Induction of Immune Responses in Mice after Oral Immunization with ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2009, p. 4491–4497 0099-2240/09/$08.00⫹0 doi:10.1128/AEM.02672-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 75, No. 13

Induction of Immune Responses in Mice after Oral Immunization with Recombinant Lactobacillus casei Strains Expressing Enterotoxigenic Escherichia coli F41 Fimbrial Protein䌤 Jian-Kui Liu,1 Xi-Lin Hou,1 Chun-Hua Wei,1 Li-Yun Yu,1,2* Xiao-Jie He,1 Gui-Hua Wang,2 Jong-Soo Lee,3 Chul-Joong Kim3 College of Animal Science and Technology, Heilongjiang August First Land Reclamation University, Daqing 163319, Heilongjiang Province, China1; College of Life Science and Technology, Heilongjiang August First Land Reclamation University, Daqing 163319, Heilongjiang Province, China2; and National Lab of Oral Vaccine, College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Korea3 Received 22 November 2008/Accepted 26 April 2009

In an effort to develop a safe and effective vaccine for the prevention of enterotoxigenic Escherichia coli (ETEC) F41 infections, we have developed a surface antigen display system using poly-␥-glutamate synthetase A (PgsA) as an anchoring matrix. The recombinant fusion proteins comprised of PgsA and fimbrial protein of F41 were stably expressed in Lactobacillus casei 525. Surface localization of the fusion protein was verified by immunoblotting, immunofluorescence microscopy, and flow cytometry. Oral inoculation of recombinant L. casei 525 into specific-pathogen-free BALB/c mice resulted in significant mucosal immunoglobulin A (IgA) titers that remained elevated for >16 weeks. High levels of IgG responses in sera specific for F41 fimbriae were also induced, with prominent IgG1 titers as well as IgG2a and IgG2b titers. The helper T-cell (Th) response was Th2-cell dominant, as evidenced by increased mucosal and systemic interleukin-4-producing T cells and a concomitant elevation of serum IgG1 antibody responses. More than 80% of the mice were protected against challenge with a 2 ⴛ 104-fold 50% lethal dose of standard-type F41 (C83919). The induced antibodies were important for eliciting a protective immune response against F41 infection. These results indicated that the use of recombinant L. casei 525 could be a valuable strategy for future vaccine development for ETEC. LAB are considered safe, they exhibit adjuvant properties, and they are weakly immunogenic (7, 9, 10, 12, 23, 41). In addition, extracellularly accessible antigens expressed on the surfaces of bacteria are better recognized by the immune system than those that are intracellular (18). It is now realized that the delivery of antigen to mucosal surfaces can induce a strong local immune response in mucosaassociated lymphoid tissue. For the surface display of antigens on Lactobacillus casei, we have developed an expression vector using the poly-␥-glutamate synthetase A (PgsA) gene product as an anchoring matrix. PgsA is a transmembrane protein derived from the poly-␥-glutamic acid synthetase complex (the PgsBCA system) of Bacillus subtilis (5, 6); in this system, the N terminus of the target protein was fused to the PgsA protein, and the resulting fusion protein was expressed on the cell surface (32). In this study, the F41 fimbrial gene of ETEC was inserted into the vector pHB:pgsA and displayed on the surface of L. casei. The oral vaccination of mice with the recombinant L. casei strain elicited systemic and mucosal immune responses. These immune responses against F41 provided protective immunity in mice challenged with virulent live infectious C83919 postimmunization. Moreover, we showed that mice orally immunized with recombinant L. casei anchoring F41 induced a Th2-type response to ETEC F41. The results of this study suggest a potential use for our surface expression system against other pathogens that are transmitted to mucosal systems.

Enterotoxigenic Escherichia coli (ETEC) strains colonize the small intestine, secrete enterotoxins, and cause diarrhea. Colonization is facilitated by pili (fimbriae). Pili facilitate the adherence of ETEC to intestinal mucosa (27). Pilus adhesins that are known to be important in ETEC infections of neonatal animals are K88, K99, 987P, FY, and F41 (26, 28, 29, 38). F41 is less prevalent than K88, K99, or 987P and is usually accompanied by K99 (25). There is, however, strong suggestive evidence that F41 can mediate colonization by adhesion. Variants of a K99- and F41-positive porcine ETEC strain that have lost the K99 gene (29) and still carry the gene for and produce F41 are still virulent for newborn pigs (13). The previously conventional vaccine variability in levels of protective immunity may have been due to the lack of stimulation of appropriate mucosal immunity, since these vaccines were delivered parenterally. Mucosal immunization has proven to be an effective approach against the colonization of pathogens and their further spread to the systemic circulation (15, 34). Therefore, it is necessary to develop efficient and safe antigen vectors that will be able to trigger mucosal and systemic immune responses. One promising approach relies on the use of live bacterial vehicles (22). For mucosal immunization, lactic acid bacteria (LAB) are more attractive as delivery vehicles than other live vaccine vectors (e.g., Shigella, Salmonella, and Listeria spp.) (1, 3, 20, 21) because

* Corresponding author. Mailing address: College of Life Science and Technology, Heilongjiang August First Land Reclamation University, Daqing 163319, China. Phone: 86 4596819292. Fax: 86 4596819666. E-mail: [email protected]. 䌤 Published ahead of print on 15 May 2009.

MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. Escherichia coli XL1-Blue was used for the construction of vectors. It was cultivated in Luria-Bertani

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medium or on Luria-Bertani agar plates and grown at 37°C. L. casei was grown at 37°C in DeMan-Rogosa-Sharpe (MRS) broth (Difco Laboratories, Detroit, MI), where appropriate antibiotics were added. The minimal surface display plasmid pHB:pgsA-F41 was constructed by PCR amplification using T7-PgsBCA and standard-type F41 (C83919) as templates under the control of the HCE promoter, as described below. A PCR-amplified 1,116-bp DNA fragment with 5⬘-CGCGGTACCATGAAAAAAGAACTG-3⬘ and 5⬘-CGCGGATCCTTTAGATTTTAGTTTGTC-3⬘ encoding the membrane protein PgsA was digested with KpnI-BamHI and inserted into pHCE1LB, creating plasmid pHB:pgsA. For the construction of plasmid pHB:pgsA-F41, a PCR-amplified 834-bp DNA fragment with 5⬘-CGCGGATCCATGAAAAAGA CTCTGA-3⬘ and 5⬘-CGCAAGCTTTTAACTATAAATAACG-3⬘ encoding the fimbrial protein of F41 was digested with BamHI and HindIII and inserted into pHB:pgsA. L. casei 525 was isolated from Korean food. The transformation of L. casei 525 was performed by electroporation. The sample was subjected to a 2.2-kV, 200-⍀, 25-␮F electric pulse in a 0.2-cm cuvette, using a gene pulser (Bio-Rad, Richmond, CA). Cell wall fractionation, immunoblotting, immunofluorescence microscopy, and flow cytometry. Recombinant L. casei 525 cells were grown at 37°C, and cell fractionations and protein extractions were performed as previously described (32). For the immunodetection of fusion proteins, mouse anti-PgsA (1:1,000) and mouse anti-F41 (1:800) were used. Horseradish peroxidase-conjugated antimouse immunoglobulin G (IgG) was used as a secondary antibody. After washing the membranes with washing buffer, the membranes were treated with avidin and biotin complex (Vectastain ABC kit; Vector Lab) following the manufacturer’s instructions. The visualization of immunobinding was carried out with diaminobenzidine solution (Vector Lab). The typical band was observed by the naked eye. For immunofluorescence microscopy, cells labeled with anti-F41 polyclonal antibodies and fluorescein isothiocyanate (FITC)-conjugated antimouse antibodies were examined using a Carl Zeiss Axioskop 2 fluorescence microscope. Photographs were taken with an Axiocam high-resolution camera using identical exposure times. For flow cytometry, L. casei 525 cells were cultured in MRS broth overnight at 37°C. The cell pellets were sequentially incubated with mouse anti-F41 polyclonal antibodies (1:800) and FITC-conjugated anti-mouse IgG secondary antibodies (1:800; Sigma). Finally, 10,000 cells were analyzed with FACSCalibur (Becton Dickinson, Oxnard, CA) equipped with Cell Quest software. Determination of plasmid copy number and stability tests. The quantitative measurement of the plasmid copy number was performed according to previously described methods (2). The plasmid copy number could then be deduced from the determined quantity of plasmids in the samples. The stability of pHB: pgsA-F41 in L. casei 525 without antibiotic selection was monitored as described previously (16). The experiment was done in triplicate. Immunizations and sample collection. Specific-pathogen-free mice (BALB/c, female, 5 weeks old) were obtained from Vital River Laboratories, Beijing, China. The BALB/c mice were placed into individual cages with autoclaved food and water available ad libitum. All animals were housed in an aseptic room. Mice were acclimated to the new environment for 1 week after arrival before being used for immunization. To study the possibility of the surface-displayed F41 antigen to induce mucosal immunity in mice, the method of Lee et al. (17) was employed. Six-week-old female BALB/c mice (120 heads) were divided into two groups (60 heads/group). Sixty mice were kept for the negative controls, and the other 60 mice were immunized orally with live L. casei 525 that expresses recombinant F41 protein from plasmid pHB:pgsA-F41. L. casei 525 harboring the parental plasmid pHB:pgsA was used as a negative control. The recombinant live L. casei 525 cells displaying F41 antigen on their surfaces were resuspended in 100 ␮l sterile phosphate-buffered saline (PBS) at a concentration of 5 ⫻ 109 cells for the oral route. The suspension was administered daily via intragastric lavage on days 0 to 4, 7 to 11, 21 to 25, and 49 to 53. Blood samples were collected from the tail vein on days 0 (preimmune), 14, 28, 42, 56, 70, and 84. Fecal samples were collected every week. Sera were prepared from the blood and stored at ⫺20°C until they were analyzed. To obtain bronchoalveolar and intestinal lavage samples, six to eight mice were killed on days 56, 70, 84, and 112. Bronchoalveolar and intestinal lavage fluids were obtained by washing the respective organs three times with 0.5 ml of ice-cold saline containing protease inhibitors. Samples were centrifuged at 2,500 ⫻ g for 20 min at 4°C, and the supernatants were stored at ⫺20°C until they were analyzed. ELISA. Specific antibody IgG titers in serum and specific antibody secretory IgA (sIgA) titers in intestinal and bronchoalveolar lavage fluid samples and fecal samples were determined by an enzyme-linked immunosorbent assay (ELISA) as previously described (17, 41). Briefly, ELISA plates were coated overnight at 4°C

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FIG. 1. Western blot analysis of PgsA-F41 fusion protein with antiF41 (A) and anti-PgsA (B) polyclonal antibodies. Lanes 1 and 2, membrane and cell wall fraction of L. casei 525 harboring pHB:pgsA and pHB:pgsA-F41, respectively. Protein bands of 72 kDa, corresponding to the expected size of PgsA-F41, were detected.

with purified F41 fimbrial protein in carbonate buffer (pH 9.6). The plates were washed five times in PBS containing 0.05% Tween 20 and then saturated with PBS containing 5% skim milk at 37°C for 1 h. The serially diluted serum samples, intestinal and bronchoalveolar lavage fluid samples, and fecal samples were added in duplicate and incubated for 1 h at 37°C. After the plates were washed five times with PBS containing 0.05% Tween 20, horseradish peroxidase-conjugated goat anti-mouse IgG, IgA, or subclass-specific goat anti-mouse immunoglobulin (IgG1, IgG2a, and IgG2b) antibodies (Sigma) were added to each well and incubated for an additional h at 37°C. After another round of washing was completed, substrate solution containing tetramethyl benzidine and H2O2 was added. The reaction was allowed to proceed for 15 min at room temperature before it was terminated by adding stop solution (H2SO4). The absorbance was measured at 450 nm using an ELISA autoreader (Molecular Devices). End-point titers were defined as the maximum dilutions, giving an A450 measurement of 0.1. This cutoff value represents the mean optical density plus 2 standard deviations of 10 normal mouse serum samples tested at a 1:50 dilution. Statistical comparison was made by the Mann-Whitney U test. ELISPOT assay. Lymphoid cell isolation and culture were performed according to previously described methods (39). Gamma interferon (IFN-␥)-producing or interleukin-4 (IL-4)-producing cells were quantified using an enzyme-linked immunospot (ELISPOT) assay kit for mouse IFN-␥ or IL-4 as recommended by the manufacturer (DaKeWe Biotech Company Limited, ShenZhen, China). Briefly, the wells of 96-well sterile microfiltration plates were treated with 70% ethanol and washed with PBS. The plate was coated with 10 ␮g/ml anti–mouse IFN-␥ or IL-4 monoclonal antibody in PBS overnight at 4°C. The plate was washed and then blocked with RPMI 1640 plus 10% fetal calf serum for 1 h at 37°C. Splenic and Peyer’s patch CD4⫹ T cells (5 ⫻ 106 cells ml⫺1) were added to each well. The lymphocytes were stimulated with 10 ␮g/ml of the ETEC F41 fimbriae for 2 to 3 days at 37°C. Control wells contained unstimulated cells. After incubation was complete, the plates were washed and incubated with 2.5 ␮g/ml biotinylated anti–mouse IFN-␥ or IL-4 antibody. After the wells were washed, spot-forming cells (SFC) were visualized upon the addition of the chromogenic substrate 3-amino-9-ethylcarbazole. The numbers of SFC were counted using the KS ELISPOT compact system (Carl Zeiss, Oberkochen, Germany). All assays were performed in triplicate. F41 adhesive fimbrial challenge experiment. The challenge experiment was performed on day 75 (3 weeks after the last booster immunization) and on day 114 (9 weeks after the last booster immunization) with 200 ␮l of C83919, which had a titer of 6 ⫻ 108 CFU (an approximately twofold 50% lethal dose [LD50]) or 6 ⫻ 1012 CFU (an ⬃2 ⫻ 104-fold LD50). Deaths were recorded, and surviving mice were maintained for 20 days postchallenge. The healthy mice without diarrhea signs were judged protected. Statistical analysis. The Mann-Whitney U test was used to evaluate differences between variations in antibody titers and to compare cytokine production levels. The difference in survival was analyzed by the log rank test with SPSS software. A significant difference was taken to exist when P was ⬍0.05.

RESULTS Expression of PgsA-F41 fusion protein on the cell surface. The PgsA-F41 fusion protein was analyzed by Western blotting. Figure 1 shows the analysis of L. casei 525 cells harboring plasmid pHB:pgsA-F41. The respective molecular sizes of the PgsA and F41 proteins were approximately 41 kDa and 31 kDa, respectively, and that of the PgsA-F41 fusion protein was therefore approximately 72 kDa. The clear band of the fusion

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MOUSE IMMUNE RESPONSE TO ETEC F41-EXPRESSING L. CASEI

FIG. 2. Systemic anti-F41 protein response following mucosal immunization. The sera of 12 mice in the orally immunized groups were taken at random. The kinetics of anti-F41 serum IgG responses are shown. Endpoint titers were calculated as the reciprocals of serum dilutions yielding the same optical density as a 1/50 dilution of a pooled preimmune serum. The data are presented as means ⫾ standard deviations. Statistical comparisons between groups were made by the Mann-Whitney U test.

protein is observed at the estimated molecular size, indicating the successful expression of the fusion protein. The localization of the fusion protein expressed on the surface of L. casei 525 was verified by immunofluorescence microscopy and flow cytometric analysis. Immunofluorescence labeling of the cells was performed using mouse anti-F41 antibody as the primary antibody and FITC-conjugated goat anti-mouse IgG as the secondary antibody. The green fluorescence of the immunostained PgsA-F41 fusion protein was observed on L. casei 525 cells harboring the plasmid pHB:pgsA-F41, whereas cells harboring the control plasmid pHB:pgsA were not immunostained (data not shown). Analyses by flow cytometry also revealed that the cells displaying PgsA-F41 showed a significantly greater intensity of fluorescence signals than the control cells (data not shown). These data demonstrate that F41 protein was expressed on the surface of L. casei 525 using PgsA as a membrane-anchored protein display motif. Copy number and stability of pHB:pgsA-F41. Extraction of the plasmid DNA of pHB:pgsA-F41 from L. casei 525 yielded small quantities of DNA, suggesting that the copy number of the plasmids was low in the host. By comparison of the hybridization signals of the plasmid target in known amounts of total

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DNA and purified plasmid, we deduced that pHB:pgsA-F41 is a low-copy-number plasmid in L. casei 525. We estimate the copy number to be around three copies per cell (data not shown). The stability of pHB:pgsA-F41 in L. casei 525 without antibiotic selection was monitored over 80 generations of growth in MRS broth medium. No loss of the plasmid was observed over this time, indicating a very high stability. Systemic and mucosal immunogenicities of the hybrid protein expressed on L. casei. In order to characterize the immunogenicity of F41 antigen surface-displayed on L. casei 525, BALB/c mice were immunized orally with L. casei 525 expressing the F41 protein on the cell surface and control group with L. casei 525 host cells harboring the control plasmid pHB: pgsA. Serum samples were used for evaluating the systemic immune response by ELISA. During the first two series of immunization, very low levels of IgG antibody were detected (Fig. 2). The higher IgG levels were detected shortly after the third immunization (day 28, P ⬍ 0.01). After the fourth immunization, further increases in the IgG titers were observed (day 70, P ⫽ 0.002). At the end of immunization, the mean serum IgG titers for the experimental groups were more than 1,000 times higher than those in the control groups. To better characterize antibody responses against F41, the levels of antigen-specific IgG subclasses (IgG1, IgG2a, and IgG2b) were assessed by ELISA. Pooled immune sera collected on days 28 and 70 after the first inoculation were used. This serum IgG1 anti-F41 titer rose prior to the IgG2a titer shortly after immunization (4 weeks) and exceeded the IgG2a antibody titer 2.8-fold (Fig. 3), although elevated serum IgG1 antigen-specific titers were equivalent to IgG2a titers 10 weeks after oral immunization (Fig. 3). The mean titers of these subtypes were significantly different from the baseline titers in the control group (P ⬍ 0.01). The levels of Th1-promoting cytokines were less than the levels of Th2-promoting cytokines, the number of splenic IL-4 SFC exceeded the number of IFN-␥ SFC by 2.8-fold, and the number of Peyer’s patch IL-4 SFC exceeded the number of IFN-␥ SFC by 4.5-fold (P ⬍ 0.01) (Fig. 4). Collectively, this evidence corroborates the Th cell subset analysis, showing that this construct induces the development of Th2 cells, which in turn, supports IgG1 and sIgA anti-F41 antibody responses.

FIG. 3. Antigen-specific serum IgG subclass antibody responses during the 4th week (A) and 10th week (B) after first oral immunization with the pHB:pgsA-F41 vaccine. Serum IgG, IgG1, and IgG2a titers were determined by using the ELISA procedure. No serum IgG titers against F41 fimbriae were elicited in mice orally immunized with the pHB:pgsA construct. Each value represents the average of duplicate samples from each of 12 mice/group. The error bars represent standard deviations.

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FIG. 6. F41 fimbria-specific IgA responses in fecal pellets were induced after the oral immunization of mice with a single dose of L. casei 525/pHB:pgsA-F41 and L. casei 525/pHB:pgsA vector as the control. The error bars represent standard deviations. FIG. 4. Oral immunization with the pHB:pgsA-F41/L. casei 525 vaccine promotes the induction of Th2 cells in Peyer’s patch and splenic CD4⫹ T-cell populations. Freshly isolated splenic (A) and Peyer’s patch (B) CD4⫹ T cells harvested 4 weeks after first vaccination were tested for cytokine secretion by the cytokine ELISPOT method. Each value is expressed as the average number of SFC/106 CD4⫹ T cells from quadruplicate cultures ⫾ the standard deviation (SD). Values were corrected for spontaneous release by cells obtained from normal, uninfected mice. A representative example of the results from three separate experiments is shown. Splenic and Peyer’s patch IL-4 SFC were significantly greater in number than IFN-␥ SFC (P ⬍ 0.01). CD4⫹ T cells in splenic (C) and Peyer’s patch (D) cells harvested 10 weeks after first vaccination were examined for cytokine secretion by the cytokine ELISPOT method. Each value is expressed as the average number of SFC/106 CD4⫹ T cells from quadruplicate cultures ⫾ the SD. Significantly more F41-specific splenic or Peyer’s patch IL-4 SFC were detected than IFN-␥ SFC (P ⬍ 0.01).

To assess mucosal immune responses, specific sIgA levels in intestinal and bronchoalveolar lavage fluids were determined by ELISA. Fluids collected on days 56, 70, 84, or 112 and fecal samples collected during week 2, 4, 6, 8, 11, or 16 after the first inoculation were examined using purified F41 fimbrial protein as the coating antigen. Oral immunizations elicited F41-specific mucosal IgA responses at the site of the inoculation as well as the remote mucosal site (Fig. 5 and 6, respectively). Not surprisingly, greater antibody titers were detected at the locality of immunization. In contrast, only background levels of antibodies were detected in the control animals. Efficacy of the F41 antigen expressed in the recombinant live L. casei. On day 75 (3 weeks after the last booster immuniza-

tion), 12 immunized mice per group were orally challenged with 6 ⫻ 108 CFU (an ⬃2⫻ LD50) or 6 ⫻ 1012 CFU (an ⬃2 ⫻ 104-fold LD50) of C83919. The postinfection survival rates of the vaccinated and control mice are compared in Fig. 7A (6 ⫻ 108 CFU) and C (6 ⫻ 1012 CFU). While 50% and 100% of the control mice died after challenge with the C83919 strain, respectively, immunization fully protected the mice regardless of the challenge dose. In the case of the higher challenge dose (Fig. 7C), the difference in survival was statistically significant (P ⫽ 0.0064), as calculated by the log rank test with SPSS software. In order to prove that the protection of mice was due to specific immunity and not to a nonspecific cellular immune response mediated by the presence of the vaccine strain in the host organs, the same experiment with a longer time interval between the last booster immunization and the challenge was repeated. In a pilot study, L. casei 525/pHB:pgsA-F41 (with a dose corresponding to that used for the vaccination experiments) was shown to be cleared from the organs (liver, spleen, lung, and Peyer’s patches) of mice within 2 weeks postimmunization (data not shown). The 12 immunized mice per group vaccinated as described above were orally challenged with 6 ⫻ 108 CFU or 6 ⫻ 1012 CFU of C83919 on day 114 (⬃6 weeks after the vaccine strain had been cleared from the immunized mice). While 40% and 100% of the control mice died after challenge with the C83919 strain, respectively, more than 80% of the mice in the oral immunization group survived after challenge with C83919. Immunization significantly increased

FIG. 5. Anti-F41 mucosal IgA antibody responses. Bronchoalveolar (A) and intestinal (B) lavage fluids, harvested from mice sacrificed on days 56, 70, 84, and 112 postimmunization, were analyzed by ELISA in triplicate. Fluids from the control animals are shown as open bars. The error bars represent the standard deviations.

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FIG. 7. Survival of BALB/c mice immunized with L. casei 525/pHB:pgsA-F41 following a subsequent challenge with the standard-type strain C83919. Survival of the immunized mice is represented with solid lines, while that of the control mice is represented with dashed lines. Twelve mice per group were challenged orally with 6 ⫻ 108 (A) or 6 ⫻ 1012 (C) CFU of C83919 on day 75 (3 weeks after the last immunization). Twelve mice per group were challenged orally with 6 ⫻ 108 (B) or 6 ⫻ 1012 (D) CFU of C83919 on day 114 (9 weeks after the last immunization). Six of 12 (A) and 7 of 12 (B) control mice died after challenge with the C83919 strain (6 ⫻ 108 CFU), but the mice in the immunity group were fully protected. Oral immunization with pHB:pgsA-F41 provided immunity to C83919 strain (6 ⫻ 1012 CFU)-challenged mice, as demonstrated by the survival of 11 of 12 (75 days; C) and 10 of 12 (114 days; D) mice. In contrast, mice vaccinated with pHB:pgsA vector only (n ⫽ 12) died.

the survival of the mice (depicted in Fig. 7B and D) with both the lower (P ⫽ 0.036) and the higher (P ⫽ 0.002) challenge doses. DISCUSSION For mucosal vaccination purposes, LAB are attractive delivery vehicles because they are considered safer than many other live vaccine carriers (e.g., Salmonella spp., E. coli, and vaccinia virus) (35) and show adjuvant effects. The recombinant live oral vaccines expressing pathogen-derived antigens on the surfaces of bacteria can be an alternative method for providing protection because ETEC replicate mainly in the villi of the small intestines. Mucosal immunization offers a number of advantages over other routes of antigen delivery, including convenience, cost effectiveness, and the induction of both local and systemic immune responses (7, 9, 10, 12, 23, 41). The goal is to provide the first line of defense by effectively eliminating pathogens at the mucosal surface. But the surface display of antigens on the bacterial surface has been problematic because large antigens perturb membrane topology. True surface ex-

posure of antigens requires a transmembrane anchor that is long enough to cross the cell wall (17). At least 100 amino acids are needed to properly cross the cell wall (19). In this report, we have developed a surface display system using the PgsA protein as the transmembrane anchor to present heterologous proteins on L. casei 525. We adopted PgsA protein derived from the PgsBCA enzyme complex of B. subtilis. To investigate the feasibility of using LAB as carriers of immunogenic peptides to the mucosal immune system, we immunized specific-pathogen-free mice orally with the recombinant L. casei 525 strain. Although the amount of fusion F41 fimbrial proteins expressed on the surface of L. casei 525 was not much higher than that of F41 fimbrial proteins expressed on E. coli (data not shown), the oral immunization of mice with these recombinant L. casei 525 strains resulted in systemic and local immune responses. The mucosal delivery of vaccines induces mucosal immunity more efficiently than parenteral immunization (31). The surfaces of the respiratory, gastrointestinal, and urogenital tracts, which are not covered by skin, are referred to as mucosa (33). In total, the mucosa cover an area

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that is about 200 times larger than the skin. In addition, IgA is the primary immunoglobulin isotype induced at the mucosal surface. sIgA in mucosal secretions provides protection against bacterial and viral pathogens and neutralization of microbial toxins (8, 24, 40, 42). Thus, it must be very effective in dealing with the large amounts of foreign antigens. The most important part of the immediate defense against pathogens in the mucosa is the innate immune system. The mucosa and associated lymphoid tissue are attractive targets for vaccine strategies because they harbor the early stages of infection. Protective immunity against ETEC depends mainly on the induction of the sIgA antibody response at the lumen of the small intestine and a Th2-dependent immune response (4, 30), and protective immunity to ETEC is antibody dependent. However, conventional oral immunization with purified ETEC fimbriae fails to elicit protective immunity as a consequence of antigenic alteration by the gastrointestinal (GI) tract (34), unless unaltered ETEC fimbriae can reach the inductive lymphoid tissues in the GI tract. We confirm the capacity of L. casei 525 to deliver F41 antigen to mucosa to activate lymphoid cells in the GI tract by using the stabilized pHB:pgsA-F41 construct that expresses F41 antigen on the surface of the cell. The results clearly demonstrate that the oral immunization of BALB/c mice with pHB:pgsA-F41 was sufficient to elicit elevated sIgA responses in mucosal tissues as well as increases in systemic IgG antibody responses to F41. In contrast, the pHB:pgsA negative control strain failed to elicit specific antibody responses to F41 antigen in serum and mucosal tissues. We observed increases in IgG2a and IgG2b antibody responses in serum and dramatic increases in IgG1. IgG1 and IgG2a seem to play an important role in the neutralization of exotoxins produced by F41 like the neutralization of exotoxins produced by Corynebacterium diphtheriae and Clostridium tetani (15). This observation would suggest that Th2 cell mechanisms may be contributing to this immune response. This idea of Th2-type involvement is further suggested by the sustained increases in sIgA antibody responses and IL-4. Thus, the oral administration of recombinant L. casei 525 displaying F41 antigens on the surface induced both systemic and mucosal immune responses against F41. Since protection can clearly be defined in this model as significant mucosal IgA titers that remained elevated for ⬎16 weeks, this retention of immunogenicity is important to provide immune protection to orally challenged mice with the standard-type ETEC strain C83919. Additional parameters need to be more clearly defined, i.e., the optimum oral immunization procedure, the optimum numbers of bacterial organisms and doses required for protection, the amount of time colonizing bacteria are present in the gut, and the effects of the continuous expression of a LAB-associated antigen. These will be the subject of additional work with this model. The previous investigators have shown that parenteral vaccination against K99 protects against strains expressing both K99 and F41, but vaccination against F41 does not protect against strains also expressing K99 (11, 36). Parenteral immunization is highly effective in protecting against systemic infection, in part due to the production of circulating IgG and its transudation into many extravascular sites, especially during inflammatory processes. However, parenteral immunization can fail to protect against the invasion of mucosa. This failure


is attributable to insufficient IgG transudation into mucosal secretions and the inability of systemic immunogen presentation to induce mucosal IgA production, the principal isotype present in external secretions. In contrast, mucosal immunization elicits both mucosal IgA and circulating IgG (14). On the mucosal surface, IgA represents a first line of defense by neutralizing invading pathogens (37). Our observations support the concept of a mucosal immune system in which mucosally situated IgA and IgG plasma cell progenitors are stimulated selectively by mucosal immunization and predict that mucosal immunization can provoke both mucosal and circulating antibody responses better than parenteral immunization (14). Whether mucosal immunization with recombinant L. casei 525 expressing F41 antigen on its surface can provide an effective means for eliciting protective immune responses against K99 and F41 needs our further clarification. In conclusion, we have demonstrated that F41 fimbrial protein exposed on the surface of the L. casei nonpathogenic strain, resisting gastric acidity, delivered orally to animals, elicits both systemic and mucosal immune responses. The data suggest that antibodies can provide protective immunity to ETEC. It will be of interest to determine whether systemic and mucosal immune responses are also obtained following the passive protection of suckling infant mice or calves against F41. ACKNOWLEDGMENTS This project was supported by the fund of the 11th Five-Year Plan in Key Science and Technology Research of the agricultural bureau in Heilongjiang province of China (HNKXIV-08-06-03) and the Key Science and Technology Research of Daqing in Heilongjiang province, China (SGG2006-011). REFERENCES 1. Abd El Ghany, M., A. Jansen, S. Clare, L. Hall, D. Pickard, R. A. Kingsley, and G. Dougan. 2007. Candidate live, attenuated Salmonella enterica serotype Typhimurium vaccines with reduced fecal shedding are immunogenic and effective oral vaccines. Infect. Immun. 75:1835–1842. 2. Alpert, C. A., A. M. Crutz-Le Coq, C. Malleret, and M. Zagorec. 2003. Characterization of a theta-type plasmid from Lactobacillus sakei: a potential basis for low-copy-number vectors in lactobacilli. Appl. Environ. Microbiol. 69:5574–5584. 3. Altboum, Z., M. M. Levine, J. E. Galen, and E. M. Barry. 2003. Genetic characterization and immunogenicity of coli surface antigen 4 from enterotoxigenic Escherichia coli when it is expressed in a Shigella live-vector strain. Infect. Immun. 71:1352–1360. 4. Alves, A. M., M. O. Lasaro, D. F. Almeida, and L. C. Ferreira. 1998. Immunoglobulin G subclass responses in mice immunized with plasmid DNA encoding the CFA/I fimbria of enterotoxigenic Escherichia coli. Immunol. Lett. 62:145–149. 5. Ashiuchi, M., C. Nawa, T. Kamei, J. J. Song, S. P. Hong, M. H. Sung, K. Soda, T. Yagi, and H. Misono. 2001. Physiological and biochemical characteristics of poly-␥-glutamate synthetase complex of Bacillus subtilis. Eur. J. Biochem. 268:5321–5328. 6. Ashiuchi, M., K. Soda, and H. Misono. 1999. A poly-␥-glutamate synthetic system of Bacillus subtilis IFO 3336: gene cloning and biochemical analysis of poly-␥-glutamate produced by Escherichia coli clone cells. Biochem. Biophys. Res. Commun. 263:6–12. 7. Bermu ´dez-Humara ń, L. G. 2009. Lactococcus lactis as a live vector for mucosal delivery of therapeutic proteins. Hum. Vaccin. 5:1–4. 8. Brandtzaeg, P. 2003. Role of secretory antibodies in the defence against infections. Int. J. Med. Microbiol. 293:3–15. 9. Cortes-Perez, N. G., F. Lefe`vre, G. Corthier, K. Adel-Patient, P. Langella, and L. G. Bermu ´dez-Humara ń. 2007. Influence of the route of immunization and the nature of the bacterial vector on immunogenicity of mucosal vaccines based on lactic acid bacteria. Vaccine 25:6581–6588. 10. Daniel, C., F. Sebbane, S. Poiret, D. Goudercourt, J. Dewulf, C. Mullet, M. Simonet, and B. Pot. 2009. Protection against Yersinia pseudotuberculosis infection conferred by a Lactococcus lactis mucosal delivery vector secreting LcrV. Vaccine 27:1141–1144. 11. Duchet-Suchaux, M. 1988. Protective antigens against enterotoxigenic Escherichia coli O101:K99,F41 in the infant mouse diarrhea model. Infect. Immun. 56:1364–1370.

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