Polyhydroxyalkanoates (PHAs) are naturally occurring organic polyesters that are of interest for industrial and biomedical applications. These polymers are synthesized by most bacteria in times of unbalanced nutrient availability from a variety of substrates and they are deposited intracellularly as insoluble spherical inclusions or PHA granules. The granules consist of a polyester core, surrounded by a boundary layer with embedded or attached proteins that include the PHA synthase, phasins, depolymerizing enzymes, and regulatory proteins. Apart from ongoing industrial interest in the material PHA, more recently there has also been increasing interest in applications of the PHA granules as nano-/micro-beads after it was conceived that fusions to the granule associated proteins (GAPs) provide a way to immobilize target proteins at the granule surface. This review gives an overview of PHA granules in general, including biogenesis and GAPs, and focuses on their potential use as nano-/micro-beads in biotechnological and biomedical applications.

Cattle may provide a suitable model for testing ways of improving tuberculosis vaccine efficacy in human infants. A vaccination and challenge study was undertaken in calves to determine the optimal time to vaccinate neonatal animals with Mycobacterium bovis bacillus Calmette-Guérin (BCG) for protection against tuberculosis and to determine whether revaccination with BCG was beneficial. Calves (10 per group) were vaccinated with BCG within 8 h of birth or at 6 weeks of age, when immune responses to antigens of environmental mycobacteria were detectable, or vaccinated at birth and revaccinated at 6 weeks. A control group was not vaccinated. BCG vaccination at birth induced strong antigen-specific gamma interferon (IFN-␥) and interleukin-2 (IL-2) responses and antigen-specific activation in CD4؉ , CD8 ؉ , and WC1 ؉ ␥␦ T-cell subsets from blood. The proportions of animals per group with macroscopic tuberculous lesions after challenge were 0/10 for BCG at birth, 1/9 for BCG at 6 weeks, 4/10 for the revaccinated group, and 10/10 for the nonvaccinated group. There was no significant difference in the levels of protection between groups vaccinated at birth or at 6 weeks, while animals vaccinated both at birth and at 6 weeks had significantly less protection than those vaccinated only at birth. The revaccinated calves that subsequently developed tuberculous lesions had significantly stronger IFN-␥ and IL-2 responses to bovine purified protein derivative after the BCG booster than those in the same group that did not develop lesions. The results indicated that BCG vaccination at birth induced a high level of immunity and that the sensitization of very young animals to antigens of environmental mycobacteria by 6 weeks of age did not affect the effectiveness of BCG. However, BCG revaccination of these young animals was contraindicated.

No abstract

New improved vaccines are needed for control of both bovine and human tuberculosis. Tuberculosis protein vaccines have advantages with regard to safety and ease of manufacture, but efficacy against tuberculosis has been difficult to achieve. Protective cellular immune responses can be preferentially induced when antigens are displayed on small particles. In this study, Escherichia coli and Lactococcus lactis were engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which displayed a fusion protein of Mycobacterium tuberculosis, antigen 85A (Ag85A)-early secreted antigenic target 6-kDa protein (ESAT-6). L. lactis was chosen as a possible production host due its extensive use in the food industry and reduced risk of lipopolysaccharide contamination. Mice were vaccinated with PHB bead vaccines with or without displaying Ag85A-ESAT-6, recombinant Ag85A-ESAT-6, or M. bovis BCG. Separate groups of mice were used to measure immune responses and assess protection against an aerosol M. bovis challenge. Increased amounts of antigen-specific gamma interferon, interleukin-17A (IL-17A), IL-6, and tumor necrosis factor alpha were produced from splenocytes postvaccination, but no or minimal IL-4, IL-5, or IL-10 was produced, indicating Th1-and Th17-biased T cell responses. Decreased lung bacterial counts and less extensive foci of inflammation were observed in lungs of mice receiving BCG or PHB bead vaccines displaying Ag85A-ESAT-6 produced in either E. coli or L. lactis compared to those observed in the lungs of phosphate-buffered saline-treated control mice. No differences between those receiving wild-type PHB beads and those receiving recombinant Ag85A-ESAT-6 were observed. This versatile particulate vaccine delivery system incorporates a relatively simple production process using safe bacteria, and the results show that it is an effective delivery system for a tuberculosis protein vaccine. Mycobacterium bovis, the causative agent of bovine tuberculosis (TB), infects a wide range of hosts, including domestic livestock and wildlife, and also causes TB in humans. Bovine TB poses a public health risk, particularly in regions where pasteurization of milk is not routine. This is of particular concern because more than 94% of the world's population lives in such regions, and M. bovis is the causative agent for up to 10% of TB cases in humans in these regions (14). Bovine TB also has a considerable economic impact on the agricultural industry. The human TB vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) is only partially effective in both cattle and humans (2, 12). Development of an effective vaccine protecting against bovine TB would provide a cost-effective TB control strategy as well as have applicability for control of human TB caused by Mycobacterium tuberculosis.A number of new TB vaccines are entering human clinical trials, including recombinant BCG, virus-vectored vaccines, and recombinant protein vaccines (20). One of the major constraints in developing effective recombinant protein vaccines is t...

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

Vaccine delivery systems based on display of antigens on bioengineered bacterial polyester inclusions can stimulate cellular immune responses. The food-grade Gram-positive bacterium Lactococcus lactis was engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which abundantly displayed the hepatitis C virus core (HCc) antigen. In mice, the immune response induced by this antigen delivery system was compared to that induced by vaccination with HCc antigen displayed on PHB beads produced in Escherichia coli, to PHB beads without antigen produced in L. lactis or E. coli, or directly to the recombinant HCc protein.Vaccination site lesions were minimal in all mice vaccinated with HCc PHB beads or recombinant protein, all mixed in the oil-in-water adjuvant Emulsigen, while vaccination with the recombinant protein in complete Freund's adjuvant produced a marked inflammatory reaction at the vaccination site. Vaccination with the PHB beads produced in L. lactis and displaying HCc antigen produced antigen-specific cellular immune responses with significant release of gamma interferon (IFN-␥) and interleukin-17A (IL-17A) from splenocyte cultures and no significant antigen-specific serum antibody, while the PHB beads displaying HCc but produced in E. coli released IFN-␥ and IL-17A as well as the proinflammatory cytokines tumor necrosis factor alpha (TNF-␣) and IL-6 and low levels of IgG2c antibody. In contrast, recombinant HCc antigen in Emulsigen produced a diverse cytokine response and a strong IgG1 antibody response. Overall it was shown that L. lactis can be used to produce immunogenic PHB beads displaying viral antigens, making the beads suitable for vaccination against viral infections.The food-grade Gram-positive bacterium, Lactococcus lactis has been increasingly considered as a production host for recombinant therapeutic proteins (6, 9, 49). The recent advances toward the development of efficient gene expression systems in L. lactis and the established safety profile of L. lactis based on long-term use in dairy food processing has led to new potential applications in protein production, therapeutic drug delivery, and vaccine delivery (5,27,30,38).Recently, it was shown that L. lactis can be engineered to produce spherical polyhydroxybutyrate (PHB) inclusions which display the Staphylococcus aureus protein A-derived IgG binding region, the Z domain, and that these can be isolated for in vitro use in purification of IgG (26). This was achieved by establishing the PHB biosynthesis pathway in L. lactis and by overproducing a Z domain-PHB synthase fusion protein which remained attached to the PHB inclusion surface. The PHB synthase represents the only essential enzyme required for PHB inclusion formation (39,40). This strategy utilized protein engineering of the PHB synthase from Ralstonia eutropha for the display of various protein-based functions, such as technical enzymes, binding domains, or a fluorescent protein, at the surfaces of PHB beads as had been previously established in recombinant Escherichi...

No abstract

Polyhydroxyalkanoates (PHAs) are biological polyesters that can be naturally produced by a range of bacteria as water-insoluble inclusions composed of a PHA core coated with PHA synthesis, structural, and regulatory proteins. These naturally self-assembling shell–core particles have been recently conceived as biomaterials that can be bioengineered as biologically active beads for medical applications. Protein engineering of PHA-associated proteins enabled the production of PHA–protein assemblies exhibiting biologically active protein-based functions relevant for applications as vaccines or diagnostics. Here we provide an overview of the recent advances in bioengineering of PHA particles toward the display of biomedically relevant protein functions such as selected disease-specific antigens as diagnostic tools or for the design of particulate subunit vaccines against infectious diseases such as tuberculosis, meningitis, pneumonia, and hepatitis C.