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Infection and Immunity, November 2005, p. 7274-7280, Vol. 73, No. 11
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.11.7274-7280.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Partially Assembled K99 Fimbriae Are Required for Protection

Miguel A. Ascón,2 Javier Ochoa-Repáraz,1 Nancy Walters,1 and David W. Pascual1*

Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717-3610,1 Division of Infectious Diseases, Department of Medicine, Johns Hopkins University, Baltimore, Maryland 212052

Received 7 June 2005/ Returned for modification 11 July 2005/ Accepted 28 July 2005


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ABSTRACT
 
Antibodies to K99 fimbriae afford protection to F5+ bovine enterotoxigenic Escherichia coli (ETEC). Previous studies show that murine dams immunized with Salmonella vaccine vectors stably expressing K99 fimbriae confer protection to ETEC-challenged neonatal pups. To begin to address adaptation of the K99 scaffold to display heterologous B- and T-cell epitopes, studies were conducted to determine how much of the assembled K99 fimbria is required to maintain protective immunity. Sequential deletions in the K99 gene clusters were made, resulting in diminished localization of the K99 fimbrial subunit in the outer membrane. As placement of the K99 fimbrial subunit became progressively contained within the vaccine vector, diminished immunoglobulin A (IgA) and IgG1 antibody titers, as well as diminished Th2-type cytokine responses, were observed in orally immunized mice. Deletion of fanGH, which greatly reduced the export of the fimbrial subunit to the outer membrane, showed only partial reduction in protective immunity. By contrast, deletion of fanDEFGH, which also reduced the export of the fimbrial subunit to the outer membrane but retained more subunit in the cytoplasm, resulted in protective immunity being dramatically reduced. Thus, these studies showed that retention of K99 fimbrial subunit as native fimbriae or with the deletion of fanGH is sufficient to confer protection.


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INTRODUCTION
 
Enterotoxigenic Escherichia coli (ETEC) continues to be problematic for both humans and livestock. This disease primarily affects adult travelers to areas where it is endemic (9, 22) and particularly children under 3 years of age living in these regions (3). Livestock-specific ETEC strains afflict newborn calves and piglets, which may lead to mortality (31, 34). E. coli becomes virulent upon the acquisition of plasmids encoding genes for adhesins and enterotoxins, including heat-stable and heat-labile enterotoxins responsible for the induced diarrhea (5, 31, 46). The adhesins impart host specificities (27, 30), while antibodies to these adhesins confer strain-specific protection against ETEC infections in humans and livestock (13, 27, 31, 33, 41).

In attempts to effect protective immunity against ETEC, oral immunizations with purified fimbriae have resulted in poor induction in human volunteers' secretory immunoglobulin A and serum IgG responses (13, 28). This loss of protective value by the purified fimbriae is believed to be, in part, due to gastric proteases, even at neutral pH (28, 43). To protect against the antigen-altering properties of the gastrointestinal tract, purified fimbriae are microencapsulated for intragastric immunization of rabbits (12), but only minimal serum and secretory IgA responses are induced. However, direct immunization of microencapsulated fimbria into rabbit duodenum does improve fimbria-specific Peyer's patch and splenic B-cell responses (40). These studies indicate that ETEC fimbriae must be sufficiently protected to withstand the host's gastrointestinal tract. To deliver fimbriae for effective immunization, we adapted an attenuated Salmonella vaccine vector to express ETEC fimbriae (1, 38, 54).

In past studies, Salmonella vaccine vectors have proven to be versatile for expressing passenger antigens (4, 8, 51), adept at delivering these antigens to mucosal inductive sites (52), and effective for inducing broad-based immune responses to recombinant and Salmonella antigens (48, 50). Typically, Salmonella vaccine vectors stimulate T helper (Th) 1-type responses, while they are thought less effective for inducing Th2-type responses (37, 51, 56). In our own studies, we demonstrated that the Salmonella enterica serovar Typhimurium vectors expressing ETEC fimbriae provide an effective means of inducing elevated mucosal and systemic immune responses against wild-type ETEC (1, 38, 54).

Evidence from oral immunization studies of mice with Salmonella vaccines expressing either human ETEC colonization factor antigen I (CFA/I) (38, 39, 54) or bovine ETEC K99 (1) fimbriae have shown enhanced production of mucosal IgA and serum IgG1 fimbria-specific responses. This enhanced humoral immunity conferred by these Salmonella vaccines suggests that Salmonella vectors, mimicking the natural expression of ETEC fimbriae, offer a mode of immunization favorable for protective immunity. To enable eventual development of a single dose, multivalent vaccine for livestock diseases, we questioned whether fully assembled fimbriae are required for immune protection. Because of the size of the K99 gene clusters, it would be desirable to determine which genes are essential to maintain K99 immunogenicity. Such reduction would then allow the genetic incorporation of T- and possibly B-cell epitopes from other infectious agents. To forward this effort, we showed that as the K99 fimbrial subunit loses its ability to be transported to the outer membrane by the Salmonella vaccine vector, the protective value of the fimbria diminishes. Consequently, the loss of fanGH may facilitate such genetic modification of fanC.


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MATERIALS AND METHODS
 
Plasmids and bacteria constructs. The original bacteria, media, and culture conditions were previously described (1). The pMAK99-asd+ plasmid was constructed by subcloning the eight K99 genes, fanABCDEFGH, as a 7-kb BamHI fragment into plasmid pYA292 that contains the asd gene (1). The other recombinant plasmids were constructed considering the following eight K99 genes' characteristics (22, 26): (i) K99 genes are highly regulated by three separate gene clusters: region I, containing fanA to fanD; region II, containing fanE and fanF; and region III containing fanG and fanH, and (ii) each region is regulated independently by the global regulators: cyclic AMP, AMP receptor protein, and Lrp (leucine-responsive protein), indicating that each region is expressed independently from a different promoter.

The pMAK99.1-asd+ recombinant plasmid was constructed by deleting the 0.9-kb BsgI DNA fragment, which encodes for minor component proteins (FanG and FanH) of K99 fimbriae present along the shaft of K99 fimbriae structures. The pMAK99.2-asd+ recombinant plasmid was constructed deleting the 4.2-kb XmaI DNA fragment, which encodes for FanG and FanH, the outer membrane protein (FanD) involved in export and assembly of fimbriae component, a chaperon protein (FanE) that carries fimbria components from cytoplasm to outer membrane, and a minor component protein (FanF) of K99 fimbriae present at the top and along the shaft of the K99 fimbriae structure (22, 26). Each ligated plasmid construct was electroporated into E. coli H681 {Delta}asd strains. Clones containing the selected plasmid constructs were detected and screened by colony immunoblotting, in vivo antigen/antibody slide agglutination, and plasmid miniprep-restriction map protocols. The purified plasmids obtained were then electroporated into Salmonella enterica serovar Typhimurium H683 {Delta}aroA {Delta}asd strain (1, 17) to obtain the desired balanced lethal Salmonella vaccine constructs. All strains were cultured by using both Luria-Bertani (LB) and Minca media without antibiotics or diaminopimelic acid, as previously described (1). Neither H681 nor H683 grows on these media unless the asd+ allele is supplied in trans. The Salmonella control strain H647 was grown in media without diaminopimelic acid supplementation.

Electron microscopy. The fimbrial antigen of Salmonella construct AP112 (fanABCDEFGH) was immunostained using a nonembedding technique. Briefly, carbon-Formvar-coated 200-mesh copper grids were placed on 20-ml droplets of the purified Salmonella suspension and incubated for 3 min at room temperature. Grids were then floated on a 60-µl droplet of phosphate-buffered saline (PBS) and 1% bovine serum albumin solution for 3 min to block nonspecific binding. After washing twice on 60-ml droplets of PBS, grids were placed on 30-µl droplets of 1:50 dilution of rabbit anti-K99 antibody in PBS (prepared in-house against total recombinant E. coli K99 fimbriae, as previously described [1]) for 15 min at 37°C, then washed three times in PBS, and placed on 30 µl droplets of goat anti-rabbit IgG conjugated to 10-nm gold particles (Vector Labs, Inc., Burlingame, CA) diluted in PBS for 20 min at 37°C. Grids were finally washed three times in PBS and distilled water, air dried, and observed in a JEOL 100X transmission electron microscope (TEM).

The K99 antigen expressed by Salmonella constructs AP114 and AP116 was immunostained using the postembedding technique. Briefly, overnight cell cultures of AP114 (fanABCDEF) and AP116 (fanABC) strains were fixed in the growth medium by adding an equal volume of Timm's fixative solution (0.5% glutaraldehyde, 3% paraformaldehyde in 0.1 M Na cacodylate, pH 7.2), and incubated for 30 min at room temperature. The pelleted cells were resuspended in fresh fixative, and fixation was continued overnight. Fixed cells were rinsed four times in cacodylate buffer and dehydrated in acetone concentration series of 50 to 95% (vol/vol). Then cells were embedded in LR White resin and polymerized at 50°C for 2 days. Immunolabeling was performed on ultrathin sections (cut from blocks on a Reichert Jung Ultracut) mounted on carbon-Formvar-coated 200-mesh copper grids, as described above in the nonembedding technique. Finally, sections were counterstained in uranyl acetate and lead citrate for examination in the TEM.

Membrane fractionation for detection of K99 fimbrial subunit. Strains AP112, AP114 and AP116 were cultured in Minca medium (1) for 24 h at 37°C to similar cell densities. Bacterial fractions enriched in K99 fimbria were obtained by sequential detergent extraction of cell envelopes (2, 15). Briefly, after disruption of cells using a French Press (Aminco-SLM Instruments Inc., Urbana, IL) and centrifuged (20,000 x g, 30 min), the soluble fractions contained the cytoplasmic proteins, and the cell pellets contained both the inner and outer membrane fractions. To obtain the inner membrane fractions, the cell pellets were solubilized with 1% Sarkosyl (Sigma-Aldrich Chemical Co., St. Louis, MO) and centrifuged (20,000 x g; 30 min). The soluble fraction contained the inner membrane fraction, and the remaining membrane pellets were suspended in 0.5 M Tris-HCl (pH 6.8) with 10% sodium dodecyl sulfate (SDS) and centrifuged (20,000 x g; 30 min). The outer membranes from the different Salmonella strains were present in the supernatant fraction. To obtain the periplasmic fractions of these Salmonella vaccines, a commercial extraction procedure (PeriPrep periplasting kit, Epicenter Biotechnologies, Madison, WI) was adapted (14).

To determine which cellular fractions contained the K99 fimbrial subunit, the enriched cytoplasmic, inner membrane, periplasmic, and outer membrane fractions were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot analysis. Samples were pretreated with formic acid (90%) for a few seconds to avoid the aggregation of its monomers similar to that previously described (6). After acid exposure, the extracts were dialyzed against water and concentrated. Following 15% SDS-PAGE, the separated proteins were transferred to a 0.2 µm pore-size nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was incubated with 1:1,000 dilution of rabbit polyclonal anti-K99 antibody (made in-house) overnight at 4°C. After four washes in PBS-Tween 20, a second incubation with 1:1000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Southern Biotechnology Associates, Birmingham, AL) in PBS-Tween 20 was done. Detection of K99 fimbrial subunit was achieved upon development with the horseradish peroxidase substrate 4-chloro-1-naphthol chromogen and H2O2 (Sigma-Aldrich Chemical Co.). The apparent molecular weights were determined by comparing electrophoretic mobilities with those of molecular mass weight standards (Kaleidoscope prestained standards, Bio-Rad Laboratories).

Quantification of K99 subunit by each Salmonella construct. To measure levels of K99 fimbrial subunit produced by strains AP112, AP114, and AP116, each was grown for 2 days on Minca agar plates for 48 h at 37°C; bacteria were harvested in PBS and centrifuged at 10,000 x g for 5 min; and pellets were resuspended in 300 ml of B-Per reagent (Pierce Chemical Co.; Rockford IL) with Protease Inhibitor Cocktail (Sigma-Aldrich Chemical Co.) and then vortexed for 1 min. The lysed cells were centrifuged at 13,000 rpm for 5 min., and the supernatant was collected. Protein concentrations were determined using the BCA Protein Assay (Pierce), and protein concentrations were adjusted to equivalence. Microtiter wells (Maxi-sorp; NUNC, Roskilde, Denmark) were coated with 1.0 µg/ml of monoclonal antibody to K99 fimbrial subunit (clone M2110124; Fitzgerald Industries International, Inc., Concord, MA) overnight at room temperature. After blocking with PBS and 1% bovine serum albumin, various dilutions of extracts from strains AP112, AP114, and AP116 were incubated 3 h at 37°C. After washing, 1:1,000 rabbit anti-K99 fimbria serum (produced in-house) was added and allowed to incubate for 90 min at 37°C. After washing, 1:1,000 horseradish peroxidase-conjugated AffiniPure donkey anti-rabbit IgG (heavy and light chains) (Jackson ImmunoResearch Laboratories, Inc.; West Grove, PA) was added and allowed to incubate for 90 min at 37°C. After washing, specific reactivity was developed with ABTS [azinobis(ethylbenzthiazolinesulfonic acid] substrate (Moss, Inc.; Pasadena, MD), and microtiter wells were read at an optical density of 415 nm (OD415), and values were extrapolated to a standard curve developed using native K99 fimbriae.

Oral immunization with Salmonella constructs. Isolated colonies of either Salmonella vaccine AP112, AP114, or AP116 were grown on Minca agar plates for 24 h at 37°C; bacteria were harvested in PBS and centrifuged at 10,000 x g for 5 min; and pellets were resuspended in 1.0 ml of PBS. The density of bacteria was adjusted by optical density measurement at 600 nm and confirmed by serial dilutions on LB agar plates. Groups of CD-1 and BALB/c mice, pretreated with a 50% saturated sodium bicarbonate solution, received a single oral dose of 5 x 109 CFU (contained in 0.2 ml) of Salmonella vaccines.

Antibody titers. Serum, colostrum, and fecal K99-specific endpoint titers were determined by an enzyme-linked immunosorbent assay (ELISA) using purified K99 fimbriae antigen, as previously described (1). Endpoint titers were expressed as the reciprocal dilution of the last sample dilution giving an absorbance ≥0.1 optical density) units above the OD415 of negative controls after 1 h incubation. To obtain colostrum, dams were anesthetized with sodium pentobarbital followed by subcutaneous injection of 6.25 U/kg oxytocin (VEDCO, Inc., St. Joseph, MO). Mammary glands were warmed by the application of soaked gauze in water. Under a slight vacuum, collection of colostrum was done at one second intervals for each teat. Dams were subsequently reunited with their respective litters.

T-cell ELISPOT. Lymphocytes from Peyer's patches and spleen were isolated by conventional methods, as previously described (39). Greater than 95% viability was noted for isolated lymphocytes, as determined by trypan blue exclusion. Lymphocytes were resuspended in complete medium (RPMI 1640, Bio-Whittaker, Walkersville, MD) containing 10% fetal calf serum (HyClone, Logan UT), plus the supplements (Life Technologies, Grand Island, NY), HEPES buffer (10 mM), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml). Subsequently, gamma interferon (IFN-{gamma})-, interleukin- (IL-)4-, IL-5-, IL-6-, and IL-10-specific cytokine-forming cells (CFC) were enumerated in cell suspensions by cytokine specific ELISPOT assays, as previously described (38, 39).

Neonatal challenge. CD-1 dams were orally immunized with Salmonella vaccine construct AP112, AP114, AP116, or H647, as described above. After 3 weeks, mice were mated, allowed to come to parturition, and pups were allowed to receive colostrum and milk for the first 24 h. Pups were then challenged with 103 bovine ETEC B41 bacteria (1, 10, 11) and monitored for survival for 10 days.

Statistics. The Kaplan-Meier method (GraphPad Prism, GraphPad Software, Inc., San Diego, CA) was applied to obtain the survival fractions following infection with a lethal dose of wild-type bovine ETEC. Using the Mantel-Haenszel log rank test, the P value for statistical differences between surviving neonates derived from dams orally vaccinated with the Salmonella vector and the various Salmonella-K99 vaccines were discerned at the 95% confidence interval. All other data were analyzed by Student t test, and significant values were recorded at P < 0.05.


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RESULTS
 
Deletions in the K99 gene cluster influences localization and production of the fimbrial subunit. Since expression of ETEC fimbriae produced enhanced mucosal and systemic antibody responses (1, 38, 54), we questioned whether retention of fimbrial antigens in various compartments within the Salmonella vaccine vector would impact protective immunity. Therefore, three balanced-lethal asd plasmids were generated to evaluate how the sequential reduction in the transport of K99 fimbrial subunit to the outer membrane of the Salmonella vaccine vector could impact its immunogenicity. Introduction of these plasmids into an attenuated S. enterica serovar Typhimurium {Delta}aroA {Delta}asd strain resulted in Salmonella-K99 strains AP112 (pMAK99-asd+), AP114 (pMAK99.1-asd+ {Delta}fanGH), and AP116 (pMAK99.2-asd+ {Delta}fanDEFGH) (Fig. 1A).



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  		FIG. 1. (A) Expression of K99 fimbriae by Salmonella vaccine vectors as examined by IEM at 12,000x. IEM of the AP112 strain showed extracellular expression of K99 fimbrial antigen stained with rabbit anti-K99 fimbriae antibody and gold-anti-rabbit antibody. IEM of the AP114 strain sectioned at 90 nm showed periplasmic expression of K99 subunit (note gold particles as black dots along the circumference of the Salmonella cells). IEM of AP116 strain sectioned at 90 nm showed periplasmic and cytoplasmic expression of K99 subunit (note gold particles localized within the Salmonella cells). (B) Differential expression of the K99 fimbrial subunit as a consequence of deletions in the K99 genes. Depicted are Western blots of K99 fimbrial subunit from strains AP112, AP114, and AP116 and compared to migration of purified K99 subunit. Compared to AP112 by densitometric measurements of Western blots from two experiments, the K99 fimbrial subunit was reduced in AP114 (*, P =0.027) and AP116 (*, P =0.004) outer membrane (OM) fractions, but these strains retained expression in cytoplasmic (C), inner membrane (IM), and periplasmic (P) fractions; however, AP114 C fractions did show significant reduction compared to AP112 (*, P =0.013) and to AP116 (, P =0.021). (C) Quantification of K99 antigen in homogenized cells from the different Salmonella strains using a capture-ELISA method (see Materials and Methods). The various Salmonella constructs underwent extraction, and protein concentrations were normalized to equivalence. Depicted are the means (ng of K99 fimbria/ml/mg total protein) ± SD of the extrapolated values obtained from standard curves obtained with purified K99 fimbriae. *, P < 0.001, **, P < 0.024 for amount of K99 fimbria in AP112 versus AP114, AP116, or H647. There was no significant difference in K99 fimbria content between AP114 and AP116.

All strains, as confirmed by immunoelectron microscopy (IEM) (Fig. 1A), Western blot analysis (Fig. 1B), and K99-specific ELISA (Fig. 1C), stably expressed K99 fimbria. Cytoplasmic inner membrane, periplasmic and outer membrane fractions were enriched by conventional methods and showed that relative to the production by the AP112 strain (Fig. 1B) the deletion of fanGH in AP114 reduced the retention of the K99 fimbrial subunit in the cytoplasm and reduced its export to the outer membrane. Unlike AP114, AP116 strain showed equivalent retention of cytoplasmic K99 fimbrial subunit as did the AP112 strain, but it also showed significantly reduced export of the fimbrial subunit to the outer membrane (Fig. 1B). For each strain, the K99 fimbrial subunit was retained in the inner membrane and periplasmic fractions (Fig. 1B). The specific amounts of K99 fimbriae derived from these mutant strains were reduced compared to levels detected in the AP112 strain (Fig. 1C).

Mucosal and serum antibody responses diminish upon progressive retention of K99 subunit. To assess the effectiveness of each Salmonella construct in delivering the K99 fimbrial subunit to mucosal inductive tissues, outbred CD-1 female mice (n = 8/immunization group) were orally immunized, and mucosal secretions and serum were collected and analyzed for anti-K99 fimbria antibody titers. Six weeks postimmunization and approximately 3 weeks after mating, the AP112-vaccinated females elicited the greatest IgA titers in colostrum, serum, and fecal samples (Fig. 2A) compared to AP114-, AP116-, and H647-vaccinated females (P < 0.001). Maximal increases in serum IgG, IgG1, and IgG2b titers were obtained for AP112-vaccinated females (Fig. 2B). However, serum IgG1 and IgG2b antibody responses were sequentially reduced in mice orally immunized with AP114, AP116, and H647 strains. Serum IgG2a anti-K99 titers were equivalent for mice vaccinated with AP112 or AP114 strains, but were reduced for AP116-vaccinated mice (Fig. 2A). Interestingly, the IgG2b titers were not reduced between AP114- and AP116-vaccinated mice. The observed increases of IgA and IgG1 K99-specific titers by AP112-immunized mice suggest that Th2 cells supported this immune response.



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  		FIG. 2. K99 subunit-specific IgA and IgG responses by mice immunized with Salmonella-K99 vaccines. (A) Depicted are the means ± standard deviation of K99 fimbria-specific IgA log2 endpoint titers in colostrum, serum, and fecal samples that developed 6 weeks after oral immunization of CD-1 mice with 5 x 109 CFU of each Salmonella construct. The greatest IgA antibody titers were found in colostrum followed by serum and fecal samples, and the greatest titers for K99 fimbria were induced by AP112 strain followed by AP114 and AP116 strains. *P < 0.001 for IgA levels by AP112- versus AP114-, AP116-, or H647-vaccinated mice. (B) Depicted are the means ± standard deviation of K99 antigen-specific IgG and IgG subclasses log2 endpoint titers in serum that developed 6 weeks after oral immunization of same mice with the described Salmonella construct. The greatest antibody titers were induced by the AP112 strain followed by the AP114 and AP116 strains. For mice orally immunized with strain AP112, elevated IgG1, IgG2a, and IgG2b anti-K99 fimbrial titers were induced. As progressive deletions in the transport of the K99 fimbrial subunit were sequentially reduced, progressive decreases in the ratio and magnitude of IgG1 and IgG2b titers were observed. *, P < 0.001 for IgG and IgG subclass responses by AP112- versus AP114-, AP116-, and H647-vaccinated mice.

Reduced Th2 cell responses upon intracellular expression of K99 antigen. In light of these antibody responses in mucosal secretions and serum, it was important to determine the cause of the sequential decreases in IgA, IgG1, and IgG2b antibody titers. We had originally established that fimbrial expression by this Salmonella vaccine vector elicits a biphasic CD4+ Th cell response when interleukin-4- and IL-5-dependent responses precede the late IFN-{gamma}-dependent phase (38). To substantiate this finding using the Salmonella-K99 construct, cytokine responses were evaluated 1 week subsequent oral immunization with the various Salmonella constructs using cytokine ELISPOT method.

To enable CD4+ T-cell analysis, Peyer's patch and splenic lymphocytes from inbred BALB/c mice were harvested 1 week after oral immunization with each Salmonella-K99 construct. The results showed that the Salmonella AP112 vaccine strain induced elevated IL-4, IL-5, and IL-6 responses in the Peyer's patches (Fig. 3A) and elevated IL-4 and IL-6 responses in the spleen (Fig. 3B). Minimal to no IFN-{gamma} responses for any of the vaccine strains for either the Peyer's patches or spleens were obtained 1 week postvaccination (Fig. 3); this observation was consistent with that previously observed with mice orally immunized with Salmonella-CFA/I (38). Significant reduction in Peyer's patch IL-4 and IL-5 was observed for AP116 and H647 vaccine strains compared to AP112 responses (P < 0.001). Splenic IL-4 and IL-6 responses were significantly reduced in the AP114, AP116, and H647 strains compared to responses induced by AP112. These results, together with the reduced K99-specific IgA and IgG antibodies induced by AP114-vaccinated mice, show that the AP114 vaccine was still able to elicit elevated antibody responses.



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  		FIG. 3. Oral vaccination with AP112 or AP114 induces elevated mucosal Th2 cell responses. Depicted are cytokine responses of isolated lymphocytes following oral immunization of BALB/c mice with each Salmonella-K99 construct, and data are expressed as the means ± standard deviation for (A) Peyer's patches and (B) splenic cytokine-forming cell (CFC) responses. Mice orally immunized with strains AP112 or AP114 showed elevated IL-4, IL-5, and IL-6 CFC in Peyer's patches compared to mice vaccinated with AP116. Different CFC responses were observed in the spleen. Both AP112 and AP116 showed elevations in IL-6 CFC, but IL-4 CFC were significantly greater in AP112-vaccinated mice compared to all other vaccine groups. Minimal IFN-{gamma} responses were detected in either tissue. These results showed that Salmonella strains AP112 and AP114 induced predominantly fimbria-specific mucosal Th2 cell responses. Data are representative of three experiments. *, P < 0.001; **, P = 0.004.

Reduced protection upon challenge with wild-type bovine ETEC. Although a shift in the type of Th cells elicited as a consequence of K99 fimbrial expression, immune IgA and IgG anti-K99 fimbria antibodies were still produced. To assess whether these differences could impact protective immunity, a Duchet-Suchaux neonatal ETEC challenge model was employed (1, 10, 11). Groups of CD-1 female mice were orally immunized with one of the Salmonella-K99 constructs or with vector (strain H647) only; they were subsequently mated, and pups were allowed to come to parturition. Neonates from each group were allowed to obtain colostrum and milk for the first 24 h after their birth and then were orally challenged with wild-type B41 ETEC. The challenged pups were monitored for survival for 10 days (Fig. 4).



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  		FIG. 4. Passive immune protection of pups orally challenged with the wild-type ETEC strain. Dams orally immunized with Salmonella strain AP112 provided the best passive immunity to wild-type ETEC-challenged pups, as demonstrated by ~90% survival (n = 71), while the passive protection diminished in mice orally vaccinated with either the AP114 strain (79% survival, n = 68) or the AP116 strain (30% survival, n = 73). In contrast, pups from dams vaccinated with the Salmonella control strain H647 showed a survival rate of only 6% (n = 69). *, P < 0.001.

As a result, pups from the AP112-vaccinated group showed a 90% survival rate (P = 0.0003). Furthermore, pups from the AP114- and the AP116-vaccinated groups showed 80% (P = 0.0005) and 30% P = 0.0006) survival rates, respectively. In contrast, the control H647-vaccinated dams were unable to provide protection, with only 6% of neonatally challenged pups surviving. These data showed that the combination of cytoplasmic retention of the K99 fimbrial subunit and its loss of export to the outer membrane severely impacts the ability of strain AP116 to stimulate protective immunity.


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DISCUSSION
 
Evidence obtained through this study shows that fully and partially assembled K99 fimbriae influence the induction of Th2 cell responses as early as seven days subsequent oral immunization. Also evidenced in this study is the magnitude of the antibody responses and type of Th cell responses elicited. It appears that the AP112 strain mimics immunization with soluble proteins in conjunction with mucosal adjuvants (47, 51, 55). However, while oral immunization with soluble proteins in conjunction with mucosal adjuvants can elicit desirable antibody responses when given in multiple doses, a single oral immunization with strain AP112 sufficiently attains similar antibody responses. The magnitude of the mucosal and systemic antibody titers obtained by the AP112 strain also resembles that obtained with mice orally immunized with our Salmonella-CFA/I construct (38, 39). This collective evidence suggests that fimbrial placement on the cell surface of Salmonella vaccine vectors more closely resembles native ETEC fimbrial expression.

Surprisingly, the amount of fimbrial expression, while impacted by the deletions in the K99 genes, still has the ability to induce protection with each mutant strain. Interestingly, the level of protection obtained upon immunization with the AP112 strain, which mimics native K99 fimbriae expression, and obtained upon immunization with the AP114 strain, for which the transport of the K99 fimbrial subunit to the outer membrane was substantially reduced, differed by only 10%. Whether it was the impaired transport of the K99 fimbrial subunit or the reduced production of K99 fimbriae could not be distinguished in this study; however, the level of detectable fimbria production differed by ~100-fold between the AP112 and AP114 strains, and yet a high level of protection was still achieved. The secretory IgA and IgG antibody titers were reduced in mice immunized with AP114, but these differences were only modest and not as dramatically different from those obtained with strain AP116.

In past studies, passenger antigen expression is frequently limited to the intracellular or periplasmic compartments within Salmonella vaccine vectors (4, 18, 19, 29, 51, 53, 56), which tends to bias the host toward CD4+ Th1 cell responses. As such, humoral responses are blunted compared to those induced by oral immunizations with soluble protein in conjunction with mucosal adjuvants. It is not surprising that diminished antibody responses were obtained with the AP116 strain. The failure of the AP116 strain to provide greater protective immunity may be due to alterations in protein folding or the lack of chaperone proteins that contribute to the K99 assembly and possibly to immunity to K99 fimbriae. Although the total production of the K99 fimbrial subunit was nearly the same as that produced by strain AP116, there was increased retention of the subunit in the cytoplasm of strain AP116. Yet, by comparison, the protection level afforded by the AP116 strain was substantially impaired compared to the level induced by the AP114 strain.

These results are encouraging, suggesting that fully assembled K99 fimbriae are not required and offer two possibilities regarding insertions of peptides into the K99 fimbrial subunit. First, the K99 genes can be modified to permit peptide insertions into varied regions within the fimbrial subunit. If assembly is partially interrupted, it still suggests that protective immunity to K99 can be maintained as long as protective B-cell epitopes are not disrupted. Second, modifications of the K99 subunit could be performed using the AP114 strain. Again, concerns expressed for the modification of AP112 apply. In many respects, as evidenced by antibody and cytokine levels (18, 24, 25, 45, 53), the fanGH deletions partially mimic cell surface K99 fimbrial expression.

What is optimal for attaining maximal humoral immunity may not be favorable for stimulating cell-mediated immunity (19, 24, 45). Passenger antigen expression in the cytoplasmic and periplasmic compartments may be more favorable for development of cell-mediated rather than humoral immunity (18, 24, 45). The consequences that a single antigen expressed in the various compartments within a Salmonella vaccine vector have upon host immunity are evident (19, 24, 45). Possibly, the mode of vaccine expression may influence peptide loading (7, 21) and peptide density (32, 44) in antigen-presenting cells, which in turn influences the balance of Th1/Th2 cell responses. As demonstrated here, extracellular expression/secretion of K99 fimbriae is more efficient than periplasmic expression/secretion (19, 24, 25, 29, 36, 49, 53) and intracellular expression (16, 24, 35) systems commonly used in Salmonella vaccines provoking Th2-type responses. Conversely, cytoplasmic expression is more efficient in stimulating Th1-type responses (24, 45, 51, 53).

ETEC fimbria is host-specific (27, 28, 30), and we have also found that neither K99+ nor CFA/I+ ETEC is able to infect adult mice (data not shown), presumably, because of the unavailability of host receptors on the murine intestinal epithelium, confirming what has been reported previously (11). Rather, the Salmonella vaccine vector has the ability to target host mucosal inductive sites, e.g., Peyer's patches (52), therefore enabling the vaccine to reach these sites. This ability to reach mucosal inductive tissues may lead to elevations in mucosal and serum antibody responses and clearly demonstrates that effective delivery of vaccines to mucosal inductive sites can result in appropriate Th cell responses.

The observations described offer new possibilities for the development of novel mucosal vaccine formulations that exploit the placement of passenger antigens on or within Salmonella vaccine vectors. Understanding the mechanisms that provide the Th2-promoting properties of fimbria-expressing vaccines may allow us to design specific Salmonella vector vaccines that are more effective at generating protective secretory IgA antibody responses against other enteric pathogens. Particularly, it would be of interest to learn the influence of adapted B- and T-cell epitopes into this fimbria.


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ACKNOWLEDGMENTS
 
This work was supported by USDA grant NRI 990-1997, U.S. Public Service Grant AI-41123, USDA Formula Funds, and the Montana Agricultural Station.

We thank S. Brumfield for assistance with the immunoelectron microscope studies, Teri Hoyt for her technical assistance, and Nancy Kommers for help in the preparation of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717-3610. Phone: (406) 994-6244. Fax: (406) 994-4303. E-mail: dpascual{at}montana.edu. Back

Editor: J. T. Barbieri


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Infection and Immunity, November 2005, p. 7274-7280, Vol. 73, No. 11
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.11.7274-7280.2005
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