Development and use of polyhydroxybutyrate biopolyester as particulate vaccine beads : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Manawatu, New Zealand
3-hydroxybutyric acid) (PHB) is the most commonly produced polyhydroxyalkanoate formed naturally inside many genera of bacteria and archaea when nutrients are limited and a carbon-source is available in excess. These water-insoluble biopolyester spherical beads in the size range of 20-800 nm can be recombinantly produced by insertion of the required PHB biosynthesis genes into alternative bacterial hosts and then culturing the organisms under suitable conditions. A gene fusion can also be made to enable production of PHB beads which display the selected proteins abundantly at the surface of the bead.
Vaccines are needed which stimulate cell-mediated immunity and are effective at reducing intracellular infections such as tuberculosis, neosporosis and many viral infections. These diseases are responsible for a huge burden to human and animal health. Particulate vaccines target antigen presenting cells and cellular immune responses to protein antigens are enhanced when particulate vaccines are used.
This thesis describes the development of a novel vaccine delivery system in which PHB beads were engineered to display vaccine antigen on the surface of the beads.
Investigations were made into the process of vaccine bead design, production and validation to enable their use in vaccine trials. PHB synthesis genes from Cupriavidus necator were inserted into production strains to enable production of PHB. Escherichia coli was initially used as a bacterial production host and then Lactococcus lactis was introduced as an alternative, due to its lack of lipopolysaccharide, previous use as a production host for recombinant proteins and history of safe use for a range of human foods and products. To expand the repertoire of PHB vaccine beads, different vaccine antigens were used: hepatitis C core antigen and mycobacterial antigens (antigen-85A and 6 kDA early secretory antigenic target). Antigen specific cellular immune responses were produced in mice vaccinated with PHB vaccine beads and protection against tuberculosis was observed in mice immunized with these vaccines.
Preliminary studies into the mechanism of uptake of PHB beads by dendritic cells (DCs) showed PHB beads were taken up readily by DCs, with maturation of DCs and subsequent secretion of interleukin-12.