State Serum Institute in Copenhagen, Denmark.

Serum samples.Sera of group I (positive for both C. pneumoniae and C. trachomatis by microimmunofluorescence [MIF]) were selected from women who were culture positive for C. trachomatis. Group II consisted of sera from normal healthy women who had been screened for antichlamydial antibodies (measured by MIF) and found positive for C. trachomatis only. Sera from patients examined for C. pneumoniae infection or ornithosis where only anti-C. pneumoniae antibodies had been found constituted group III. This group also included 11 sera from apparently healthy donors (asymptomatic patients). Sera without antichlamydial antibodies (measured by MIF) at routine screening constituted the fourth group.

MIF.Prototype strains were grown in yolk sacs of embryonated hens’ eggs. Semipurified yolk sac material was treated with 0.1% formalin and then used as antigen. C. pneumoniaeIOL-207 and C. psittaci 6BC were used. A pool of C. trachomatis serovars D through K was also included. One small dot of each antigen in a group of three was placed on microscope slides. Twelve such antigen clusters in two rows were included on each slide. Different dilutions of patient serum were placed on each antigen cluster. Using a fluorescein-labelled anti-human immunoglobulin G (IgG) conjugate, it was possible to measure antibodies to the different species of Chlamydia. End-point titrations of reactive sera were performed and geometric mean titers were calculated for positive sera. The protocol has previously been described (22).

Sequence analysis.DNA and protein sequences were analyzed by using programs in the Wisconsin Genetic Computer Group sequence analysis software package (4). Chlamydia Omp2 sequences were obtained through the EMBL-GenBank-DDBJ sequence database and the SwissProt database.

Cloning of gene fragments.In order to generate gene fragments, oligonucleotides specific for selected regions of theomp2 genes were produced and used as primers for PCR (DNA-Technology, Aarhus, Denmark) (Table1). To facilitate subsequent cloning of amplified DNA, EcoRI and BamHI restriction endonuclease sites were introduced in primers. PCR withChlamydia template DNA was done as described by the manufacturer (Boehringer Mannheim GmbH, Mannheim, Germany). The PCR protocol used included 30 cycles (30 s at 94°C, 30 s at 47°C, and 60 s at 72°C). Products were directly ligated into pCR II vector, and recombinant DNA was transformed into INVαF′Escherichia coli as described (Invitrogen, San Diego, Calif.). Isolation of recombinant plasmid was done by alkaline lysis (25). Positive clones were obtained and control sequenced as described by Hattori and Sakaki (14), by using [α-³²P]dATP (Amersham International, Little Chalfont, Buckinghamshire, United Kingdom) and T7 DNA polymerase (Pharmacia, Uppsala, Sweden). To express recombinant proteins, omp2fragments were moved into the expression vector pEV40 (15, 25). This was performed by using vector and/or primer-defined restriction endonuclease sites (EcoRI and BamHI). Recombinant pEV40 vectors were electrotransformed into E. coli pl248. Plasmid preparation and restriction enzyme digestion were used to detect clones containing omp2 fragments.

Production and purification of fusion proteins.Generation of fusion proteins from recombinant pEV40-E. coli pl248 is controlled by a heat-labile promoter, and by induction at 42°C, fragments of Omp2 were produced. The pEV40 plasmid carries a nucleotide fragment encoding a hexamer histidine tag at the N-terminal end of the fusion protein, and by affinity chromatography under denaturing conditions using Ni²⁺-nitrilotriacetic acid resin fusion proteins were purified (Qiagen Inc., Valencia, Calif.). The purifications were done essentially as described by the manufacturer (Qiagen Inc.). Briefly, the recombinant culture was induced (3 h, 42°C), harvested by centrifugation (6,000 × g), and resuspended in a guanidine-hydrochloride buffer (6 M guanidine-HCl, 0.1 M NaH₂PO₄, 10 mM Tris, 14 mM β-mercaptoethanol [pH 8]). Upon centrifugation, the supernatant was loaded onto a column of Ni²⁺-nitrilotriacetic acid resin and washed, and by applying a linear pH gradient of urea buffer, the fusion protein was purified and eluted from the column (8 M urea, 0.1 M NaH₂PO₄, 10 mM Tris, 14 mM β-mercaptoethanol [pH 8 to pH 4.5]). The eluate was neutralized by a one-tenth volume of Tris buffer (1 M, pH 8). Samples of eluates were diluted in sodium dodecyl sulfate (SDS) buffer (125 mM Tris [pH 6.8], glycerol [10%, wt/vol], SDS [2.3%, wt/vol], β-mercaptoethanol [5%, wt/vol]), boiled, and analyzed by SDS-polyacrylamide gel electrophoresis and Coomassie blue staining.

ELISA.The antigenicities of purified fusion proteins were measured by ELISA by using the selected panel of sera. Prior to the determination of antigenicity, the optimal coating concentration of each protein was determined by using a monoclonal antibody directed against the 6-mer histidine fragments of the antigen. MaxiSorb microtiter plates (Nunc, Roskilde, Denmark) were then coated with 50 μl of recombinant protein per well (2 to 10 μg/ml), suspended in 50 mM carbonate buffer (pH 9.6), and incubated for 2 h. The plates were washed twice in phosphate-buffered saline (PBS) (pH 7.4) containing 0.05% Tween 20 (PBS–T) (Sigma, St. Louis, Mo.), and excess binding capacity was blocked by adding 200 μl of 20% fetal calf serum in PBS–T. In order to absorb any cross-reacting antibodies directed against fetal calf serum the patient sera were diluted in the blocking agent (1/200 and 1/1,000). Antigen-coated microtiter plates and preabsorbed sera were stored overnight (4°C). Plates were then washed four times in PBS–T, and diluted patient sera were added (50 μl/well). Following a 2-h incubation period, plates were washed again, four times in PBS–T. Antigen-directed Ig was detected by incubation with 50 μl of peroxidase-labelled goat anti-human Ig, diluted 1/3,000 in PBS–T (Bio-Rad, Richmond, Calif.). After 1 h of incubation, microtiter plates were washed four times in PBS–T followed by four times in PBS. Fifty microliters of tetramethylbenzidine (Sigma) was added as described by the manufacturer. The color development was terminated after 20 min by adding an equal amount of 1 M HCl. The optical density at 450 nm (OD₄₅₀) was measured on a Bio-Kinetics Reader by using the KC3 software program (Bio-Tek Instruments, Winooski, Vt.). All procedures were carried out at room temperature (20°C).

Accession numbers.EMBL-GenBank-DDBJ DNA sequences are m23001, m61116, and x53511 and SwissProt protein sequences are P23700 ,P23701 , P26758 , P18151 , P23603 , P21264 , and P18586 .