Development of Sustained-Release Subunit Nanovaccines Against Anaplasma marginale

Abstract

Anaplasma marginale is a prevalent tick-transmitted intraerythrocytic riskettsial pathogen of cattle causing bovine Anaplasmosis. This disease is a major threat to global beef and cattle industry and causes huge economic losses each year, as there is no widely accepted vaccine against A. marginale available at present. Thus, development of novel sustained-release subunit nanovaccines that can induce protective immunity against A. marginale with single administration is in great demand. However, current understandings of potential protective epitopes on the outer membrane of A. marginale are poor. In addition, the expression methods of these antigen candidates still await developing and their immunogenicity need to be evaluated.This thesis demonstrates a novel approach of combining bioengineering together with nanotechnology to address these problems. The major outcomes of the work presented in this thesis are as follows: (i) two immunogenic outer membrane proteins of A. marginale, VirB9-1 and VirB9-2 were chosen as antigen candidates and expression methods using Escherichia coli expressing systems were developed. (ii) purification methods for the expressed recombinant VirB9-1 and VirB9-2 were developed and high yield products were obtained; (iii) specially designed mesoporous silica nanoparticles were applied as both adjuvant and nanocarrier for VirB9-1 and VirB9-2 and the adsorption conditions were optimised; (iv) development of highly immunogenic nanovaccine formulation consisting of the two protein antigens combined with mesoporous silica nanoparticles, and the immunogenicity of the two antigens and the adjuvanting effect of the nanoparticle were investigated using murine models.This work introduces, to the best of our knowledge, the first high yield soluble expression of recombinant VirB9-1 in E. coli. Optimised purification methods are introduced and both purified and partially-purified VirB9-1 are demonstrated to adsorb to specially designed mesoporous silica nanoparticles at high loading capacity and induce high level immune responses in vivo. In addition, high yield insoluble expression of recombinant VirB9-2 in E. coli is also introduced in this thesis and a novel method that circumvents the protein refolding process by directly adsorbing VirB9-2 to mesoporous silica nanoparticles under denatured conditions is developed.This work contributes to better understandings of immunogenic outer membrane proteins of A marginale and the adjuvanting effect of mesoporous silica nanoparticles. These promising findings can serve as a solid foundation for further developments of novel nanovaccines against A. marginale, and therefore benefit worldwide beef and cattle industry.