Chimeric Dr Fimbriae with a Herpes Simplex Virus Type 1 Epitope as a Model for a Recombinant Vaccine

Bacterial strains, plasmids, enzymes, and reagents.Overexpression was carried out in E. coli strain BL21(DE3), which does not express Lon and OmpT proteases (Novagen, Nottingham, United Kingdom). Restriction enzymes were purchased from BioLabs. The reagents for PCR were obtained from DNA-Gdańsk II s.c. (Gdańsk, Poland). Isopropyl-β-d-thiogalactopyranoside (IPTG), agarose, and other reagents were purchased from Sigma.

Bacterial cells were grown on solid medium (Luria agar [LA], containing, per liter, 5 g of yeast extract, 10 g of tryptone-pancreatic digest of casein, 10 g of sodium chloride, and 15 g of agar) or liquid broth (Luria broth [LB] medium, containing, per liter, 5 g of yeast extract, 10 g of tryptone-pancreatic digest of casein, and 10 g of sodium chloride) supplemented with the appropriate antibiotics (Sigma).

Plasmid pBJN406, containing an 11.4-kb HindIII-HindIII DNA fragment (previously constructed by B. Nowicki) (11, 12) carrying the whole dra operon, which is responsible for the uropathogenic properties of bacteria, was used for PCR amplification.

Plasmid pCC90, corresponding to the Dr hemagglutinin operon, which had its promoter region and its regulatory genes upstream of draB gene deleted, was from S. Moseley, University of Washington (Seattle). This plasmid was constructed in order to eliminate differences in protein expression among mutants as a result of methylation-dependent phase variation, which occurs in the promoter regions of adhesins (2, 21). In the first step, an EcoRI restriction site was inserted by site directed mutagenesis 45 bp upstream of the draB gene. The resulting 2.8-kb EcoRI fragment was deleted, and the EcoRI-HindIII fragment containing the truncated dra operon was cloned into the pACYC177 vector, allowing transcription of the dra gene cluster from the kanamycin resistance gene promoter. The plasmid carries the origin of replication from plasmid p15A, enabling it to coexist in cells with vectors, such as pBR322 and pUC19, that carry a ColE1 compatibility group origin.

Plasmid pCC90D54stop, which contains the dra gene cluster with a deletion of the region upstream of draB and a mutated draE gene (Dr-D54stop mutant; by site-directed mutagenesis, the GAC triplet of the draE gene encoding Asp-54 was replaced with a stop codon), was from S. Moseley.

Plasmid pET30 Ek/LIC, an expression vector with a strong T7 promoter, a kanamycin resistance gene, and a pBR322 origin of replication, was from Novagen.

Plasmid DNAs were isolated from E. coli cultures by using the Mini-prep Plus kit (A&A Biotechnology, Gdynia, Poland).

PCR amplification.The draE gene with its signal sequence, encoding bacterial DraE adhesin, was prepared by PCR amplification using the DNA of plasmid pBJN406 as a template. The primers enabled the introduction of a DNA sequence encoding an HSV-1 epitope into the draE gene. The draE gene with the HSV-1 epitope was amplified in two DNA fragments: DNA fragment I, encoding the N-terminal region of the DraE adhesin with the HSV-1 epitope, and DNA fragment II, encoding the C-terminal region of the DraE adhesin with the HSV-1 epitope. The sequences of the primers designed on the basis of the draE gene with its signal sequence (GenBank accession no. AF329316 ) are shown in Fig. 1B.

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FIG. 1.

(A) Construction strategy for a recombinant draE gene with inserted DNA encoding an antigenic sequence (for example, an HSV-1 gD epitope). DNA fragments encoding the N- and C-terminal regions of the DraE adhesin present in chimeric DraE-HSV protein are shown (boxes labeled draE-N and draE-C, respectively). Between them, the location of the DNA fragment (33 bp) of the draE gene deleted in chimeric draE-HSV (the site of HSV epitope sequence insertion) is indicated. The 5′ overhanging sequences of primers 1-DraE-HSV and 2-DraE-HSV are also indicated. The BamHI site is used to connect two amplification products (DNA fragments I and II) and to facilitate later manipulations and insertion of other heterologous peptides into the DraE adhesin. (B) Sequences of the PCR primers used to amplify the recombinant draE-HSV1 gene. Underlining indicates the recognition sequences for appropriate restriction endonucleases permitting cloning into a pET30LIC/Ek plasmid. Boldfaced portions of primer sequences are complementary to the nucleotide sequence of the 5′ or 3′ end of the draE gene. The 5′ overhanging sequences of the 2-DraE-HSV and 3-DraE-HSV primers encoding the 11-amino-acid HSV-1 gD epitope are italicized.

PCRs were performed on a Perkin-Elmer 2400 thermocycler. PCRs were carried out in 50-μl volumes containing 1 μl of DNA template (plasmid pBJN406; 0.25 μg/μl), 5 μl of 10× reaction buffer (100 mM Tris-HCl [pH 8.8], 20 mM MgCl₂, 500 mM KCl, 1% Triton X-100), 5 μl of deoxynucleoside triphosphates (10 mM), 2 μl of each primer (10 μM), and 1 U of hyperthermostable Pwo DNA polymerase (DNA Gdańsk II s.c.). Reactions were started with 300 s of denaturation at 94°C, followed by 10 cycles, each consisting of 30 s at 94°C, 40 s at 52°C, and 40 s at 72°C, and then by 25 cycles, each consisting of 30 s at 94°C, 40 s at 68°C, and 40 s at 72°C, with an additional extension step at 72°C (300 s). After amplification, 10-μl samples were subjected to electrophoresis in a standard 2% agarose gel to confirm the presence of the amplified products. The specific 192-bp DNA fragment I and 357-bp DNA fragment II were obtained.

Cloning.The amplified DNA fragment I (encoding the N-terminal part of the DraE adhesin with the HSV-1 epitope) and the amplified DNA fragment II (encoding the C-terminal part of the DraE adhesin with the HSV-1 epitope) were purified from an agarose gel band by using the DNA Gel-Out kit (A&A Biotechnology). DNA fragment I was digested with the KpnI and BamHI endonucleases (BioLabs), purified using the DNA Clean Up kit (A&A Biotechnology), and cloned directionally into KpnI and BamHI sites of the pET30LIC/Ek vector, producing recombinant plasmid pETDraE-HSV1 (the gene is out of frame in this construct). That plasmid was then digested with the NdeI endonuclease and ligated in the presence of the KpnI endonuclease to provide the gene in frame. The resulting recombinant plasmid encoding the N-terminal part of the DraE adhesin with the HSV-1 epitope was designated pDraE-HSV2. After that, the resulting plasmid was digested with the BamHI and HindIII endonucleases, permitting cloning of DNA fragment II, which was digested with the same restriction enzymes. The resulting recombinant plasmid encoding the whole draE gene with the HSV-1 epitope was designated pDraE-HSV3. The recombinant plasmids were selected by electrophoretic mobility analysis and confirmed by restriction analysis. The draE-HSV-1 gene of the pDraE-HSV3 plasmid was sequenced using the ABI Prism 377 automatic system, and the nucleotide sequence was analyzed using the GeneDoc and ClustalX computer programs.

Expression of the DraE adhesin with the HSV-1 epitope.The pDraE-HSV3 recombinant plasmid was transformed into E. coli BL21(DE3). One liter of LB medium supplemented with 20 μg of kanamycin/ml was inoculated with 20 ml of an overnight culture of E. coli strain BL21(DE3) containing the recombinant pDraE-HSV3 plasmid. The inoculated culture was grown with agitation in a 3-liter flask at 30°C to an optical density at 600 nm (OD₆₀₀) of 0.2, induced by addition of IPTG (final concentration, 0.1 mM), and grown as described above for an additional 6 h. Then the culture was harvested by 10 min of centrifugation at 6,000 × g and 4°C and was stored at −20°C for further use.

For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of proteins during cultivation, 1-ml samples were collected and the cells were harvested by centrifugation. Next, the pellets were resuspended in 100 μl of phosphate-buffered saline (PBS [pH 7.5], containing 80 mM sodium hydrogen orthophosphate, 20 mM sodium dihydrogen orthophosphate, and 100 mM sodium chloride). A 10-μl portion of each cell solution was then used directly for SDS-PAGE in 15% polyacrylamide gels stained with Coomassie brillant blue R-250 (Sigma).

Purification of chimeric Dr-HSV fimbriae and Dr fimbriae.Recombinant plasmid pDraE-HSV3 encoding the DraE-syg adhesin (with the signal sequence) with the HSV-1 gD epitope was introduced into the DraE⁻ mutant E. coli BL21(DE3)/pCC90D54stop, which contains the dra gene cluster with deletion of a region upstream of draB and with a mutated draE gene (Dr-D54stop mutant). The recombinant E. coli BL21(DE3)/pCC90D54stopE-HSV cells expressing chimeric Dr-HSV fimbriae were grown on LA plates with appropriate antibiotics (20 μg of kanamycin/ml and 100 μg of ampicillin/ml) and IPTG (final concentration, 6 mM) at 37°C for 24 h, harvested in PBS (15 ml for 10 150-mm-diameter plates), and heat shocked at 65°C for 1 h in a water bath. At the end of the incubation, the bacterial suspension was centrifuged at 6,000 × g for 20 min, and the supernatant was collected in fresh tubes. After that, a 40% solution of ammonium sulfate in PBS was added to a final concentration of 20% and mixed overnight at 4°C to precipitate the fimbriae. The next day, the suspension was centrifuged at 3,000 × g for 30 min at 4°C. The supernatant was poured out, and the pellet was redissolved in a 40% solution of ammonium sulfate and rotated overnight at 4°C. The precipitated protein (obtained after centrifugation) was resuspended in 2 ml of PBS. The suspension was poured into a dialysis bag (molecular weight cutoff, >100,000) and dialyzed overnight against PBS. The protein was then passed through a Sepharose 4B column (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, United Kingdom), where the Dr-HSV fimbriae were eluted in the void volume. The purity of the preparation was evaluated by SDS-PAGE in a 15% polyacrylamide gel stained with Coomassie brilliant blue R-250 (Sigma). The Coomassie brilliant blue R-250-stained polyacrylamide gel was photographed and scanned. The concentration of the protein was determined by densitometric analysis using the ScanPack (version 2.0) and BioDoc (version 1.0) programs (Biometra) with a low-molecular-weight marker (Amersham Pharmacia Biotech) as a standard.

Recombinant plasmid pCC90, which contains the dra gene cluster with a deletion of the region upstream of draB, was transformed into E. coli BL21(DE3). The recombinant E. coli BL21(DE3)/pCC90 cells were used for expression and purification of Dr fimbriae by the method described above for chimeric Dr-HSV fimbriae. The recombinant strain was used as a positive control. The recombinant strain E. coli BL21(DE3)pCC90D54stop, which does not express Dr fimbriae, was used as a negative control.

Hemagglutination assay.In order to determine whether purified Dr protein and DraE-HSV protein bind to DAF, 15 μl of each preparation (at a concentration of 1 μg/μl) was mixed with 15 μl of a 3% (vol/vol) solution of human erythrocytes in PBS with 2% α-methylmannose on glass slides. The mixture was rotated for 5 min, and agglutination was observed using a light microscope. Erythrocytes mixed with the Dr-D54 stop mutant were used as a negative control.

Antisera.Rabbit anti-Dr adhesin antibodies raised against purified native Dr fimbriae have been described previously (17). HSV-Tag mouse monoclonal immunoglobulin G1 (IgG1) antibodies with specificity and affinity for the 11-amino-acid peptide derived from HSV-1 gD were from Novagen. All other anti-IgG antibodies were against the whole molecule. Anti-rabbit IgG, anti-mouse IgG, and anti-human IgG antibodies conjugated to horseradish peroxidase were purchased from Sigma. Anti-rabbit IgG conjugated to tetramethylrhodamine isothiocyanate (TRITC) and anti-mouse IgG conjugated to fluorescein isothiocyanate (FITC) were purchased from Sigma. The sera obtained from women infected with HSV-1 were from the Clinical Biochemistry Department, Medical Academy of Gdańsk. These sera were obtained from patients who can serve as reservoirs of infection for years or decades after initial HSV-1 infection. Stress triggered a reactivation event where one or more latent-phase viral gene products could reestablish the productive replication cycle in the patients studied.

Western blot analysis.Samples were mixed with sample buffer, run in a 15% bisacrylamide gel containing SDS, and transferred to a nitrocellulose membrane in a transfer buffer (pH 8.3; 12.5 mM Tris, 96 mM glycine, 10% methanol) for 2 h by using a semidry blotting apparatus (0.5 V/cm² of gel). All subsequent steps were carried out in PBS containing 0.05% Tween 20 (PBS-T) at room temperature with gentle rocking. After transfer, the membrane was blocked with 3% nonfat milk in PBS-T buffer for at least 30 min and then incubated with rabbit anti-Dr adhesin antibodies at a 1:5,000 dilution or with mouse anti-HSV-Tag antibodies at a 1:2,500 dilution for 1 h. The nitrocellulose membrane was washed three times with PBS-T and then incubated with anti-rabbit IgG antibodies or anti-mouse IgG antibodies conjugated to horseradish peroxidase at a 1:1,000 dilution for 1 h. After three washes with PBS-T, protein bands were visualized by using a diaminobenzidine (DAB) substrate for horseradish peroxidase and by adding a 0.3% stock solution of nickel chloride in water. Six milligrams of DAB (DAB tetrahydrochloride) was dissolved in 9 ml of 0.05 M Tris buffer (pH 7.6). Then 1 ml of a 0.3% stock solution of nickel chloride in water and 0.1 ml of a 3% solution of H₂O₂ in H₂O were added to Tris buffer with DAB. The membrane was incubated in this solution for 1 to 20 min, and washing in water stopped the reaction. The reaction products were slate gray to black.

Immunofluorescence microscopy.Cells from cultures grown on L-agar plates at 37°C for 24 h were harvested and washed gently in PBS. Bacterial suspensions (100 μl; 10⁵ to 10⁶ cells/ml) were incubated with 50 μl of a 1:500 dilution (anti-Dr fimbriae) or a 1:250 dilution (anti-HSV-Tag) in PBS of the primary antibody at room temperature for 1 h. The reaction mixtures were then washed three times with PBS containing 10% glycerol and incubated with 50 μl of a 1:25 dilution (anti-rabbit IgG conjugated to TRITC) or a 1:50 dilution (anti-mouse IgG conjugated to FITC) in PBS of the secondary antibody at room temperature for 1 h. The reaction mixtures were then washed again three times with PBS containing 10% glycerol. Bacterial suspensions (10 μl) were loaded onto glass slides and observed with an immunofluorescence microscope (Olympus BX-60). A bacterial suspension of E. coli BL21(DE3)/pCC90D54stop was used as a negative control. A bacterial suspension of E. coli BL21(DE3)/pCC90 (expressing wild-type Dr fimbriae) was used as a positive control.

Electron microscopy.Cells of the E. coli strain BL21(DE3) carrying the recombinant plasmids under study were grown on LA plates. With a platinum loop, a small portion of cells was scraped off the plate and very gently suspended in a 10-μl drop of PBS buffer. Carbon-coated copper grids (300 mesh) were floated for 1 to 2 min on the cell suspensions and then washed twice by being floated on 50-μl drops of PBS buffer; after that, they were negatively stained with 1.5% potassium phosphotungstate for 90 s. The preparations were analyzed with a Philips CM 100 transmission electron microscope at 60 kV at a magnification of ×3,805.

Immunization with fimbriae.The purified fimbriae were used to immunize rabbits. One rabbit was immunized with purified chimeric (DraE-HSV) fimbriae, and one rabbit was immunized with wild-type Dr fimbriae (control). Two doses of a suspension of 0.1 ml of fimbriae (0.1 mg of fimbriae/ml) mixed with equal parts of Freund's incomplete adjuvant were administered subcutanenously at 2-week intervals. Blood was collected from animals before and 3, 6, and 9 weeks after immunization.

Construction of the recombinant plasmid pDraE-HSV3 encoding the DraE-syg adhesin with the HSV-1 epitope.The first step of our strategy was production of recombinant plasmid pDraE-HSV3 encoding the DraE adhesin (with its signal sequence) with an HSV-1 gD epitope derived from HSV-1. The sequence encoding the HSV-1 epitope consisting of 11 amino acids (QPELAPEDPED) was introduced into the draE gene in place of the N-terminal region of surface-exposed domain 2 (VAKTRGQLTDA), which is implicated in the DAF binding process. Sequence analysis and site-directed mutagenesis (4) located the surface-exposed domain 2. In the expression system described here, the draE gene (with its signal sequence) with the nucleotide sequence encoding the HSV-1 epitope was prepared by PCR amplification, with primers designed to clone the target gene into pET30LIC/Ek. The draE gene with the HSV-1 epitope was amplified by use of plasmid pBJN406 (harboring the dra gene cluster) in two DNA fragments: DNA fragment I, encoding the N-terminal region of the DraE adhesin with the HSV-1 epitope, and DNA fragment II, encoding the C-terminal region of the DraE adhesin with the HSV-1 epitope. The internal primers 2-DraE-HSV and 3-DraE-HSV enabled the introduction of the nucleotide sequence encoding the HSV-1 epitope into the draE gene with the signal sequence by PCR amplification (Fig. 1). The amplified draE-HSV gene was cloned in two DNA fragments into plasmid pET30LIC/Ek under the control of a strong T7 promoter (Novagen). The genetic construct obtained, harboring a single copy of the HSV-1 insert, retained the open reading frame, as confirmed by restriction and DNA sequence analyses. The chimeric fimbrial protein DraE-syg-HSV encoded by the recombinant plasmid has a molecular mass of 17,258 kDa and a calculated pI of 4.5. The nucleotide sequence encoding the cloned HSV-1 epitope contains a recognition sequence for BamHI that facilitates later manipulations and insertions of heterologous peptides into the DraE adhesin.

Expression of the DraE-HSV adhesin with and without the signal sequence. E. coli strain BL21(DE3) transformed with plasmid pDraE-HSV3 was grown at 30°C in 800 ml of LB medium containing 20 μg of kanamycin/ml to an OD₆₀₀ of 0.2. IPTG was then added to a final concentration of 0.1 mM. The culture was then cultivated further at 30°C. The cells were harvested after 6 h of cultivation. Optimization of the expression conditions revealed that a 6-h induction and growth at 30°C in LB medium led to substantial overproduction of the DraE-HSV adhesin with (17,258 kDa) and without (15,622 kDa) the signal sequence. Bands corresponding to a ∼17-kDa protein and a 15.5-kDa protein were observed in SDS-PAGE of crude extracts of E. coli BL21(DE3)/pDraE-HSV3 culture after IPTG induction (Fig. 2, lanes 2 to 6). These bands were absent in the control crude extract of E. coli BL21(DE3)/pET30LIC/Ek culture (Fig. 2, lane 1). Western blot analysis of E. coli BL21(DE3)/pDraE-HSV3 whole-cell lysates with anti-Dr (Fig. 3A) and anti-HSV (Fig. 3B) antibodies revealed the presence of chimeric proteins with and without the signal sequence, with the expected molecular masses.

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FIG. 2.

SDS-PAGE in a 15% polyacrylamide gel. The gel was stained with Coomassie brilliant blue R-250 (Sigma). Results of overexpression of the DraE-HSV adhesin with and without the signal sequence, encoded by plasmid pDraE-HSV3, are shown. Lane M, low-molecular-weight calibration kit (Amersham Pharmacia Biotech), with markers at 94.0, 67.0, 43.0, 20.1, and 14.4 kDa; lane 1, whole-cell lysate of E. coli BL21(DE3) harboring plasmid pET30LIC/Ek; lanes 2 to 6, whole-cell lysates of E. coli BL21(DE3) harboring plasmid pDraE-HSV3 obtained 2, 3, 4, 5, and 6 h after induction of expression with IPTG, respectively.

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FIG. 3.

Western blot analysis of overexpression of the DraE-HSV adhesin, with and without the signal sequence, encoded by plasmid pDraE-HSV3. (A) A rabbit anti-Dr (anti-fimbrial) serum was used as the primary serum, and a peroxidase-coupled anti-rabbit serum was employed as the secondary serum. (B) A mouse anti-HSV serum was used as the primary serum, and a peroxidase-coupled anti-mouse serum was employed as the secondary serum. Lanes M, low range color marker (Sigma) for 45.0, 29.0, 20.0, 14.2, and 6.5 kDa; lanes 1, whole-cell lysate of E. coli BL21(DE3) harboring plasmid pET30LIC/Ek; lanes 2 to 6, whole-cell lysate of E. coli BL21(DE3) harboring plasmid pDraE-HSV3 obtained 2, 3, 4, 5, and 6 h after induction of expression with IPTG, respectively.

Construction of two-plasmid expression system of chimeric Dr-HSV fimbriae (pDraE-HSV3, encoding the DraE-syg-HSV protein, and pCC90D54stop, containing the dra operon with a mutated draE gene).To create a model expression system for chimeric Dr fimbriae with a gD epitope derived from HSV-1, recombinant plasmid pDraE-HSV3 was introduced into a DraE⁻ mutant [E. coli BL21(DE3)/pCC90D54stop] that contained the dra gene cluster with deletion of a region upstream of draB and with a mutated draE gene (Dr-D54stop mutant). When both plasmids pDraE-HSV3 and pCC90D54stop were present in a given strain, complementation occurred: the recombinant E. coli BL21(DE3)/pDraE-HSV3/pCC90D54stop strain showed expression of chimeric Dr-HSV fimbriae that in quality and quantity were similar to those produced by E. coli BL21(DE3)/pCC90 containing the dra gene cluster with deletion of a region upstream of draB (as described below).

Expression of chimeric Dr-HSV fimbriae and immunological detection of the HSV-1 epitope inserted into the DraE adhesin.Expression of chimeric Dr-HSV fimbriae and wild type Dr fimbriae was performed as described in Materials and Methods. We obtained about 2 mg each of the DraE-HSV and DraE proteins. The products were >90% pure. The concentrations of the proteins were determined by densitometric analysis using the ScanPack (version 2.0) and BioDoc (version 1.0) programs (Biometra) with a low-molecular-weight marker (Amersham Pharmacia Biotech) as a standard.

The ability of E. coli strain BL21(DE3) containing plasmids pDraE-HSV3 and pCC90D54stop to express chimeric Dr-HSV fimbriae was assayed by immunoblotting, hemagglutination, immunofluorescence microscopy, and electron microscopy. The E. coli strain expressing wild type Dr fimbriae was used as a positive control in the experiments. As a negative control we used the E. coli strain containing the dra gene cluster with a mutated draE gene. In Western blots the purified fimbria-specific rabbit anti-Dr antibody (Fig. 4A), the mouse anti-HSV antibody (Fig. 4B), and sera obtained from women infected with HSV-1 (see Fig. 8) recognized chimeric DraE-HSV proteins without signal sequences. Immunofluorescence staining (anti-Dr-anti-TRITC and anti-HSV-anti-FITC staining) of E. coli cells expressing chimeric Dr-HSV fimbriae showed that the specific antibodies recognized chimeric DraE-HSV protein (Fig. 5C and E). This experiment proved that the foreign HSV-1 epitope was exposed on the surfaces of the host cells. The surface localization and organelle assembly of the chimeric DraE-HSV adhesin were investigated by electron microscopy (Fig. 6). The results obtained revealed that chimeric DraE-HSV proteins were assembled into fimbrial organelles and that the shape and structure of these fimbriae were the same as those of native DraE fimbriae (Fig. 6; compare panels A and B). The microscopic data proved that the subunit proteins produced in the cytoplasm as precursors with the N-terminal signal sequence were secreted into the periplasma and then exported across the inner membrane on the surfaces of bacterial cells. Electron microscopy for E. coli BL21(DE3)/pCC90D54stop cells (negative control) showed bacterial cell surfaces without any fimbriae (data not shown).

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FIG. 4.

Production and purification of fimbriae from recombinant strains assayed by Western blotting. (A) A rabbit anti-Dr (anti-fimbrial) serum was used as the primary serum, and a peroxidase-coupled anti-rabbit serum was employed as the secondary serum. (B) A mouse anti-HSV serum was used as the primary serum, and a peroxidase-coupled anti-mouse serum was employed as the secondary serum. Purified fimbrial proteins were prepared as described in the text. Lanes M, low-range color marker (Sigma) for 45.0, 29.0, 20.0, 14.2, and 6.5 kDa; lanes 1, elution fraction of chimeric Dr-HSV fimbriae (collected from the Sepharose 4B column); lanes 2, elution fraction of wild-type Dr fimbriae (collected from the Sepharose 4B column); lanes 3 to 5, dialysis fractions of purified chimeric Dr-HSV fimbriae after induction with 3, 4, and 6 mM IPTG, respectively; lanes 6, dialysis fraction of purified wild-type Dr fimbriae; lanes 7, whole-cell lysate of the E. coli BL21(DE3) host harboring plasmid pCC90D54stop (carrying the dra operon with a deleted region upstream of draB and a mutated draE gene).

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FIG. 5.

(A, C, E, and G) Immunofluorescence microscopy of E. coli BL21(DE3)/pCC90 cells incubated with a rabbit anti-Dr serum and an anti-rabbit serum conjugated with TRITC (positive control) (A), E. coli BL21(DE3)/pCC90D54stopE-HSV cells incubated with a mouse anti-HSV serum and an anti-mouse serum conjugated with FITC (C), E. coli BL21(DE3)/pCC90D54stopE-HSV cells incubated with a rabbit anti-Dr serum and an anti-rabbit serum conjugated with TRITC (E), and E. coli BL21(DE3)/pCC90D54stop cells incubated both with a rabbit anti-Dr serum and an anti-rabbit serum conjugated with TRITC and with a mouse anti-HSV serum and an anti-mouse serum conjugated with FITC (negative control) (G). (B, D, F, and H) Phase-contrast micrographs of the bacterial cells shown in panels A, C, E, and G, respectively.

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FIG. 6.

Electron micrographs of negatively stained preparations of E. coli BL21(DE3) cells harboring plasmid pCC90 (encoding the dra operon with a deleted region upstream of draB) (A) and E. coli BL21(DE3) cells harboring plasmid pCC90D54stop (encoding the dra operon with a deleted region upstream of draB and a mutated draE gene) and plasmid pDraE-HSV3 (encoding the DraE-HSV adhesin) (B). Magnification, ×3,805.

Hemagglutination assay.We mixed equal volumes of purified chimeric Dr-HSV fimbriae or wild type Dr fimbriae (as a positive control) isolated from the recombinant bacterial strains and the erythrocyte suspension to determine hemagglutination ability in the presence of d-mannose. This experiment showed that the purified DraE adhesin gave a positive hemagglutination reaction. Introduction of the HSV-1 epitope into the DraE adhesin in place of the N-terminal region of surface-exposed domain 2 abolished the hemagglutination capacity of the DraE-HSV fimbrial protein.

Immunogenicity of chimeric Dr-HSV fimbriae.Chimeric Dr-HSV fimbriae purified from the bacterial host containing plasmids pDraE-HSV3 and pCC90D54stop were used to immunize a rabbit. Another rabbit was immunized with wild type Dr fimbriae as a control. The serum from the rabbit immunized with the chimeric fimbriae was able to recognize the HSV-1 epitope in chimeric fimbriae (DraE-HSV protein) and in a 31.1-kDa target HSV-1 protein (Novagen) by use of an immunoblotting technique, but the serum obtained from the control rabbit immunized with the wild type fimbriae was not able to recognize the HSV-1 epitope (Fig. 7).

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FIG. 7.

Immunoblots showing the immune responses of rabbits immunized with wild-type purified Dr fimbriae (serum dilution, 1:5,000) (A) and purified chimeric Dr-HSV fimbriae (serum dilution, 1:500) (B). Lanes M, low-range color marker (Sigma) for 45.0, 29.0, 20.0, 14.2, and 6.5 kDa; lanes 1, dialysis fraction of purified wild-type Dr fimbriae; lanes 2, dialysis fraction of purified chimeric Dr-HSV fimbriae after induction with 6 mM IPTG; lane 3, HSV-Tag extract containing a 31.1-kDa target protein (Novagen), used as a positive control.