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Appl Environ Microbiol. 2009 December; 75(24): 7739–7744.
Published online 2009 October 16. doi:  10.1128/AEM.01965-09
PMCID: PMC2794117

Bacterial Polyester Inclusions Engineered To Display Vaccine Candidate Antigens for Use as a Novel Class of Safe and Efficient Vaccine Delivery Agents[down-pointing small open triangle]

Abstract

Bioengineered bacterial polyester inclusions have the potential to be used as a vaccine delivery system. The biopolyester beads were engineered to display a fusion protein of the polyester synthase PhaC and the two key antigens involved in immune response to the infectious agent that causes tuberculosis, Mycobacterium tuberculosis, notably antigen 85A (Ag85A) and the 6-kDa early secreted antigenic target (ESAT-6) from Mycobacterium tuberculosis. Polyester beads displaying the respective fusion protein at a high density were successfully produced (henceforth called Ag85A-ESAT-6 beads) by recombinant Escherichia coli. The ability of the Ag85A-ESAT-6 beads to enhance mouse immunity to the displayed antigens was investigated. The beads were not toxic to the animals, as determined by weight gain and absence of lesions at the inoculation site in immunized animals. In vivo injection of the Ag85A-ESAT-6 beads in mice induced significant humoral and cell-mediated immune responses to both Ag85A and ESAT-6. Vaccination with Ag85A-ESAT-6 beads was efficient at stimulating immunity on their own, and this ability was enhanced by administration of the beads in an oil-in-water emulsion. In addition, vaccination with the Ag85A-ESAT-6 beads induced significantly stronger humoral and cell-mediated immune responses than vaccination with an equivalent dose of the fusion protein Ag85A-ESAT-6 alone. The immune response induced by the beads was of a mixed Th1/Th2 nature, as assessed from the induction of the cytokine gamma interferon (Th1 immune response) and increased levels of immunoglobulin G1 (Th2 immune response). Hence, engineered biopolyester beads displaying foreign antigens represent a new class of versatile, safe, and biocompatible vaccines.

Bioengineered nano-/microstructures manufactured by microorganisms are becoming increasingly attractive because of their functional properties suitable for applications in various fields, particularly the medical sciences (9, 25, 29). Biopolyester beads comprising polyhydroxyalkanoate (PHA) are produced as intracellular inclusions by a wide range of bacteria and archaea when a carbon source is available in excess (30). PHA synthesis requires the key enzyme, polyester synthase, to catalyze the stereoselective polymerization of (R)-3-hydroxyacyl-coenzyme A to PHA. Self-assembly of polyester chains results in the formation of polymer granules with a hydrophobic core, and the PHA synthase protein remains covalently attached at the surface (28). These spherical granules range in size from 50 to 300 nm and accumulate in the intracellular space (34).

Such biopolyester beads can be engineered to display the PHA synthase protein and its fusion partners on the surface at a high density (24). There have been recent examples where biopolyester beads were specifically engineered, produced in bacteria, and then harvested for their potential applications as life science tools. For example, biopolyester beads have been produced which display the immunoglobulin G (IgG) binding domain ZZ from protein A (6) for use as an alternative to protein A latex beads for a variety of diagnostic tests. Another study produced beads which displayed green fluorescent protein to enable tracking following in vivo administration (23). Beads have been developed with covalently attached enzymes, suggesting an application in immobilization and stabilization of biocatalysts (22). Recently, biopolyester beads have been produced which display immobilized antibody single-chain fragments as well as multiple binding functions, including the binding of inorganic compounds (4, 11, 14).

Our interest in these biopolyester beads is to explore their properties for use as vaccine delivery agents. Potential advantages associated with using these beads as vaccine delivery agents include their size, versatility, and inherent biocompatibility with living tissues. Particles smaller than 2 μm in size are readily phagocytosed by macrophages and dendritic cells (20), suggesting the value of using nano-/microsized particles as vaccine delivery systems. The concept of using nano-/microparticles for delivering vaccines has already been explored; for example, biodegradable biocompatible polyesters polylactide and poly-d,l-lactide-co-glycolic acid have been used as vaccine delivery systems (31) or carriers of adjuvant systems (15). Employing PHA beads for delivery of vaccines may present additional advantages, such as low cost, ease of production, and mode of surface functionalization. Novel vaccines are required for a variety of infectious diseases, including tuberculosis, for which no truly efficacious vaccine has yet been designed (16). A number of antigens have been considered for developing new tuberculosis vaccines (3, 19, 33). Early secreted antigenic target 6-kDa protein (ESAT-6) is found in Mycobacterium bovis and Mycobacterium tuberculosis but not in the vaccine strain Mycobacterium bovis BCG (12). This antigen is recognized immunologically in tuberculosis-infected humans (27), cattle (26), and mice (5). The Ag85 complex is composed of three homologous proteins, Ag85A, Ag85B, and Ag85C (1). Ag85A has been used in a number of immunization studies and has been shown to elicit an immune response and, in some cases, enhanced protection (10, 13).

This paper describes the development and microbial production of bioengineered biopolyester beads displaying on their surfaces a functional antigen comprising a fusion protein of polyester synthase, Ag85A, and ESAT-6 and subsequent evaluation of antigen-specific immune responses in immunized mice.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

Escherichia coli DH5α (Invitrogen, CA) was grown in Luria broth (Difco, Detroit, MI) supplemented with 1% (wt/vol) glucose and ampicillin (75 μg/ml). Medium for growth of E. coli BL21(DE3) (Invitrogen) in addition contained chloramphenicol (30 μg/ml).

Plasmids, oligonucleotides, and construction of plasmids for production of Ag85A-ESAT-6 displaying polyester beads.

All plasmids and oligonucleotides are listed in Table Table1.1. DNA sequences of new plasmid constructs were verified by DNA sequencing. In addition to the polyester synthase gene (phaC), PHA biosynthesis requires the enzymes PhaA and PhaB for precursor synthesis, and these enzymes were encoded by plasmid pMCS69. The plasmid DK1.2-Ag85A-ESAT-6 containing a hybrid gene comprised of the coding region (without the secretory signal sequence) of Ag85A (N-terminal component) and the coding region of ESAT-6 (C-terminal component) was a kind gift from Lynne Slobbe, University of Otago, New Zealand (35). A DNA fragment, encoding the Ag85A-ESAT-6 fusion protein and including a translation initiation site and start codon, was isolated from this plasmid by PCR analysis using primers Ag85A-SpeI and ESAT-6-SpeI and ligated into the vector pCWE SpeI at the SpeI restriction site. The resultant construct, pCWE SpeI-Ag85A-ESAT-6, had the mycobacterial fusion gene at the N terminus of PhaC and downstream of plac. Bacteria transformed with this plasmid did not produce detectable levels of polyester, and consequently a second construct was made using the pET-14b-based plasmid pHAS, which contains the T7 promoter. The DNA segment comprising the Ag85A-ESAT-6-phaC hybrid gene was obtained by hydrolysis of pCWE SpeI-Ag85A-ESAT-6 with XbaI and ClaI and subcloned into pHAS using the restriction sites XbaI and ClaI of pHAS to generate the plasmid pHAS-Ag85A-ESAT-6. This construct was used to produce biopolyester beads displaying the vaccine candidate antigen Ag85A-ESAT-6 evaluated in this study.

TABLE 1.
Bacterial strains and plasmids used in this study

In vivo functionality of the polymer-synthesizing enzyme PhaC.

To assess whether the PhaC fusion partner still catalyzes polyester synthesis and mediates intracellular granule formation, the polyester content of bacterial cells harboring the various plasmids was assessed by gas chromatography-mass spectroscopy (GC-MS) analysis. The amount of accumulated polyester corresponds to the in vivo PhaC activity. Polyester content was quantitatively determined by GC-MS after conversion of the polyester into 3-hydroxymethyl ester by acid-catalyzed methanolysis.

Isolation of polyester granules.

Polyester granules were isolated as previously described (24). Briefly, bacteria were disrupted and the whole-cell lysate was centrifuged at 4,000 × g for 15 min at 4°C to sediment the polyester beads. Beads were purified via glycerol gradient ultracentrifugation.

Analysis of proteins attached to the polyester beads.

The concentration of proteins attached to the beads was determined using the Bio-Rad protein assay, (Bio-Rad, CA). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using NuPAGE gels (Invitrogen) and stained with SimplyBlue safe stain (Invitrogen). The amount of Ag85A-ESAT-6 PhaC fusion protein relative to the amount of total proteins attached to the beads was detected using Gel Doc XR and analyzed using Quantity One software (version 4.6.2) (Bio-Rad, Hercules, CA). Proteins of interest were excised from the gels and subjected to tryptic peptide fingerprinting using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Enzyme-linked immunosorbent assay (ELISA).

MaxiSorp plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with purified Ag85A-ESAT-6 beads or wild-type beads, diluted in carbonate-bicarbonate coating buffer, pH 9.6 (Sigma-Aldrich), ranging from 1 mg/ml to 0.015 mg/ml protein concentration over serial dilutions. Plates were washed with phosphate-buffered saline (PBS) containing 0.05% (vol/vol) Tween 20 (PBST) and blocked with 3% (wt/vol) bovine serum albumin (BSA) in PBS for 2 h at 25°C. Plates were then washed in PBST and incubated with mouse antibody to ESAT-6 (Abcam, Cambridge, United Kingdom). Following washing with PBST, plates were incubated for 1 h at room temperature with anti-mouse IgG-horseradish peroxidase (HRP) conjugate (Sigma-Aldrich) diluted in 1% (wt/vol) bovine serum albumin in PBS. After further washing, o-phenylenediamine (OPD) substrate (Sigma-Aldrich) was added and incubated for 30 min at room temperature. The reaction was stopped with 0.5 M H2SO4, and the absorbance was recorded at 495 nm on a VersaMax microplate reader.

Flow cytometry.

Twenty-five micrograms of purified Ag85A-ESAT-6 beads or wild-type beads were washed twice in ice-cold flow cytometry buffer (PBS, 1% [wt/vol] fetal calf serum [FCS], 0.1% [wt/vol] sodium azide) and detected with mouse anti-ESAT-6 antibodies (Abcam, Cambridge, United Kingdom). Following washing in flow cytometry buffer, beads were stained with rat anti-mouse fluorescein isothiocyanate-labeled antibody (BD Pharmingen, CA), incubated for 30 min on ice in the dark, and washed again. A BD FACSCalibur (BD Biosciences, CA) was used to collect at least 10,000 events for each sample, and they were analyzed using CellQuest software.

Immunization of mice.

All animal experiments were approved by the AgResearch Grasslands Animal Ethics Committee (Palmerston North, New Zealand). Groups of female C57BL/6 mice (purchased from the animal breeding facility of the Malaghan Institute of Medical Research, Wellington, New Zealand) aged 6 to 8 weeks old were immunized by the subcutaneous route three times at 2-week intervals with wild-type beads, Ag85A-ESAT-6 beads, or recombinant Ag85A-ESAT-6 protein either alone or mixed with 20% (vol/vol) Emulsigen (MVP Laboratories, Omaha, NE) adjuvant (100 μl/injection). Nonimmunized or PBS-immunized control animals were included in each set of experiments.

Immunological assays.

Three weeks after the last immunization, all mice were anesthetized intraperitoneally using 87 μg ketamine (Parnell Laboratories, Alexandria, NSW, Australia) and 2.6 μg xylazine hydrochloride (Bayer, Leverkusen, Germany) per gram of body weight. Blood was collected by cardiac puncture, allowed to clot, and centrifuged prior to collecting and freezing serum at −20°C until it was assayed. Mice were euthanized, the spleens were removed, and a single-cell suspension was prepared by passaging through an 80-gauge wire mesh sieve. Spleen red blood cells were lysed using a solution of 17 mM Tris-HCl and 140 mM NH4Cl. After washing, the cells were cultured in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 2 mM glutamine (Invitrogen), 100 U/ml penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen), 5 × 10−5 M 2-mercaptoethanol (Sigma), and 5% (wt/vol) FCS (Invitrogen) in triplicate wells of flat-bottomed 96-well plates at a concentration of 5 × 105 cells/well in a 200-μl volume. The cells were incubated with medium alone or in medium containing either Ag85A or ESAT-6 (5 μg/ml) or a combination of both antigens. Concanavalin A (ConA; Sigma; final concentration of 5 μg/ml) was used as a positive control. Cells were incubated at 37°C and 10% CO2 in air.

Measurement of IFN-γ.

Culture supernatants were removed after 4 days of incubation and frozen at −20°C until assayed. Levels of gamma interferon (IFN-γ) in culture supernatants were measured by ELISA according to the manufacturer's recommendations (BD Biosciences) using a commercial pair of antibodies and standards (BD Pharmingen). Briefly, MaxiSorp plates (Nunc) were coated overnight with monoclonal antibody, washed, and blocked with PBS containing 10% (wt/vol) FCS. After washing, culture supernatants or standards were added to the wells and the plates were incubated. After further washing, biotinylated detection antibody was added, incubated, and washed prior to the addition of an avidin HRP conjugate. The assay used o-phenylenediamine substrate and was read at 495 nm on a VersaMax microplate reader. Standard curves were constructed using SoftMax Pro software, and cytokine values were determined from the curve. The limit of detection for IFN-γ was 0.05 ng/ml.

Measurement of serum antibody.

Antibody in sera was measured by ELISA. MaxiSorp (Nunc) plates were coated overnight with 5 μg/ml Ag85A or ESAT-6 and then blocked using 10% (wt/vol) FCS in PBS. After washing in PBST, dilutions of serum (from 1/10 to 1/10,000) were added and incubated. Following washing, anti-mouse IgG1-HRP or IgG2c-HRP (ICL, Newberg, OR) was added and the plates were incubated. Plates were washed, and tetramethylbenzidine was used as a substrate prior to reading at 450 nm on a VersaMax microplate reader. Monoclonal anti-ESAT-6 or anti-Ag85A antibodies (Abcam) were titrated and included as a positive control for the IgG1 plates. Results were expressed in titers representing the reciprocal of the serum dilution which gave half the maximal optical density (OD).

Statistical analysis.

Analyses of the IFN-γ and antibody responses were performed by using the Kruskal-Wallis one-way analysis of variance.

RESULTS

Microbial production and characterization of biopolyester beads displaying Ag85A-ESAT-6 at their surfaces.

Plasmids pHAS-Ag85A-ESAT-6 and pHAS were introduced into E. coli BL21(DE3) cells harboring plasmid pMCS69, and the transformants were cultured to produce Ag85A-ESAT-6 and wild-type beads, respectively. GC-MS analysis of respective cells confirmed the presence of the polyester, polyhydroxybutyrate, indicating functionality of the polyester synthase domain in the tripartite fusion protein (data not shown). The presence of intracellular polyester inclusions was further confirmed by fluorescent microscopy using Nile Red staining as previously described (21).

The proteins associated with the Ag85A-ESAT-6 beads and the wild-type beads were separated by SDS-PAGE (Fig. (Fig.1).1). The beads displayed high levels of the respective overproduced protein as indicated by a prominent protein with an apparent molecular mass of 102 and 63 kDa for Ag85A-ESAT-6-PhaC and PhaC, respectively. The identity of these proteins was confirmed by tryptic peptide fingerprinting using MALDI-TOF MS. Densitometry analysis of the gels indicated that the Ag85A-ESAT-6-PhaC protein accounted for 20% of the total protein associated with the Ag85A-ESAT-6 beads. The presence of antigenic ESAT-6 at the surfaces of the Ag85A-ESAT-6 beads was assessed by ELISA. The results shown in Fig. Fig.22 indicated that Ag85A-ESAT-6 beads bound to the anti-ESAT-6 antibody in a dose-dependent manner. Specific binding of anti-ESAT-6 antibody to Ag85A-ESAT-6 beads was measured using flow cytometry, and the results showed that >98% of Ag85A-ESAT-6 beads bound anti-ESAT-6 antibodies (Fig. (Fig.33).

FIG. 1.
SDS-PAGE analysis of proteins attached to the polyester beads. Lane A, molecular weight markers; lane B, beads isolated from E. coli harboring plasmid pHAS-Ag85A-ESAT-6; lane C, beads isolated from E. coli harboring plasmid pHAS. The presence of the Ag85A-ESAT-6-PhaC ...
FIG. 2.
The display of the protein ESAT-6 on the bead surface was assessed by ELISA. Beads were diluted from 0.2 to 80 μg/ml on an ELISA plate and then incubated with anti-ESAT-6 antibody. Bead-bound antibody was detected using an HRP-conjugated secondary ...
FIG. 3.
Flow cytometry analysis of bioengineered beads displaying ESAT-6. Wild-type beads or Ag85A-ESAT-6 beads were incubated with anti-ESAT-6 antibody, and bound antibody was detected using fluorescein isothiocyanate-conjugated secondary antibody. Fluorescent ...

Immunization of mice with Ag85A-ESAT-6 beads stimulates the generation of an antigen-specific humoral and cellular immune response.

Mice were immunized with wild-type beads, Ag85A-ESAT-6 beads alone, Ag85A-ESAT-6 beads formulated in Emulsigen, or recombinant Ag85A-ESAT-6 protein alone by the subcutaneous route. Following immunization, no overt toxicity was observed in any of the animals. Weights of mice did not differ significantly between groups during the time course of the experiment, and mice in all groups gained weight (data not shown). Mice immunized with polyester beads developed small lumps (2.5 mm in diameter) at the immunization sites but no abscess or suppuration was observed, and all mice were healthy throughout the trial, with normal behavior and good-quality fur (data not shown).

Immune responses were determined in immunized animals to assess the potential of the polyester beads to act as vaccine delivery agents. A dose response experiment, where mice were immunized with various doses of beads displaying Ag85A-ESAT6, determined that beads displaying 30 μg of Ag85A-ESAT-6 are sufficient to generate both a significant antibody response (Fig. (Fig.4)4) and an IFN-γ response (Table (Table2)2) in mice. In addition, this dose of Ag85A-ESAT-6 beads induced significantly higher antibody titers and IFN-γ responses compared to a 30-μg dose of recombinant Ag85A-ESAT-6 protein alone (P < 0.01). A similar dose of Ag85A-ESAT-6 beads was used in a second experiment which included nonimmunized control animals and compared beads formulated with and without the adjuvant Emulsigen. Antigen-specific serum antibody responses were significantly higher in both vaccine groups given Ag85A-ESAT-6 beads compared to nonimmunized mice (P < 0.01), and the highest antibody responses were observed in mice immunized with Ag85A-ESAT-6 beads in Emulsigen (see Fig. Fig.6).6). Antibody responses for the IgG1 isotype were stronger than responses for IgG2 in both experiments (Fig. (Fig.44 and and55).

FIG. 4.
Antibody responses in mice immunized three times with 0- to 90-μg beads displaying Ag85A-ESAT-6 or 30 μg recombinant Ag85A-ESAT-6. Sera were obtained 3 weeks after the final immunization. Levels of Ag85A-ESAT-6-specific antibodies of the ...
FIG. 5.
Antibody responses in nonimmunized mice or in mice immunized three times with 30 μg of wild-type beads, Ag85A-ESAT-6 beads, or Ag85A-ESAT-6 beads with Emulsigen. Sera were obtained 3 weeks after the final immunization. Levels of Ag85A-ESAT-6-specific ...
FIG. 6.
IFN-γ responses in nonimmunized mice or in mice immunized three times with 30 μg of wild-type beads, Ag85A-ESAT-6 beads, or Ag85A-ESAT-6 beads with Emulsigen. Splenocytes were cultured for 3 days with 5 μg Ag85A or ESAT-6, and ...
TABLE 2.
Mean IFN-γ responses from groups of immunized mice

A marker of the development of cell-mediated immunity was assessed by measuring the release of the key cytokine IFN-γ in splenocytes restimulated in vitro with proteins used for immunization. Immunization with Ag85A-ESAT-6 beads significantly enhanced the cell-mediated immune response to Ag85A-ESAT 6 (Table (Table2)2) and to individual component antigens, namely Ag85A and ESAT-6 (P < 0.01) (Fig. (Fig.6).6). This enhancement was improved by formulating the beads in Emulsigen.

DISCUSSION

This study assessed the potential use of engineered bacterial polyester inclusions as a particulate vaccine-delivery system utilizing the natural intracellular production of polyester beads by bacteria as a one-step production system, which does not require adsorption or conjugation of an antigen to a polymeric particle. A hybrid gene encoding a tripartite fusion protein, Ag85A-ESAT-6-PhaC, was successfully overproduced in recombinant E. coli mediating formation of polyester beads with the fusion protein attached to its surface. Surface display of the M. tuberculosis antigen Ag85A-ESAT-6 was confirmed by ELISA and flow cytometry.

Polyester beads produced in this study were injected in mice to analyze the respective humoral and cell-mediated immune response. Immunization using beads which displayed 30 μg Ag85A-ESAT-6 were shown to induce significantly higher antibody and IFN-γ responses than immunization with 30 μg recombinant Ag85A-ESAT-6 protein alone. Formulation of the beads in the adjuvant Emulsigen further enhanced the specific and significant immune response. The type of immune response which develops in response to a vaccine can be crucial for various diseases. A humoral antibody response characterized by IgG1 in mice is most useful for diseases caused by extracellular pathogens, whereas a cell-mediated response characterized by increased IFN-γ and IgG2 antibody is most valuable in diseases caused by intracellular pathogens, of which tuberculosis is a classic example. Tuberculosis causes approximately 2 million human deaths each year (8), and novel vaccine strategies against this pathogen are urgently required (16) because the live, attenuated M. bovis BCG vaccine currently used has shown highly variable protection (16). This study has shown that the use of engineered polyester beads which displayed mycobacterial antigens resulted in a specific and significant cell-mediated immune response. The lack of adverse side effects, such as weight loss, and no abscess or suppuration at the injection site suggested that the polyester beads are safe and nontoxic. Further studies are planned to test these vaccines for protection of mice against challenge with M. tuberculosis.

The versatility and potential of the bead antigen delivery system to elicit different complementary facets of the immune response could be applied to the development of multivalent vaccines (7). Furthermore, the immune responses might be enhanced by the codisplay of antigen and immunostimulatory molecules on the same biopolyester bead. Immunostimulatory molecules could be used to enhance particular components of the immune response, such as Toll-like receptor agonists for cellular immunity (17).

Delivery of drugs or vaccines using biocompatible particulate vehicles is an area currently gaining significant momentum. Cell-free vaccine delivery systems are often particulate (e.g., emulsions, micro-/nanoparticles, and liposomes), and their dimensions are similar to those of pathogens, which the immune system has evolved to inactivate (18). However, such particulate systems have to be chemically synthesized and processed to enable adsorption of the separately produced and purified protein antigen (7). In comparison, this study utilizing engineered bacteria as a production host for polymeric carriers of protein subunit vaccines seems to provide an affordable and efficient alternative.

In summary, our findings showed that bioengineered polyester beads are safe and efficient delivery systems for immunization purposes. This unique approach to designing and producing these polyester beads for antigen display allows tailoring of vaccines for incorporation of specific antigens or immunostimulants without the need to produce, purify, and conjugate recombinant proteins (18).

Acknowledgments

We thank Jessica Koach and Gina Pedersen (AgResearch) for expert technical help and Michel Denis (AgResearch) for assistance in writing the manuscript.

Natalie Parlane was supported in part by a grant-in-aid from AGMARDT. Financial assistance for the work was provided by the Foundation for Research, Science and Technology NZ.

Footnotes

[down-pointing small open triangle]Published ahead of print on 16 October 2009.

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