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The mucosae serve as portals of entry for many pathogens. Because of our growing understanding of pathogenic mechanisms and host-pathogen relationships, there is increased interest in stimulating mucosal immunity as a first line of defense against colonization and establishment of disease. In order to render potential vaccine antigens immunogenic, a variety of approaches have been taken to stimulate effective mucosal immunity. These approaches include mucosal adjuvants and nonliving and live delivery systems (7, 12, 18). Avirulent Salmonella enterica serovar Typhimurium expressing foreign gene products has been used as a delivery system for a number of vaccine antigens (4). Live, avirulent Salmonella induces a diverse response including both mucosal and systemic immunity. One of the historical problems with mucosal responses to oral vaccines has been the lack of long-term mucosal memory.

The hagB gene codes for a hemagglutinin from the periodontopathogen Porphyromonas gingivalis and is a potential virulence factor (15, 19). We have previously shown that mice immunized intragastrically with Salmonella serovar Typhimurium expressing the hagB gene exhibit a vigorous serum immunoglobulin G (IgG) and IgA response to purified, recombinant HagB as well as a significant mucosal IgA response in saliva, gut secretions, and vaginal washes (5). The primary response peaks around 5 or 6 weeks after primary immunization. When mice are boosted at 14 weeks, a more rapid and intense recall response in serum and secretions is seen (16). The objectives of this study were to examine the Salmonella delivery system in terms of the duration of the immune response and to determine the long-term ability to mount a systemic and mucosal recall response.

Bacterial strains, plasmids, media, and culture conditions. Salmonella serovar Typhimurium 4072, an SR-11 derivative (pStSR100 gyrA1816 cya-1 crp-1 asdA1 [zhf-4::Tn10]), and plasmid pYA292 (10) were provided by Roy Curtiss III (Washington University, St. Louis, Mo.). The vaccine strain 4072/pDMD1 was constructed by electroporation with plasmid pDMD1, which expresses the hagB gene of P. gingivalis, as previously described (5). Strains were routinely grown at 37°C in Luria-Bertani (LB) medium (23). Cultures were maintained at 80°C as glycerol stocks.

Purification of HagB. Histidine-tagged HagB was purified using the QIA Express system (Qiagen Inc., Valencia, Calif.). A Tru9I-XbaI fragment of a hagB clone (carried on p18AX1) was subcloned into the expression vector pQE31. The recombinant plasmid was designated pQE31-TX1. Positive subclones were selected on colony blots by using absorbed antiserum to HagB (6). Cultures (500 ml) were grown with aeration at 37°C in LB broth to an A₆₀₀ of 0.8 and then induced with 1 mM isopropyl -D-thiogalactoside for 5 h. The cells were lysed for 1.5 h at room temperature in 6 M guanidine-HCl-0.1 M NaH₂PO₄-0.01 M Tris (pH 8.0) (buffer A). The supernatant was mixed with 8 ml of Ni-nitrilotriacetic acid resin for 1.5 h. The resin was loaded into a 1.6-cm-diameter column and washed with 10 column volumes of buffer A, followed by 5 column volumes of 8 M urea-0.1 M NaH₂PO₄-0.01 M Tris (pH 8.0) (buffer B). The column was then washed with buffer B adjusted to pH 6.3 until the A₂₈₀ was <0.01. Attempts to refold eluted HagB by gradual dialysis were unsuccessful and resulted in precipitation of the protein. Refolding was accomplished while HagB was bound to the column. The column was equilibrated with refolding buffer (0.5 M NaCl, 10 mM Tris, 20% glycerol [pH 7.4]) containing 6 M urea. The column was then washed with a linear gradient of 6 M to 0 M urea at a flow rate of 12 ml/h over a period of 1.5 h. The histidine-tagged HagB was eluted with 250 mM imidazole and dialyzed against phosphate-buffered saline. The purified HagB appeared as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reacted with HagB antiserum on Western blots (not shown). Routine yields were 8 to 10 mg per 500-ml culture.

Mouse immunization and sample collection. Female BALB/c, VAF/Plus mice, 6 to 8 weeks of age (Charles River, Wilmington, Mass.), were housed in the Infectious Disease Isolation Unit at the University of Florida Animal Resource Center and given food and water ad libitum. Groups of six mice were immunized with Salmonella serovar Typhimurium strain 4072/pDMD1. The strain was grown as a static culture in LB broth overnight at 37°C, diluted 1/20 in fresh LB broth, grown for ca. 4 h at 37°C to an optical density at 600 nm of 0.8, after which the culture was centrifuged and resuspended in sterile 0.1 M NaHCO₃ to a density of 10¹⁰ CFU/ml. The food supply was removed and the bedding was changed 4 h prior to immunization. Mice were immunized by gastric intubation with 10⁹ cells (0.1 ml of 10¹⁰ cells/ml) in three doses on days 1, 3, and 5 of week 0. Boosting was carried out in the same manner.

Immunoassay methods. Samples were assayed for IgG and IgA antibody to HagB on microwell plates as described previously (5) using an enzyme-linked immunosorbent assay coated with purified HagB protein. The salivary IgA anti-HagB antibody levels were normalized to amylase activity levels, and the antibody levels in vaginal washes were normalized to the total IgA to account for variable dilution encountered in secretions. The amylase activity was determined using a colorimetric enzyme assay (3).

Anti-HagB responses in serum. Mice immunized at week 0 and week 52 (Fig. 1) showed a low but measurable residual serum IgG response at week 51, just prior to boost, and a recall response at weeks 55, 57, and 59. Mice in group II, which were also boosted at week 14, showed a strong IgG recall response after the first boost and recall responses of up to ca. 1,000 ng/ml following the boost at week 52. Even though they did not exceed the peak responses seen at the earlier boost at week 14, the levels were higher than the week 6 levels and much higher than the 1-year recall levels seen in group I mice. With serum IgA (Fig. 2), antibody levels in group I mice following the boost at week 52 were higher than those for IgG. Much higher recall levels were seen in mice of group II than in mice of group I, although in this case the recall levels were comparable to those measured following the boost at week 14. Detectable levels of anti-HagB were measured at week 51 prior to boost in both groups, and they were low compared to the recall levels.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Serum IgG anti-HagB levels following oral immunization and boosting (arrows) with Salmonella serovar Typhimurium 4072/pDMD1. Error bars, standard errors of the means.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Serum IgA anti-HagB levels following oral immunization and boosting (arrows) with Salmonella serovar Typhimurium 4072/pDMD1. Error bars, standard errors of the means.

Anti-HagB responses in secretions. Recall responses in vaginal washes of single-immunized mice (Fig. 3) were comparable to primary responses, while in mice immunized both at week 0 and week 14 (group II), there were significant residual levels at week 51 as well as higher recall responses following boosting at week 52. 

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Vaginal wash IgA anti-HagB levels following oral immunization and boosting (arrows) with Salmonella serovar Typhimurium 4072/pDMD1. Error bars, standard errors of the means.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   Salivary IgA anti-HagB levels following oral immunization and boosting (arrows) with Salmonella serovar Typhimurium 4072/pDMD1. Error bars, standard errors of the means.