Bacterial Polyester Inclusions Engineered To Display Vaccine Candidate Antigens for Use as a Novel Class of Safe and Efficient Vaccine Delivery Agents

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.