A Synthetic Biology Approach to Engineering Protein Assemblies for use in Vaccine Design.

  • Freya King

Student thesis: Phd


Technological advancements have allowed the field of vaccine design to develop rapidly to meet the on-going need to protect against infectious disease. Protein-based formulations are currently at the forefront of vaccine-based research and several virus-like particle (VLP)-based vaccines have entered clinical trials. VLPs are self-assembling structures that form the viral capsid but do not contain nucleic acids, hence are non-infectious. These structures are established antigen presentation platforms with the ability to display a range of antigens to the host immune system to protect against a range of pathogens. Here we investigate a synthetic platform, I3-01, for use as a self-assembling antigen presenting platform. I3-01 was engineered to form a cage-like structure with similar dimensions to a VLP, comprising 20 trimers of the thermostable enzyme 4-hydroxy-2-oxoglutarate aldolase from Thermotoga maritima. The I3-01 platform was purified and the structure was determined to 8Å resolution by cryoelectron microscopy; the structure confirmed it matched the designed model within the limits of experimental error. The potential of I3-01 as a vaccine platform was tested by assessing the ability of the assembly to present antigens from N. meningitidis. The antigens, adhesin NadA and the heparin binding protein NHBA, were selected because they are included in the licensed meningococcal 4CMenB vaccine. The relevant antigen domains were fused by genetic means to I3-01 but the products failed to assemble correctly. A novel fusion technology, exploiting the ability of Protein A to bind to the Fc portion of antibodies, was designed to overcome this problem. Protein A was fused to the C-terminus of I3-01; the fusion protein was expressed in E. coli and electron microscopy was used to confirm that the purified assembly formed a homogeneous population of particles which closely resembled the unmodified I3-01. In order to test that the I3-01-ProteinA (I3-01-PA) assembly was able to bind Fc fusion proteins, a fusion protein consisting of ovalbumin (OVA) fused to the Fc fragment of mouse IgG2A was generated. A bio-layer interferometry assay confirmed that the I3-01-PA construct bound to the Fc-OVA protein with high affinity. In vitro studies compared the use of I3-01-PA as an antigen presentation platform to a VLP assembly- Hepatitis B core protein- also fused to Protein A (HBc-PA) VLP. Analysis of the uptake into THP-1 derived macrophages showed that neither platform increase uptake, compared to Fc-OVA alone. A similar observation was made using mouse bone marrow-derived dendritic cells; there was also no increase in the expression of CD40, CD80, CD86 and MHC-II following antigen uptake. This suggested that these cells were not more activated by either platform. The I3-01-PA assembly stimulated a significant increase in IL-10 and TNF- α secretion when in complex with Fc-OVA compared to Fc-OVA alone, showing an effect of the I3-01-PA assembly. However, this effect was significantly less than when the Fc-OVA antigen was in complex with the HBc-PA VLP. In order to assess the CD4 T cell response following antigen uptake, CD4+ OT-II OVA specific T cells were co-cultured with dendritic cells which had been stimulated by Fc-OVA antigen presented on both platforms. Both the I3-01-PA assembly and the HBc-PA VLP significantly increased the ability of the Fc-OVA antigen to stimulate a specific T cell proliferation, compared to Fc-OVA alone (p
Date of Award1 Aug 2021
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorJeremy Derrick (Supervisor), James Warwicker (Supervisor) & Alan Roseman (Supervisor)

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