Display of Antigens on Polyester Inclusions Lowers the Antigen Concentration Required for a Bovine Tuberculosis Skin Test

Abstract

eThe tuberculin skin test is the primary screening test for the diagnosis of bovine tuberculosis (TB), and use of this test has been very valuable in the control of this disease in many countries. However, the test lacks specificity when cattle have been exposed to environmental mycobacteria or vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG). Recent studies showed that the use of three or four recombinant mycobacterial proteins, including 6-kDa early secretory antigenic target (ESAT6), 10-kDa culture filtrate protein (CFP10), Rv3615c, and Rv3020c, or a peptide cocktail derived from those proteins, in the skin test greatly enhanced test specificity, with minimal loss of test sensitivity. The proteins are present in members of the pathogenic Mycobacterium tuberculosis complex but are absent in or not expressed by the majority of environmental mycobacteria and the BCG vaccine strain. To produce a low-cost skin test reagent, the proteins were displayed at high density on polyester beads through translational fusion to a polyhydroxyalkanoate synthase that mediates the formation of antigen-displaying inclusions in recombinant Escherichia coli. Display of the proteins on the polyester beads greatly increased their immunogenicity, allowing for the use of very low concentrations of proteins (0.1 to 3 g of mycobacterial protein/inoculum) in the skin test. Polyester beads simultaneously displaying all four proteins were produced in a single fermentation process. The polyester beads displaying three or four mycobacterial proteins were shown to have high sensitivity for detection of M. bovis-infected cattle and induced minimal responses in animals exposed to environmental mycobacteria or vaccinated with BCG. C ontrol of bovine tuberculosis (TB), which is caused by infection with Mycobacterium bovis, is critical, as this disease is of great economic and zoonotic importance. Programs for eradication in cattle are primarily based on use of the tuberculin skin test for diagnosis of the disease, with slaughter of reactor animals. This approach has been instrumental in eradication of this disease from a number of countries (1). The tuberculin skin test is a delayedtype hypersensitivity (DTH) test that was developed more than 100 years ago for the diagnosis of TB, and it has proved to be a simple, inexpensive, robust, and widely accepted test. However, the test lacks specificity when animals have been sensitized to environmental mycobacteria or have been vaccinated with the human TB vaccine, M. bovis bacille Calmette-Guérin (BCG) (2). Estimates of sensitivity for a single intradermal test in cattle using purified protein derivative (PPD) prepared from M. bovis (bovine PPD) have ranged from 63.2% to 100% (median, 83.9%), with specificity between 75.5% and 99.0% (median, 96.8%) (reviewed in reference 2). Development of a more specific skin test reagent would be highly desirable. The use of BCG vaccine alone or as part of a heterologous prime-boost combination is currently being considered by a number of countries fo...

Innovative antigen carrier system for the development of tuberculosis vaccines

“…This study evaluated the potential application of selfassembled polyester particles produced in E. coli cells as a carrier system for the delivery of mycobacterial antigens H4 and H28. A number of studies have shown that polyester particles could be genetically engineered to display antigens of interest (29,30,37). This particle-based vaccine delivery system had been shown to enhance antigen-specific immune responses (25,38).…”

“…This concept of using biopolyester particles for vaccine delivery is further underpinned by their unique favorable properties such as biocompatibility with mammalian systems (23)(24)(25)(26)(27)(28). Hepatitis C and TB antigens displayed on the particle surface have demonstrated increased immunogenicity when compared with their soluble forms, suggesting that polyester particles are capable of increasing the immunogenicity of vaccines when desired antigen candidates are displayed on their surfaces (29)(30)(31). In addition, polyester particles could serve as a depot for prolonged multivalent-antigen display to provide enhanced immunogenicity.…”

Recombinant surface engineering to enhance and expand the potential of biologically produced nanoparticles: A review

“…It is essential that any given nanoparticle intended for human therapeutic applications such as drug delivery does not elicit any kind of immunological noise. In a case study by Parlane and colleagues [72], however, the use of engineered PHAs granules was shown to increase pro-inflammatory cytokine levels for unknown reasons. In the case of exosomes, it has been suggested that care should be taken regarding the choice of exosomes [40].…”

“…A further extension of these findings is the use of granules for the effective detection of M. tuberculosis and Mycobacterium bovis in infected and non-infected animals. To distinguish infected from non-infected animals, multiple tuberculosis antigens including ESAT6, 10-kDa culture filtrate protein (CFP10), Rv3615c, and Rv3020c were fused to the PHA synthase protein and were displayed on the granules[72]. These granules served as a highly sensitive system for the identification of M. bovis-infected animals from skin tests and furthermore allowed for the discrimination between naturally M. bovis-infected cattle from BCG-vaccinated non-infected animals.A fusion protein of B7-2, a costimulatory molecule, fused to PhaP1 was expressed and displayed on granules in recombinant E. coli.…”

Bioengineered Polyhydroxyalkanoates as Immobilized Enzyme Scaffolds for Industrial Applications

“…*Kinetic parameters are not mentioned in the reference. Hay et al, 2015;Parlane et al, 2016a). Assembly of immobilized multiprotein complexes enables multi-enzymatic cascade systems with superior catalytic performance as recently reviewed (Hwang and Lee, 2019).…”