An oral delivery system based on ApxIIA#5-expressed on Saccharomyces cerevisiae was studied for its potential to induce immune responses in mice. Murine bone marrow-derived dendritic cells (DCs) stimulated in vitro with ApxIIA#5-expressed on S. cerevisiae upregulated the expression of maturation and activation markers, leading to production of tumor necrosis factor-α, interleukin (IL)-1β, IL-12p70 and IL-10. Presentation of these activated DCs to cluster of differentiation CD4+ T cells collected from mice that had been orally immunized with the ApxIIA#5-expressed on S. cerevisiae elicited specific T-cell proliferation. In addition, the orally immunized mice had stronger antigen-specific serum IgG and IgA antibody responses and larger numbers of antigen-specific IgG and IgA antibody-secreting cells in their spleens, Peyer's patches and lamina propria than did those immunized with vector-only S. cerevisiae or those not immunized. Furthermore, oral immunization induced T helper 1-type immune responses mediated via increased serum concentrations of IgG2a and an increase predominantly of IFN-γ-producing cells in their spleens and lamina propria. Our findings suggest that surface-displayed ApxIIA#5-expressed on S. cerevisiae may be a promising candidate for an oral vaccine delivery system for eliciting systemic and mucosal immunity.
- A. pleuropneumoniae
cluster of differentiation
CD11chigh conventional DC
carboxyfluorescein succinimidyl ester
enzyme-linked immunosorbent spot
generally recognized as safe
granulocyte-macrophage colony-stimulating factor
major histocompatibility complex
PBS containing triton X-100
- S. cerevisiae
standard error of the mean
tumor necrosis factor-α
yeast extract-peptone-dextrose growth medium
Saccharomyces cerevisiae, which is typically used in oral vaccines and drugs, is classified as a GRAS organism [1, 2]. Currently, there is great interest in developing mucosal, particularly oral, vaccines, because such vaccines would not only induce locally and systemically protective immune responses against infectious disease, but would also be safe and convenient to administer. Several oral delivery systems using live oral vaccines such as a Salmonella typhimurium mutant, Lactobacillus spp., or S. cerevisiae [3-5] have been attempted. Among these delivery systems, the S. cerevisiae yeast expression system has several advantages: high expression levels, ease of scale-up, low cost and the adjuvant potential of yeast cell-wall components such as β-1,3-D-glucan and mannan . Yeast-based expression systems have been developed and successfully used to produce recombinant proteins [2, 6]. These systems have been employed in pharmaceutical, livestock feed and food industry applications .
Recently, the genetic engineering technique of yeast cell-surface display has been used to display heterologous proteins on the surfaces of yeast cells [2, 7-9]. This system could be a good candidate for a live oral vaccine carrier because it stably maintains surface-expressed epitopes with a high density of proteins .
Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a highly contagious endemic disease of pigs that results in significant economic losses worldwide [10, 11]. A. pleuropneumoniae can result in various clinical signs ranging from peracute to chronic, infected pigs typically having hemorrhagic, necrotizing pneumonia, often associated with fibrinous pleuritis . The ApxII toxin, which is believed to be involved in the virulence of A. pleuropneumoniae, has been used as a vaccine protein . The antigenic determinant of ApxIIA (ApxIIA#5) has been shown to induce a strong protective immune response against A. pleuropneumoniae . ApxIIA, expressed in either S. cerevisiae or Nicotiana tabacum, has previously been reported to be capable of inducing protective immune responses against A. pleuropneumoniae in mice [3, 12, 14]. Moreover, surface-displayed expression of ApxIIA#5 on S. cerevisiae has been studied and induction of antigen-specific immune responses and protection against A. pleuropneumoniae in mice assessed . In the present study, we demonstrated that surface-displayed expression of ApxIIA#5 on S. cerevisiae has immunogenic potential as an oral vaccine that could help to improve both systemic and mucosal immune responses.
1 MATERIALS AND METHODS
1.1 Preparation of vaccines and oral vaccination of mice
Saccharomyces cerevisiae expressing surface-displayed ApxIIA#5 was prepared as previously described . Briefly, the yeast was cultured in a selective medium (uracil-deficient medium: casamino acid 5 g, yeast nitrogen base 6.7 g, glucose 20 g, adenine 0.03 g and tryptophan 0.03 g in 1 L of DW) for 16 hrs at 30°C and then transferred and cultured in basic medium (YEPD: yeast extract 10 g, bacto peptone 20 g and glucose 20 g in 1 L of DW) for 3 days at 30°C. Yeast harboring a control vector or yeast expressing surface-displayed ApxIIA#5 was washed in saline and diluted to a titer of 5 × 108 cells/mL in PBS.
Five-week-old female C57BL/6 mice (Central Lab Animal Inc., Seoul, Korea) were used in this study, which was conducted in accordance with the policies and regulations of the care and use of laboratory animals of the Institute of Laboratory Animal Resources, Seoul National University, Korea. All the animals were provided with standard mouse chow and water ad libitum. 1.5 × 109 cells/day per mouse of surface-displayed ApxIIA#5 expressed on S. cerevisiae (vaccinated group) and vector-only S. cerevisiae (vector control group) were administered by oral gavage for two days on each occasion at 10-day intervals. Nontreated mice were also maintained as a mock control. Specimens and serum samples were collected 3 days after each immunization.
1.2 Preparation of primary cells
Murine DCs were isolated from bone marrow progenitors according to previously described procedures . The bone marrow cells were cultured in RPMI 1640 medium (Gibco Invitrogen, Karlsruhe, Germany) in the presence of 10% heat-inactivated FBS (Gibco Invitrogen), 10 ng/mL recombinant murine GM-CSF (PeproTech, London, UK) and 5 ng/mL recombinant IL-4 (PeproTech). Non-adherent cells were collected and used for further experiments on Day 10. The purity of the cells, assessed by flow cytometry using phycoerythrin-conjugated anti-CD11c mAb (Abcam, Cambridge, UK), was 91.1 ± 0.92%. Single cell suspensions were obtained from samples of SP, intestinal LP and PP for T-cell proliferation and ELISPOT assays, as previously described [16, 17].
1.3 Activation of dendritic cells by transgenic Saccharomyces cerevisiae
To examine the in vitro activation of the DCs by transgenic S. cerevisiae, immature DCs (1 × 106 cells/mL) were stimulated with surface-displayed ApxIIA#5 expressed on S. cerevisiae or vector-only S. cerevisiae (1 × 106 cells/mL). After 48 hrs, the cells were harvested for flow cytometry, and supernatants collected and stored at −80°C until the analysis of cytokine secretion by quantitative ELISA. The secreted concentrations of TNF-α, IL-1β, IL-10 and IL-12p70 were measured using the ELISA method (eBioscience, San Diego, CA, USA). The activation and upregulation of costimulatory molecules in the DCs were examined using a FACScalibur flow cytometer (BD Biosciences, San Jose, CA, USA). After harvesting, the DCs were washed and fixed with 4% paraformaldehyde in PBS, followed by labeling with fluorescein isothiocyanate-conjugated antibodies against MHC class II, CD40 and CD86 (all from Abcam). CellQuest software (BD Biosciences) was used to analyze the flow cytometry data.
1.4 Carboxyfluorescein succinimidyl ester-based T-cell proliferation assay
One week after the final administration, T cells were isolated from the spleens of mice immunized with surface-displayed ApxIIA#5 expressed on S. cerevisiae, vector-only S. cerevisiae, and those that were not immunized. The cells were labeled with CFSE according to previously described procedures . The labeled cells (5 × 106 cells) were cultured for 4 days with Apx-activated DCs (1 × 106 cells) and stained with antimouse CD4 PE monoclonal antibody (Abcam) for 45 mins at 4°C. The cells were then washed twice with Dulbecco's PBS (Gibco Invitrogen), which contains 5% FBS, and fixed with 4% paraformaldehyde. The cells were acquired on a FACScalibur flow cytometer (BD Biosciences) and then analyzed using FlowJo software (version 7.6.5, Tree Star, San Carlo, CA, USA). The percentage of CFSE-low cells was expressed as the mean ± SEM.
1.5 Enzyme-linked immunosorbent assays
Enzyme-linked immunosorbent assay was used to quantify antigen-specific IgG and IgA antibodies in the serum samples by slight modification of an assay described previously . The plates were coated with 100 pg of recombinant ApxIIA suspended in 100 μL of PBS and blocked with PBST containing 1% BSA (Amresco, Solon, OH, USA). The diluted sera (1:20) were added to the plates and horseradish peroxidase-conjugated goat antimouse IgG (H + L) (Bio-Rad, Hercules, CA, USA), horseradish peroxidase-conjugated antimouse IgA (α-chain specific; Bethyl Laboratories, Montgomery, TX, USA) or horseradish peroxidase-conjugated antimouse IgG1/IgG2a (Serotec, Oxford, UK) (1:2000 in PBST containing 1% BSA) were used as secondary antibodies. Color development was carried out using a TMB substrate (Sigma, St. Louis, MO, USA). The TMB reaction was stopped with 2 M H2SO4 and measured at 450 nm using an Emax Precision microplate reader (MDS, Sunnyvale, CA, USA).
1.6 Detection of interferon-γ-, interleukin-4-, IgG-, or IgA-producing cells by enzyme-linked immunosorbent spot assay
The frequencies of specific cytokine- and antibody-producing cells in SP, LP and PP cell suspensions were assayed with an ELISPOT assay kit for mouse IFN-γ, IL-4, IgG, or IgA according to the manufacturer's instructions (Mabtech, Stockholm, Sweden). Spots were counted using an automated reader.
1.7 Statistical analysis
Statistical significance (P-values) was calculated using Tukey's test with the statistical program Statistical Package for Social Sciences software (version 17.0; SPSS, Chicago, IL, USA). Differences were considered significant if a value of P < 0.05 was obtained. All experiments were repeated at least three times.
2.1 Activation of dendritic cells by surface-displayed ApxIIA#5 expressed on Saccharomyces cerevisiae
After optimizing the concentrations of transgenic S. cerevisiae for DCs, they were stimulated with different ratios of DCs (transgenic S. cerevisiae 4:1, 1:1 or 1:4) and the activity of the DCs determined by expression of CD86 marker. When a ratio of 1:1 was used (data not shown), surface-displayed ApxIIA#5 expressed on S. cerevisiae showed the greatest differences from vector-only S. cerevisiae. Treatment of immature DCs with surface-displayed ApxIIA#5 expressed on S. cerevisiae or vector-only S. cerevisiae (1:1) induced significant upregulation of surface MHC class II molecules and CD40 and CD86 activation markers (P < 0.05; Table 1). The DC-stimulatory potential of the surface-displayed ApxIIA#5 expressed on S. cerevisiae was also shown by induction of the cytokines TNF-α, IL-12p70, IL-1β and IL-10 (Fig. 1). Compared with vector-only S. cerevisiae, surface-displayed ApxIIA#5 expressed on S. cerevisiae was sufficient to induce strong secretion of the proinflammatory cytokines TNF-α, IL-12p70 and IL-1β and the Th2-inducing cytokine IL-10.
|CD40||MHC class II||CD86|
|Mock control||11.32 ± 2.09||26.4 ± 1.13||18.86 ± 0.64|
|Vector control||60.3 ± 3.11||65.2 ± 2.40||62.75 ± 1.41|
|Surface-displayed ApxIIA#5 expressed on S. cerevisiae||68.1 ± 0.57||76.05 ± 1.48*||72.075 ± 0.07*|
2.2 T-cell proliferation after restimulation with ApxIIA-activated dendritic cells
Dendritic cells were stimulated with recombinant ApxIIA to produce ApxIIA-activated DCs and then presented to T cells from the experimental mice. T-cell proliferation was analyzed by examining the CFSE division profiles. The mock control and the vector control groups appeared to have similar percentages of CFSE-low cells, 51.4% and 51.6%, respectively; however, the vaccinated group showed enhanced CD4+ T-cell proliferation, with 81.8% CFSE-low cells. CD4+ T-cell proliferation was four times greater in the vaccinated group than in the control groups (P < 0.001). Presentation of ApxIIA on activated DCs to T cells taken from the experimental mice after the third immunization elicited specific proliferation of CD4+ T cells (Fig. 2).
2.3 Enhancement of antigen-specific IgG and IgA antibody responses in mice orally immunized with the surface-displayed ApxIIA#5 expressed on Saccharomyces cerevisiae
To assess the potential of the surface-displayed antigen expressed on S. cerevisiae in an oral delivery system, antigen-specific antibody responses were determined in sera and cell suspensions from the SP, LP and PP of mice orally immunized with vector-only S. cerevisiae and surface-displayed ApxIIA#5 expressed on S. cerevisiae. As shown in Figure 3, high IgG and IgA antibody activities were maintained in the sera of the vaccinated group after the final immunization. The group immunized with surface-displayed ApxIIA#5 expressed on S. cerevisiae showed higher specific IgA responses to ApxIIA in sera than did those treated with vector-only S. cerevisiae (P < 0.05).
The numbers of antigen-specific IgG and IgA antibody-producing B cells increased significantly in the SP, PP and LP of the vaccinated group (P < 0.05; Fig. 4). In particular, the numbers of antigen-specific IgA antibody-producing cells in the PP were significantly higher than those in the LP and SP.
2.4 Induction of T helper 1-type responses in mice immunized via the oral route with surface-displayed ApxIIA#5 expressed on Saccharomyces cerevisiae
IgG subclasses were assessed to determine the basis of the Th1- and Th2-type immune responses induced in the serum of the mice immunized via the oral route with surface-displayed ApxIIA#5 expressed on S. cerevisiae. There were no differences among the experimental groups in the ApxIIA-specific IgG1 (Th2) subclass, whereas the ApxIIA-specific IgG2a (Th1) subclass increased significantly in the vaccinated group (P < 0.01; Fig. 3). In the SP and CD4+ T cells, IL-4 producing cells were more numerous in the vaccinated mice than in the control mice. In particular, IFN-γ-producing cells were both more predominant and more numerous in the LP, SP and CD4+ T cells of the vaccinated mice than in those of the control groups (P < 0.05; Fig. 5). Collectively, there were fewer Th2-promoting cytokine cells (IL-4) than Th1-promoting cytokine cells (IFN-γ).
In our previous study, we developed surface-displayed ApxIIA#5 expressed on S. cerevisiae and full ApxIIA-expressing S. cerevisiae and demonstrated that oral immunization of mice induced antigen-specific immune responses and protection against A. pleuropneumoniae [3, 9]. However, to develop an efficient oral vaccine, further study of the mucosal immune responses induced by transgenic S. cerevisiae was needed.
We selected surface-displayed ApxIIA#5 expressed on S. cerevisiae as an oral vaccine for porcine pleuropneumonia. In mice, it has greater specific antibody activities than other yeasts, including ApxIIA#5-secreting S. cerevisiae and full-ApxIIA expressing S. cerevisiae . As APCs, DCs induce primary immune responses and have a key role in both innate and adaptive immunity . In adaptive immune responses, the phenotype and function of DCs determine the initiation of tolerance, memory and polarized Th1 and Th2 differentiation . Stimulation of bone marrow-derived DCs with surface-displayed ApxIIA#5 expressed on S. cerevisiae in vitro indicated that this could generally induce secretion of the proinflammatory cytokines TNF-α and IL-1β, the Th1-inducing cytokine IL-12p70 and the Th2-inducing cytokine IL-10. Moreover, maturation of the APCs was confirmed by showing upregulation of CD40 and CD86 costimulatory molecules and surface MHC class II, all of which are required for efficient stimulation of T cells .
Mucosal protection requires generation of antigen-specific T cells and antibodies . In addition, following ablation of immune responses after oral and nasal immunization of mice depleted of cDCs in vivo, cDCs are reportedly essential for activation of CD4+ T cells and generation of specific antibodies . In the present study, we demonstrated that surface-displayed ApxIIA#5 expressed on S. cerevisiae helped to improve both systemic and mucosal immune responses in mice by generating antigen-specific antibodies and encouraging proliferation of CD4+ T cells, which were stimulated by DCs activated by oral vaccination.
Presentation of ApxIIA on activated DCs to CD4+ T cells from mice in the vaccinated group elicited specific T-cell proliferation. The induction of ApxIIA-specific T-cell proliferation demonstrated that ApxIIA was indeed presented on DCs and that the orally administered surface-displayed ApxIIA#5 expressed on S. cerevisiae induced cellular immune responses in mice. Both serum Ag-specific IgG and Ag-specific IgA antibody activities increased in the vaccinated group. Furthermore, both Apx-specific IgG and IgA antibody-producing cells in the PP, LP and SP were significantly more numerous in the vaccinated group than in the control group. PP and LP serve as inductive and effector sites, respectively, of intestinal mucosal immune reactions . Therefore, the present results support previous findings that surface-displayed ApxIIA#5 expressed on S. cerevisiae helps to improve mucosal immune response.
The ApxIIA-specific IgG2a subclass was significantly higher in sera of the vaccinated group than in those of the control groups. Although specific IL-4 cytokine-producing cells were considerably more numerous in the SP of the vaccinated group, specific IFN-γ-producing cells were the predominant cells produced in the LP and the SP of the vaccinated group. Consequently, the preponderance of IFN-γ responses and the ApxIIA-specific IgG2a subclass indicated the induction of a Th1-type immune response. The lymphocyte population in the PP is composed of 80% B cells and 18% T cells, and the LP lymphocyte population is composed of 60% T cells and 32% B cells . We found increased numbers of IgG- and IgA-secreting cells and IFN-γ-producing cells predominantly in the PP and the LP, respectively. These results suggest that oral administration of surface-displayed ApxIIA#5 expressed on S. cerevisiae induces both systemic and mucosal immune responses in mice. Thus, the results of this study contribute to the application of S. cerevisiae as a live oral vaccine that has been engineered by yeast cell-surface display techniques.
This study was supported by ARPC (107034-03), the BioGreen 21 Program (PJ007044), the BK21 Program for Veterinary Science and the Research Institute of Veterinary Science, Seoul National University, Korea.
All the authors have no conflicts of interest.