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Functional Pentameric Formation via Coexpression of the Escherichia coli Heat-Labile Enterotoxin B Subunit and Its Fusion Protein Subunit with a Neutralizing Epitope of ApxIIA Exotoxin Improves the Mucosal Immunogenicity and Protection against Challenge by Actinobacillus pleuropneumoniae

ABSTRACT A coexpression strategy in Saccharomyces cerevisiae using episomal and integrative vectors for the Escherichia coli heat-labile enterotoxin B subunit (LTB) and a fusion protein of an ApxIIA toxin epitope produced by Actinobacillus pleuropneumoniae coupled to LTB, respectively, was adapted for the hetero-oligomerization of LTB and the LTB fusion construct. Enzyme-linked immunosorbent assay (ELISA) with GM1 ganglioside indicated that the LTB fusion construct, along with LTB, was oligomerized to make the functional heteropentameric form, which can bind to receptors on the mucosal epithelium. The antigen-specific antibody titer of mice orally administered antigen was increased when using recombinant yeast coexpressing the pentameric form instead of recombinant yeast expressing either the LTB fusion form or antigen alone. Better protection against challenge infection with A. pleuropneumoniae was also observed for coexpression in recombinant yeast compared with others. The present study clearly indicated that the coexpression strategy enabled the LTB fusion construct to participate in the pentameric formation, resulting in an improved induction of systemic and mucosal immune responses.

The heat-labile enterotoxin of Escherichia coli (LT) is the principal disease agent of enterotoxigenic E. coli, which is the most common cause of diarrhea (24,62). LT is composed of one copy of the A subunit (LTA), which has ADP-ribosylation activity, and a homopentamer of B subunits (LTB), which binds to cell receptors at the surfaces of eukaryotic cells (39,59). The LT homopentamer consists of five identical 103-amino-acid (aa) B subunits (11.6 kDa each), which form a donut-shaped pentamer via noncovalent associations (58). LT is an immunogenic molecule that stimulates both systemic and mucosal immune responses (16), and it also has been identified as a potent immunoadjuvant, which has been demonstrated to enhance mucosal immunoglobulin A (IgA) as well as systemic antibody responses against coadministered or coupled antigens (60). However, the use of LT as an adjuvant remains limited, partly due to the toxicity of the LTA subunit (65). Thus, strategies to separate the effect of the adjuvant from its toxicity resulted in the production of LTB lacking the A subunit (10,37). Additionally, many previous studies have shown that LTB itself is a potent mucosal immunogen that induces systemic and mucosal responses (7,61) and has profound adjuvant activity via its function as a carrier protein for coadministered unre-lated antigens (63). Although the exact mechanism by which the adjuvant effects of LTB are exerted is not yet fully understood, the stimulatory activity of LTB has been suggested to depend on its ability to bind to the cell receptor, most commonly GM1, which requires the pentameric association of B subunits.
Actinobacillus pleuropneumoniae, the causal agent of porcine pleuropneumonia, results in severe and fatal hemorrhagic necrotizing pneumonia and fibrinous pleuritis in pigs. Gramnegative A. pleuropneumoniae has been classified as having two biotypes, based on the requirement for NAD, and 15 serotypes, based on surface polysaccharide antigens (1,15). Although multiple factors such as capsular polysaccharides, outer membrane proteins, Apx exotoxins, lipopolysaccharides, permeability factors, and iron-regulated proteins (1,8,12) are believed to be involved in the virulence of A. pleuropneumoniae, Apx toxins are thought to be particularly important in the induction of protective immunity, as previously demonstrated using several mutants (12,13,46,47). Apx toxins, which belong to the family of repeats in toxin (RTX) toxins secreted by several Gramnegative bacteria, can create pores in the host cell membrane. Most A. pleuropneumoniae serotypes produce one or two exotoxins among the ApxI, -II, and -III proteins. Both ApxI and ApxII of A. pleuropneumoniae are essential for full virulence and the development of clinical signs and typical lung lesions (1,12,31,38,47). Among the Apx toxins, ApxII is expressed in all but serotype 10, whereas the other two major toxins, ApxI and ApxIII, are expressed in fewer serotypes. In South Korea, more than half of all A. pleuropneumoniae isolates obtained from infected pigs have been classified as serotype 2 and determined to secrete ApxII and ApxIII (40). Thus, a vaccination strategy against ApxII could be an effective approach for reducing porcine pleuropneumonia caused by a broad range of serotypes of A. pleuropneumoniae.
A. pleuropneumoniae is a good model system for the development of a vaccination strategy leading to a mucosal immune response. First, A. pleuropneumoniae gains access to its host through mucosal surfaces of the respiratory tract. Second, we previously reported that orally administered ApxIIA or an antigenic fragment containing its neutralizing epitopes could induce an immune response against A. pleuropneumoniae infection (26,33,43,55,56). However, improved strategies for efficient antigen presentation and immune responses are still needed for enhanced protection against pathogen infection.
Thus, oral coadministration of recombinant LTB and the ApxIIA epitope is appropriate because we can expect improved protection against A. pleuropneumoniae infection due to the increased vaccine activity of the ApxIIA epitope through the adjuvant effects of LTB. Assuming that the pentameric formation of LTB is a prerequisite for the proper presentation of the antigenic part, steric hindrance due to the increased molecular size of the fusion partner needs to be overcome.
As an antigen delivery system, baker's yeast, Saccharomyces cerevisiae, is desirable because it has already been successfully used live and orally for the treatment of human as well as animal diseases (9,51). Additionally, several studies have supported the use of yeast expression systems for recombinant vaccine formulations due to the adjuvant properties of yeast derivatives, which enhance the immune response (53).
In the present study, along with characteristics of S. cerevisiae, LTB, and the protective antigenic determinant of the ApxIIA toxin of A. pleuropneumoniae, the heterologous expression of S. cerevisiae was designed to differentially coexpress LTB and the fusion subunit (LTB-ApxIIA#5) containing the neutralizing epitope (ApxIIA#5) of ApxIIA to obtain heteropentamers that contained a limited number of LTB::ApxIIA#5 subunits using a low-copy-number integrative vector and a high-copy-number episomal vector for the LTB::ApxIIA#5 and LTB subunits, respectively. Additionally, the resulting pentameric formation of heteromeric subunits was examined for the increased vaccine efficacy of the ApxIIA antigen due to the pentameric formation that can bind to the cell receptor.

MATERIALS AND METHODS
Strains and culture conditions. Plasmids were maintained and propagated in E. coli HB101 or DH5␣ according to the work of Sambrook et al. (48). S. cerevisiae 2805-a7 (pep4::HIS3 leu2-3 ura3-52) was used as the recipient cell for transformation. The S. cerevisiae culture was maintained in YEPD medium (1% yeast extract, 2% peptone, and 2% dextrose) while uracil-deficient (Ura Ϫ ) selection medium (0.67% yeast nitrogen base without amino acids [Sigma-Aldrich], 30 mg liter Ϫ1 adenine and tryptophan, 0.5% Casamino Acids, 2% dextrose, and 2% agar) and leucine-deficient (Leu Ϫ ) selection medium (0.67% yeast nitrogen base without amino acids, 0.14% yeast synthetic dropout medium [Sigma-Aldrich], 30 mg liter Ϫ1 tryptophan, 2% dextrose, and 2% agar) were used to screen transformants at 30°C. A primary inoculum was prepared from 5 ml of the Ura Ϫ selection medium and cultured for 24 h, and 10 7 cells were inoculated into a 300-ml Erlenmeyer flask containing 40 ml of YEPD medium. Expression cultures were grown at 30°C with continuous agitation (200 ϫ g), after which the cells were harvested and examined for the expression of recombinant proteins.

Vector construction.
To express different levels of LTB and LTB::ApxIIA#5, a low-copy-number integrative vector, YIplac128 (14), and a high-copy-number episomal vector, YEp352 (42), were engineered to express the LTB::ApxIIA#5 fusion proteins and LTB, respectively. We used the same promoter for the two vectors to minimize the discrepancy due to differences in promoter strength. Briefly, the amylase 1A (Ramy1A) signal peptide and the LTB gene (LTB; GenBank accession no. AB011677) were amplified from the plasmid pYEGLTB (35). The ApxIIA epitope gene (ApxIIA#5) encoding amino acid (aa) residues 439 to 801 (ApxIIA; GenBank accession no. AF363362) was amplified from the plasmid pApxIIA5 (26). The resulting two PCR amplicons were fused by overlap extension PCR to incorporate an amino acid hinge (GPGP; underlined sequence) to generate a degree of flexibility between the LTB and ApxIIA#5 using the overlap primers overlap-forward (5Ј-CAATCAGTATGGAAAACGGACC AGGACCACAAGGTTATGATTCTCGT-3Ј) and overlap-reverse (5Ј-CGAGA ATCATAACCTTGTGGTCCTGGTCCGTTTTCCATACTGATTG-3Ј). In addition, BamHI and SalI restriction sites at the 5Ј and 3Ј ends, respectively, were included for the ease of subsequent cloning using the forward primer 5Ј-GGA TCCGCATCCAGGTGCTGAAC-3Ј and the reverse primer 5Ј-GGGTCGACT GTAATAGAATCATT-3Ј. The amplified gene was cloned into pGEM-T Easy vector (Promega), analyzed by restriction enzyme digestion, and confirmed by DNA sequencing. We then cloned the gene encoding the LTB::ApxIIA#5 fusion protein into the BamHI/SalI sites in the integrative pYIGPD-TER vector having the same restriction sites between the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and the galactose-1-P uridyl transferase (GAL7) terminator (pYIGLA5). For the expression of LTB::ApxIIA#5 only, the LTB::ApxIIA#5 fusion gene was placed in the BamHI and SalI sites between the GPD promoter and the GAL7 terminator of the episomal vector pYEGPD-TER (36). In addition, for ApxIIA#5 only, the amylase 1A (Ramy1A) signal peptide and the ApxIIA neutralizing epitope gene (ApxIIA#5) were also placed between the GPD promoter and the GAL7 terminator of the episomal vector pYEGPD-TER. The restriction maps of both the integrative and episomal recombinant plasmids are shown in Fig. 1. For LTB only, transformant TYEGLTB-4 from previous studies (35) was used.
The S. cerevisiae strain was transformed as previously reported (14,20). The transformed cells were selected on either Leu Ϫ or Ura Ϫ selection medium, and the presence of the transforming plasmid was confirmed by either Southern blot analysis or back-transformation of E. coli with DNA prepared from the putative transformants.
The stability of the introduced plasmids in yeast was measured using appropriate selection media according to a previously described procedure (44). Vectors for yeast transformation and recombinant yeast strains constructed in this study are indicated in Table 1 and Table 2, respectively.
Northern blot analysis. Transformants were grown in YEPD medium, and total RNA was prepared as previously described (44). Following electrophoresis on a 1.2% agarose gel in the presence of formaldehyde and transfer to a nylon membrane, hybridization was performed in Church buffer (7% SDS, 1% bovine serum albumin [BSA], 1 mM EDTA, 250 mM Na 2 HPO 4 , pH 7.2) at 65°C. The probe was labeled with [␣-32 P]dCTP using a random labeling kit (Amersham).
Western blot analysis. Preparation of cell extracts (CFE) was conducted as previously described (41). Briefly, cells were grown for 3 days, harvested, washed twice in extraction buffer (50 mM Tris-HCl, 2 mM EDTA), and homogenized seven times in a vortex for 1 min each, with 1-min intervals of ice cooling. The homogenates were observed under a microscope to confirm that the cells had been broken and centrifuged (10 min at 10,000 ϫ g). The resultant supernatant was then filtered through a cellulose filter (pore size, 0.4 m; Amersham) to prepare the cell extract. To obtain the concentrate of culture supernatant, the culture supernatant of recombinant S. cerevisiae was collected by centrifugation at 3,000 ϫ g, concentrated using acetone (45), dialyzed against phosphatebuffered saline (PBS) twice for 4 h at 4°C, and sterilized by passing through a 0.4-m syringe filter. The protein concentrations in the filter-sterilized cell extract were measured via the Bradford assay using the Bio-Rad protein assay kit (Bio-Rad Inc.) (2).
Sample aliquots of the CFE were separated via SDS-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was conducted according to the method of Laemmli (30), with 12% acrylamide separating gels and 5% stacking gels, each containing 1% SDS. Samples were heated at 100°C for 10 min in 125 mM Tris-glycine buffer (pH 6.8) containing 1% SDS and 1% ␤-mercaptoethanol (17). Electrophoresis was performed at a constant current of 10 mA for 3 h using a running buffer of 25 mM Tris-glycine (pH 8.8) containing 0.1% SDS. After electrophoresis, the gel was stained with 0.025% Coomassie brilliant blue R-250.

ELISA quantification of recombinant LTB protein.
The quantity of expressed LTB protein in CFE was determined via quantitative enzyme-linked immunosorbent assay (ELISA) as previously described (23). Briefly, the CFE with 10-fold dilutions were used to coat a 96-well microtitration plate at a total of 100 l per well in bicarbonate buffer (15 mM Na 2 CO 3 and 35 mM NaHCO 3 ) and incubated overnight at 4°C. The wells were washed three times in PBS containing 0.05% Tween 20 (PBST), blocked for 2 h at 37°C via the addition of 300 l of 1% BSA in PBS, and again washed three times in PBST. The plates were incubated with a 1:4,000 dilution of anti-LTB antiserum (Immunology Consultants Laboratory, Inc.) in 0.01 M PBS containing 0.5% BSA for 2 h at 37°C and washed three times with PBST. The plate was then incubated with a 1:7,000 dilution of anti-rabbit IgG conjugated with horseradish peroxidase (Promega) in 0.01 M PBS containing 0.1% BSA and washed three times with PBST buffer. The plate was developed via the addition of 100 l of tetramethylbenzidine (TMB) substrate kit for peroxidase (PharMingen) for 30 min at room temperature in darkness. The plate was read at 405 nm in an ELISA reader (Packard Instrument MRA-006) and quantified via comparison with a known quantity of purified bacterial LTB. The purified bacterial LTB, obtained from the previous study (22), was serially diluted within the range of the concentrations from 0.1 ng to 10 ng. GM1 ganglioside binding assay. GM1 ganglioside has been shown to be the receptor for cholera toxin B subunit (CTB) protein in vivo (5), and for appreciable receptor binding, a pentameric structure of LTB is required (64). Thus, GM1 ganglioside receptor binding in vitro has been used to assess the native pentameric form of recombinant protein. The affinity of yeast-derived LTB for the GM1 ganglioside receptor was determined via GM1-ELISA (28). Briefly, microtiter plates were coated with 0.3 g of monosialoganglioside (Sigma-Aldrich) dissolved in bicarbonate buffer and incubated overnight at 4°C. The wells were washed, blocked, again washed three times in PBST, blocked for 2 h at 37°C via the addition of 300 l of 1% BSA in PBS, and again washed three times in PBST. The wells were then loaded with 250 ng of protein preparations of CFE and then incubated overnight at 4°C. The wells were washed three times in PBST, loaded with 100 l per well of either a 1:4,000 dilution of rabbit anti-LTB primary antibody (Immunology Consultants Laboratory, Inc.) or mouse anti-ApxIIA antiserum (26), and incubated for 3 h at room temperature, followed by three washes with PBST. The remaining steps were conducted as described in the ELISA quantification section above.
Experimental animals, immunization, and sample collections. Oral immunization and sample collections were conducted as described previously (33,55). Briefly, 5-week-old BALB/c female mice (Charles River Technology through Orient Bio, South Korea) were used throughout this study according to the policy and regulations for the care and use of laboratory animals (Laboratory Animal Center, South Korea). All animals were provided with standard mouse chow and water ad libitum.
Oral immunizations for the mice were preceded by an overnight fasting (water was provided ad libitum). Freshly harvested cells (2.5 ϫ 10 7 or 2.5 ϫ 10 8 ) were dissolved into 1 ml of 0.9% saline and orally administered at 200 l per mouse through an oral gavage four times with 10-day intervals. Serum samples were collected 3 days after each immunization. Fecal extract samples were obtained as previously described (27). Briefly, feces as weighted pellets were added to PBS containing 0.1% sodium azide (1 ml/100 mg fecal sample). The pellet was vortex mixed and centrifuged at 13,000 ϫ g for 10 min at 4°C. Supernatants were collected for subsequent assay and stored at Ϫ70°C until use.
Measurement of ApxIIA#5-specific antibody immune responses. The level of antigen-specific antibodies (IgA or IgG) in mouse fecal extract and serum samples was determined using ELISA after the plate was coated with ApxIIA#5 (33). Each well in the ELISA plate was coated with 50 l of 10 g ml Ϫ1 of the recombinant ApxIIA#5 in bicarbonate buffer by incubation overnight at 4°C and then blocked with 10% fetal bovine serum (FBS) (HyClone) in PBST for 2 h at 37°C. After washing the wells with PBS, 50 l of the individual samples, which had been serially diluted in PBS, was added to each well and incubated overnight at 4°C. Goat antibodies specific to mouse IgG (Promega) or IgA (Sigma-Aldrich) were used as a secondary antibody to determine the total amount of antigenspecific antibody, and the substrate was added for color development. The plate was read at 405 nm in an ELISA reader. To quantify the level of antigen-specific antibody with specific isotypes, control IgG was used as a standard as described previously (33).
Challenge assay. Experimental procedures using laboratory animals were approved by the Institutional Animal Care and Use Committee of the Chonbuk National University (approval number CBU 2009-0014) and followed the guidelines suggested by the committee. Control or immunized mice were challenged with A. pleuropneumoniae serotype 2 prepared as described previously (33,55). The immunized mice (n ϭ 10 in each group) were injected intraperitoneally with 200 l of an A. pleuropneumoniae preparation (ϳ1 ϫ 10 8 CFU) or buffer (control). Each mouse was then monitored every 6 h for up to 3 days. The number of mice alive at 3 days after the challenge was recorded; these mice were considered to have been protected from the challenge (34,54).

Expression of the LTB::ApxIIA#5 fusion protein and coexpression of LTB and LTB::ApxIIA#5 in recombinant yeast.
The pYEGLA5 plasmid was constructed as described in Materials and Methods (Fig. 1) and used for the transformation of the S. cerevisiae a7 strain. Over 20 colonies were selected randomly on an Ura Ϫ medium and then examined for the presence of pYEGLA5 via plasmid extraction, followed by PCR amplification of the LTB::ApxIIA#5 fusion gene and reintroduction into E. coli. Among tested transformants, the transformant with the highest expression level, TYELA5-1, was selected using Northern blot analysis and then used for the production and assembly of the recombinant LTB::ApxIIA#5 fusion protein. In addition, the pYEGA5 plasmid was constructed as described in Materials and Methods (Fig. 1) and used for the transformation of the S. cerevisiae a7 strain to express the neutralizing epitope of ApxIIA toxin, ApxIIA#5. The transformant TYEA5-1, showing the highest expression level, was selected and used for further experiments.
For coexpression of LTB and LTB::ApxIIA#5 for heteropentameric formation, a high-copy-number episomal vector, pYEGPD-TER, was engineered to construct the expression plasmid for LTB. Northern blot analysis of the transformants showed that the introduced LTB gene was strongly expressed under the control of the GPD promoter. Transformant TYELTB-1, which expressed the highest levels among the 20 transformants, was selected for subsequent transformation with the LTB::ApxIIA#5 fusion gene. After transformation of TYELTB-1 with a low-copy-number integrative vector (pYIGPD-TER) containing the LTB::ApxIIA#5 fusion gene, transformant TYCO-1 (coexpression of LTB and LTB-APXIIA#5) was selected from 20 candidates that showed similar levels of LTB::ApxIIA#5 fusion gene transcript expression (data not shown).
To produce homopentameric LTB, transformant TYEGLTB-4, which contains an episomal vector containing LTB alone, was obtained from our previous study (35). The plasmid stability of the episomal vector was good, and more than 80% of the plated cells appeared to carry the plasmid 72 h after liquid culture. In addition, no loss of an integrative plasmid was observed (data not shown).
Immunoblot analysis of recombinant yeast. Immunoblot analysis was conducted for the detection of LTB and the LTB::ApxIIA#5 fusion protein using an LTB-specific antibody. As shown in Fig. 2, when samples were loaded on a 10% denaturing SDS-polyacrylamide gel without boiling to avoid reduction of the pentameric structure into monomers, a band of ϳ60 kDa was detected in transformant TYELA5-1, which is designed to express only the LTB::ApxIIA#5 fusion protein.
Moreover, when the samples were boiled to dissociate the pentameric structure into monomers prior to electrophoresis, an 11.6-kDa band, identified as monomeric LTB, was observed in the boiled sample of the cell extract (CFE) of recombinant S. cerevisiae expressing LTB alone as expected (Fig. 2B). In addition, a band of ϳ100 kDa disappeared in the boiled TYCO-1 sample. When samples were boiled, bands of ϳ60 kDa were not observed in transformants TYCO-1 and TYELA5-1. Moreover, a new 11.6-kDa band observed in TYEGLTB-4 was not easily detectable in the boiled TYCO-1 sample (Fig. 2B).
Although immunoblot analysis using an ApxIIA-specific antibody showed several nonspecific bands, comparison with results obtained using an LTB-specific antibody was sufficient to identify the specific band with a molecular mass of ϳ100 kDa in the nonboiled sample of TYCO-1 but not in the boiled sample of TYCO-1. In addition, a band of ϳ60 kDa was observed in transformants TYCO-1 and TYELA5-1 (Fig. 2C). A band of ϳ52 kDa was observed in samples of TYEA5-1 regardless of boiling. However, no band was observed in samples of the recombinant S. cerevisiae expressing LTB alone in TYEGLTB-4 (Fig. 2D).
These results suggested that a band of ϳ100 kDa in TYCO-1 appeared to be the complex structure consisting of LTB and LTB::ApxIIA#5 fusion protein based on the crossreactivity against both LTB-and ApxIIA-specific antibodies. The disappearance of corresponding 100-kDa bands in boiled samples suggested that the LTB-mediated assembly of the corresponding recombinant LTB and LTB::ApxIIA#5 fusion protein subunits into oligomeric structures occurred. However, the presence of bands of ϳ50 kDa and ϳ60 kDa in TYCO-1 corresponding to the LTB homopentamer and the LTB::ApxIIA#5 fusion protein, respectively, suggested that a large quantity of LTB::ApxIIA#5 fusion protein subunits existed that could not assemble into an oligomeric structure, and the formation of homopolymers comprising only LTB is still possible.
It is not uncommon to observe a low affinity of the LTB antibody for the monomeric subunit in boiled samples because the antibody was raised against the pentameric form of LTB (29). Thus, compared with the distinctive band in nonboiled samples, the absence of a cross-reacting monomeric band with the LTB antibody in boiled samples of TYCO-1 and TYELA5-1 was expected. In addition, the absence of a crossreacting monomeric LTB band suggested that the cellular level of monomeric LTB molecules in TYCO-1 was not quite comparable to that of TYEGLTB-4. Moreover, the LTB part in the fusion protein appeared to be heat labile to lose the crossreactivity against LTB antibody, resulting in the lack of a band in boiled samples of TYCO-1 and TYELA5-1. Although immunoblot analysis using an ApxIIA-specific antibody showed a band of ϳ130 kDa in the nonboiled sample but not in the boiled sample of TYELA5-1, it was not easy to determine whether it was a specific pentameric form of the LTB::ApxIIA#5 fusion protein because LTB antibody did not cross-react in the nonboiled sample of TYELA5-1 ( Fig. 2A) and the corresponding band was not observed in the nonboiled sample of TYCO-1 (Fig. 2C).
GM1 ganglioside binding assay. The biological functions of LTB, including its binding to the GM1 ganglioside receptor, are dependent on the formation of a pentameric structure of monomers, and the ability of recombinant heteropentameric LTB and LTB::ApxIIA#5 fusion protein to bind to gangliosides was assessed using 96-well plates coated with GM1 ganglioside. As shown in Fig. 3, yeast-derived recombinant homopentameric LTB and hetero-oligomeric LTB and the LTB::ApxIIA#5 fusion protein of TYEGLTB-4 and TYCO-1, respectively, demonstrated a profound affinity for the GM1 ganglioside, whereas no affinity for the GM1 ganglioside was observed in TYEA5-1, TYELA5-1, and mock-transformant strains. The boiling of yeast-derived recombinant homopentameric LTB and of hetero-oligomeric LTB and the LTB::ApxIIA#5 fusion protein for 10 min induced denaturation of the oligomeric structure and abolished GM1 ganglioside binding (data not shown). Because immunoblotting indicated that the TYELA5-1 strain could express the LTB::ApxIIA#5 fusion protein and pentamerization is essential for binding to the GM1 ganglioside, the results indicated that the intracellular LTB::ApxIIA#5 fusion protein could not assemble into the native pentameric conformation. Moreover, a profound affinity for the GM1 ganglioside demonstrated by coexpression of LTB and the LTB::ApxIIA#5 fusion protein when using anti-ApxIIA antiserum (Fig. 3B) indicated that LTB and the LTB::ApxIIA#5 fusion protein can form an oligomeric configuration, and this configuration appeared to be heteropentameric to interact with the GM1 ganglioside. These results are consistent with those of the immunoblot assay showing the presence of a 100-kDa band only in the nonboiled sample of TYCO-1, but not in the boiled sample or in the sample from TYELA5-1.
To conduct quantitative estimates of recombinant LTB, ApxIIA#5, and the LTB::ApxIIA#5 fusion protein, we used ELISA. The quantity of yeast-derived recombinant LTB and ApxIIA#5 was estimated via comparison of the relative light units (RLUs) from a known quantity of bacterial LTB and ApxIIA#5 protein-antibody complexes. Quantitative ELISA revealed that yields of LTB from TYEGLTB-4 comprised 1.4 to 2.0% of CFE as inferred from comparison with known amounts of LTB (Fig. 3C). The yield of ApxIIA#5 measured using an ApxIIA-specific antibody revealed that transformants TYEA5-1 and TYELA5-1 expressing only ApxIIA#5 and the LTB::ApxIIA#5 fusion protein, respectively, produced recombinant ApxIIA#5 in a range from 0.5 to 1.2% of CFE. However, compared with those of TYEA5-1 and TYELA5-1, the yield of ApxIIA#5 in cotransformant TYCO-1 was low and comprised less than 0.4% of CFE (Fig. 3D).

Induction of systemic immune responses by oral administration of the recombinant yeast.
To evaluate the efficacy of oral administration of ApxIIA#5-expressing recombinant yeast to induce immune responses, the level of ApxIIA#5specific antibodies was measured in the sera of mice fed with the recombinant yeasts TYEA5-1, TYELA5-1, and TYCO-1. As shown in Fig. 4, ELISA revealed that the levels of ApxIIA#5-specific antibodies present in sera of mice fed TYEA5-1, TYELA5-1, and TYCO-1 were higher than those obtained from mice fed the vector-only transformant. In addition, the level of ApxIIA#5-specific antibodies present in the sera of mice fed TYCO-1 was significantly higher than those obtained from mice fed TYEA5-1 and TYELA5-1 transformants.
Specific IgA responses to ApxIIA#5 in the small intestine of mice immunized with recombinant yeasts were also examined. The antibody titer of IgA in the intestine showed a pattern similar to that of the IgG titer in sera, although significant differences were observed from the third immunization. The levels of specific IgG in mice fed TYEA5-1, TYELA5-1, and TYCO-1 were higher than those in control groups. Again, the specific IgA level in mice fed TYCO-1 was significantly higher than those obtained from mice fed TYEA5-1 and TYELA5-1.
In both specific IgG and IgA responses, antigen-specific antibodies were time dependently increased by an oral immunization with the recombinant yeast. For example, antigenspecific antibodies were obviously higher in mice fed with recombinant yeast after 40 days of feeding than in mice fed with yeast after 30 days of feeding.
Induction of protective immune responses against the A. pleuropneumoniae challenge. Because oral administration of the recombinant yeast induced systemic immune responses, the survival of immunized mice after the A. pleuropneumoniae challenge was examined. As shown in Fig. 5, survival rates of the vaccinated groups were higher than those of control groups, such that all mice in the control groups died by 30 h after the challenge whereas the survival rates were 80%, 50%, and 40% in the TYCO-1-, TYELA5-1-, and TYEA5-1-vaccinated groups, respectively, at the same time point. The final survival rates of the TYCO-1-, TYELA5-1-, and TYEA5-1vaccinated groups were 60%, 30%, and 20%, respectively, at 72 h after the challenge, whereas no mouse in either control group survived.
Statistical analysis. Statistical analysis was performed in Excel and SigmaPlot software version 10.0. All results were expressed as the mean Ϯ standard deviation (SD). Student's t test was used to assess differences between the groups. Differences were considered to be significant if probability values of P Ͻ 0.05 were obtained.

DISCUSSION
Many immunogens are coadministered with LTB because it is known to be a potent mucosal adjuvant toward coadministered unrelated antigens. The LTB subunit in a chimeric fusion protein exhibits a strong mucosal adjuvant effect as a carrier molecule, whereas the foreign antigenic determinants fused to the LTB subunit can be delivered across the mucosal epithelium to the underlying mucosa-associated lymphoid tissue to stimulate the mucosal immune system by mediating the induction of antigen-specific serum IgG and mucosal IgA. However, the application of LTB as an immunogenic molecule and as a potent immunoadjuvant depends on the formation of a pentameric association of LTB binding with GM1 ganglioside receptors on the surface of intestinal epithelial cells. Thus, LTB is of utmost interest for mucosal vaccine development, and its capability for binding to the cell receptor depending on pentameric formation has gained more attention for its application as an immunopotentiating carrier molecule (6,32,50). The biogenesis of oligomeric proteins is a complex phenomenon involving polypeptide folding into structures that allow stable subunit interactions. Studies regarding the reassociation of denatured proteins have established that the steric information required for subunit-subunit interactions is contained within the constituent polypeptide chains (49). Several studies on the expression of bacterial LTB subunits have shown that the bacterial subunit can assemble into a functional pentameric protein under physiological conditions of eukaryotes, including plants and yeast (18,35,52). In addition, some chimeric fusion proteins, i.e., LTB fused with other epitope proteins such as classical swine fever virus (32), infectious bursal disease virus (11), porcine epidemic diarrhea virus (23), synapsin peptide (50), and porcine reproductive and respiratory syndrome virus (3) proteins, could oligomerize into a pentameric assembly. However, as a fusion partner becomes larger, structural hindrance or repulsion between the fusion partner and the adjacent LTB subunits, which occurs in the geometry of the oligomerization, is likely to be the inhibitor in the pentameric assembly of the fused subunits. LTB should thus be suitable for targeting not only small antigens but also larger antigens to the mucosal epithelium.
Although it has been shown that large proteins (Ͼ100 kDa) could be fused to the C-terminal end of LTB with a retained ability to bind GM1 (21), others have shown that a C-terminal fusion to LTB inhibited pentamer formation (4,32). Although some exceptions exist depending on the intrinsic characteristics of the protein, as a fusion partner, smaller-epitope proteins are preferable because their size does not disturb the subunit interaction. Accordingly, for the chimeric fusion proteins included in the current study, which show that the LTB::ApxIIA#5 fusion protein could not assemble into a pentameric structure, size limitation can be a problem in cases where the expression of a large subunit is preferred or required for efficient protection or prevention. Thus, coexpression of LTB and the LTB::ApxIIA#5 fusion protein was suggested, as previously described (57). Based on our previous studies expressing heteromers with different subunit ratios, a differential expression system is preferred to an equimolar expression system, and hetero-oligomerization was achieved even with respect to the preferable ratio between subunits, which was as-cribed to the characteristics of the native state of the holoprotein itself. Accordingly, although the current studies on LTB and the LTB::ApxIIA#5 fusion protein differed from the previous study in that it was not possible to predict what ratio can be achieved (i.e., homopentameric oligomerization by LTB alone or heteropentameric oligomerization by LTB and the LTB::ApxIIA#5 fusion protein), the differential expression system is preferred to an equimolar expression system because of the steric hindrance among LTB::ApxIIA#5 fusion proteins. If steric hindrance can be avoided by the incorporation of LTB in the pentamer, more pentamers can be assembled with less incorporation of the LTB::ApxIIA#5 fusion protein, resulting in a differential molar ratio between LTB and the LTB::ApxIIA#5 fusion protein. Even in the differential expression, Western blot analysis indicated that considerable amounts of unassembled LTB::ApxIIA#5 fusion protein subunits still exist, suggesting that further studies are necessary to decide the optimum ratio between LTB and the LTB::ApxIIA#5 fusion protein as well as the optimal physiological environment for heteromeric assembly. If the expression levels of transformants with an identical integrative vector vary according to where the vector is integrated, these random integrative transformants can be used to obtain a broad spectrum of different combinations of LTB and the LTB::ApxIIA#5 fusion protein in vivo. Accordingly, it will be interesting to analyze other integrative transformants to determine the function-structure relationship of the heteropentamers.
Oral administration of the recombinant yeasts was conducted based on the equal number of the cells due to the difference in the amount of the expressed recombinant protein level. The expression levels of ApxIIA#5 differed among TYEA5-1, TYELA5-1, and TYCO-1. The difference based on the quantitative ELISA suggested that, compared with TYCO-1, more than two and more than four times the number of ApxIIA#5 proteins were expressed in TYELA5-1 and TYEA5-1, respectively. Higher expression levels of ApxIIA#5 in TYEA5-1 and TYELA5-1 were not uncommon because compared with TYCO-1, a high-copy-number episomal vector was used to direct the expression of ApxIIA#5 in both strains. In addition, the TYCO-1 strain was designed to express additional LTB only via the high-copy-number episomal vector, resulting in a severe metabolic burden to express two different proteins in the same cell. Thus, the lower expression level of ApxIIA#5 in TYCO-1 is not uncommon. However, several studies have shown that the antigens chemically or genetically coupled to LTB allowed the use of a very small antigen dose with great effectiveness (19,66). In the present study, the immune response to the coexpressed ApxIIA#5 in TYCO-1, despite the lower expression level, was significantly higher than that of the LTB::ApxIIA#5 fusion protein or ApxIIA#5. Concomitantly, the protection assay with TYCO-1 showed the best protection against the pathogen infection. These results indicated that the LTB::ApxIIA#5 fusion protein in heteropentameric formation can induce A. pleuropneumoniae-specific antibodies in mice. The efficacy of the coexpression of LTB and the LTB::ApxIIA#5 fusion protein as an oral antigen was significantly increased due to the heteropentameric assembly, which can be ascribed to the binding capability of the antigen to the GM1 ganglioside for an efficient mucosal entry. These better efficacies in protection are consistent with the results of VOL. 18,2011 HETEROPENTAMERIC FORMATION BY COEXPRESSION IN YEAST 2175 the ELISA showing higher specific IgG and IgA levels in mice immunized with TYCO-1 than in those immunized with TYEA5-1 and TYELA5-1. Our previous studies indicated that although oral immunization with recombinant ApxIIA protein was possible (26), improved strategies for efficient antigen presentation and immune responses are still needed for enhanced protection against pathogen infection. Thus, the pentameric formation itself is a good strategy for better delivery to achieve improved mucosal immunization, and it is expected that coexpression allowing a large fusion protein to assemble into the pentameric formation for biological function can be applied in several other biotechnological approaches, including enhanced oral vaccine development.