Design and engineering of self-assembling antigens towards particulate vaccines : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

Abstract

Natural and synthetic self-assembling polymers and proteins could be bioengineered to display and/or encapsulate antigens to serve as innovative antigen carrier systems for the induction of desirable immunities. Polyhydroxyalkanoates (PHAs) are naturally occurring polyesters synthesized as cytoplasmic polyester inclusions (polyester particles) by various bacteria. The particles have been used as an antigen delivery platform by translationally fusing antigens to the particle surface-associated protein, PHA synthase. Furthermore, it has been found that protein inclusion bodies contain a large amount of correctly folded and biologically active proteins and could be engineered to perform as an antigen carrier system. Tuberculosis (TB) is a global health issue for both humans and animals. Inaccurate diagnosis and inefficacious vaccination make TB control problematic. The Mantoux tuberculin skin test gives false positive results if humans or animals are vaccinated with the Bacille Calmette-Guérin (BCG) strain or exposed to environmental mycobacteria. BCG cannot provide effective protection against TB. Subunit vaccines have great promise to protect against infectious diseases, but they are often weak immunogenically. A strategy to circumvent this problem is the use of self-assembly particulate vaccines, which could present multiple copies of antigens and serve as a depot for prolonged multivalent antigen display to induce enhanced immunogenicity. In this thesis, four specific TB diagnostic antigens — CFP10, Rv3615c, ESAT6, and Rv3020c — were displayed on polyester particles. The results showed that polyester particles displaying TB antigens specifically distinguished TB-infected from non-infected cattle. Antigen immunogenicity was dramatically enhanced after the display on polyester particles, which lowered the antigen concentration (0.1 to 3 μg dose/inoculum) required for skin tests. Mycobacterial vaccines H4 (Ag85B-TB10.4) or H28 (Ag85B-TB10.4-Rv2660c) were bioengineered to display H4/H28 on polyester particles and/or self-assemble H4/H28 into protein inclusion bodies. The results demonstrated that polyester particle-/protein inclusion body-based particulate TB vaccines increased overall immunogenicity by enhancing humoral (for example, IgG1 and IgG2c) and cellular (for example, IFNγ and IL17A) immune responses when compared to respective soluble antigens.