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
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Date
2019
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Massey University
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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.
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Keywords
Tuberculosis vaccines, Biotechnology, Tuberculin test, Immunological adjuvants, Antigens, Tuberculosis in cattle