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.
Many bacteria are naturally capable of accumulating biopolyesters composed of 3-hydroxy fatty acids as intracellular inclusions, which serve as storage granules. Recently, these inclusions have been ...considered as nano-/microbeads with surface-attached proteins, which can be engineered to display various protein-based functions that are suitable for biotechnological and biomedical applications. In this study, the food-grade, generally-regarded-as-safe gram-positive organism Lactococcus lactis was engineered to recombinantly produce the biopolyester poly(3-hydroxybutyrate) and the respective intracellular inclusions. The codon-optimized polyhydroxybutyrate biosynthesis operon phaCAB from Cupriavidus necator was expressed using the nisin-controlled gene expression system. Recombinant L. lactis accumulated up to 6% (wt/wt) poly(3-hydroxybutyrate) of cellular dry weight. Poly(3-hydroxybutyrate) granules were isolated and analyzed with respect to bound proteins using biochemical methods and with respect to shape/size using transmission electron microscopy. The immunoglobulin G (IgG) binding ZZ domain of Staphylococcus aureus protein A was chosen as an exemplary functionality to be displayed at the granule surface by fusing it to the N terminus of the granule-associated poly(3-hydroxybutyrate) synthase. The presence of the fusion protein at the surface of isolated granules was confirmed by peptide fingerprinting using matrix-assisted laser desorption ionization-time of flight (mass spectrometry). The functionality of the ZZ domain-displaying granules was demonstrated by enzyme-linked immunosorbent assay and IgG affinity purification. In both assays, the ZZ beads from recombinant L. lactis performed at least equally to ZZ beads from Escherichia coli. Overall, in this study it was shown that recombinant L. lactis can be used to manufacture endotoxin-free poly(3-hydroxybutyrate) beads with surface functionalities that are suitable for biomedical applications.
PHAs (polyhydroxyalkanoates = biopolyester) composed of hydroxy fatty acids represent a rather complex class of storage polymers synthesized by various bacteria and archaea and are deposited as ...water-insoluble cytoplasmic nano-sized inclusions. These spherical particles are composed of a polyester core surrounded by phospholipids and proteins. The key enzymes of polyester biosynthesis and polyester particle formation are the polyester synthases, which catalyze the formation of polyesters. Various metabolic routes have been identified and established in bacteria to provide substrate for polyester synthases. Although not essential for particle formation, non-covalently attached proteins, the so-called phasins, can be found at the particle surface and are considered as structural proteins. Protein engineering of polyester synthases and phasins was used to shed light into the topology of these granule attached proteins. Biopolyesters and the respective micro-/nano-structures are currently considered as biocompatible and biodegradable biomaterials with numerous potential applications particularly in the medical field.
Q fever is an infectious zoonotic disease, caused by the Gram-negative bacterium Coxiella burnetii. Transmission occurs from livestock to humans through inhalation of a survival form of the ...bacterium, the Small Cell Variant, often via handling of animal parturition products. Q fever manifests as an acute self-limiting febrile illness or as a chronic disease with complications such as vasculitis and endocarditis. The current preventative human Q fever vaccine Q-VAX poses limitations on its worldwide implementation due to reactogenic responses in pre-sensitized individuals. Many strategies have been undertaken to develop a universal Q fever vaccine but with little success to date. The mechanisms of the underlying reactogenic responses remain only partially understood and are important factors in the development of a safe Q fever vaccine. This review provides an overview of previous and current experimental vaccines developed for use against Q fever and proposes approaches to develop a vaccine that establishes immunological memory while eliminating harmful reactogenic responses.
Coxiella burnetti is an intracellular bacterium that causes Q fever, a disease of worldwide importance. Q‐VAX®, the approved human Q fever vaccine, is a whole cell vaccine associated with safety ...concerns. Here a safe particulate subunit vaccine candidate is developed that is ambient‐temperature stable and can be cost‐effectively manufactured. Endotoxin‐free Escherichia coli is bioengineered to efficiently self‐assemble biopolymer particles (BPs) that are densely coated with either strings of 18 T‐cell epitopes (COX‐BP) or two full‐length immunodominant antigens (YbgF‐BP‐Com1) all derived from C. burnetii. BP vaccine candidates are ambient‐temperature stable. Safety and immunogenicity are confirmed in mice and guinea pig (GP) models. YbgF‐BP‐Com1 elicits specific and strong humoral immune responses in GPs with IgG titers that are at least 1 000 times higher than those induced by Q‐VAX®. BP vaccine candidates are not reactogenic. After challenge with C. burnetii, YbgF‐BP‐Com1 vaccine leads to reduced fever responses and pathogen burden in the liver and the induction of proinflammatory cytokines IL‐12 and IFN‐γ inducible protein (IP‐10) when compared to negative control groups. These data suggest that YbgF‐BP‐Com1 induces functional immune responses reducing infection by C. burnetii. Collectively, these findings illustrate the potential of BPs as effective antigen carrier for Q fever vaccine development.
Escherichia coli is engineered to assemble ambient‐temperaturestable biopolymer particles (BPs) coated with either COX epitopes or C.burnetii antigens YbgF and Com1. Respective BPs are safe and immunogenic inmouse and guinea pig (GP) models. Notably, YbgF‐BP‐Com1 immunisation effectivelyreducing C. burnetii infection. This study underscores the effectiveness of BPsas carriers in the development of Q fever vaccines.
Polyhydroxyalkanoate (PHA) beads, recombinantly produced in Escherichia coli, were functionalized to display lipase B from Candida antarctica as translational protein fusion. The respective beads ...were characterized in respect to protein content, functionality, long term storage capacity and re-usability. The direct fusion of the PHA synthase, PhaC, to lipase B yielded active PHA lipase beads capable of hydrolyzing glycerol tributyrate. Lipase B beads showed stable activity over several weeks and re-usability without loss of function.
Objectives
To overcome laborious and costly procedures often associated with therapeutic protein production and purification, in vivo polyester immobilized sortase is explored for the production of ...human tumor necrosis factor alpha (TNFα) and human interferon alpha 2b (IFNα2b) by
Escherichia coli
.
Results
Hybrid genes encoding PhaC-Sortase-TNFα or PhaC-Sortase-IFNα2b fusions (with a LPETG recognition signal immediately before TNFα or IFNα2b), mediated intracellular production of polyester (polyhydroxyalkanoate, PHA) beads in
Escherichia coli
. Upon isolation of respective PHA beads, pure soluble TNFα or IFNα2b was released by activating sortase via addition of CaCl
2
and triglycine. TNFα and IFNα2b each were recognized by corresponding conformational antibodies in an ELISA assay.
Conclusions
In vivo polyester immobilized sortase could be exploited for production and purification of high-value therapeutic proteins without laborious and costly downstream processing.
ABSTRACT
A major obstacle to tuberculosis (TB)‐subunit–vaccine development has been the induction of inadequate levels of protective immunity due to the limited breadth of antigen in vaccine ...preparations. In this study, immunogenic mycobacterial fusion peptides Ag85B‐TB10.4 and Ag85B‐TB10.4‐Rv2660c were covalently displayed on the surface of self‐assembled polyester particles. This study investigated whether polyester particles displaying mycobacterial antigens could provide augmented immunogenicity (i.e., offer an innovative vaccine formulation) when compared with free soluble antigens. Herein, polyester particle–based particulate vaccines were produced in an endotoxin‐free Escherichia coli strain and emulsified with the adjuvant dimethyl dioctadecyl ammonium bromide. C57BL/6 mice were used to study the immunogenicity of formulated particulate vaccines. The result of humoral immunity showed the antibodies only interacted with target antigens and not with PhaC and the background proteins of the production host. The analysis of T helper 1 cellular immunity indicated that a relatively strong production of cellular immunity biomarkers, IFN‐γ and IL‐17A cytokines, was induced by particulate vaccines when compared with the respective soluble controls. This study demonstrated that polyester particles have the potential to perform as a mycobacterial antigen–delivery agent to induce augmented antigen‐specific immune responses in contrast to free soluble vaccines.—Chen, S., Sandford, S., Kirman, J. R., Rehm, B. H. A. Innovative antigen carrier system for the development of tuberculosis vaccines. FASEB J. 33, 7505–7518 (2019). www.fasebj.org
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