ENGINEERING PROTEIN ASSEMBLIES FOR USE IN VACCINE DESIGN

Abstract

Virus-like-particles (VLP) are self-assembling non-infectious bio-nanoparticles, and are widely used in medicine and nanotechnology research. VLPs mimic the structure of the native virus nucleocapsid and are attractive platforms to present multivalent antigens on their surface, notably in vaccines. They do not contain viral genetic material and are therefore non-infectious, an advantage over live or attenuated viruses. However, display of antigens on VLPs by genetic fusion or chemical coupling often leads to capsid disassembly, misfolding and a decrease in thermal stability, which hampers the generation of a protective immune response. Computational methods were used to predict mutations to improve thermal stability of Hepatitis B core capsid VLP (HBc VLP); however, of these, only Y6F led to a modest increase in Tm of ~1oC without compromising particle assembly. In an alternative approach, in order to overcome the limitations of antigen fusion, HBc VLP was fused to two Protein A domains (HBc-PA-DD), allowing the facile coupling of any protein or antibody containing the CH2 and CH3 domains from IgG antibody. The engineered VLP (HBc-PA-DD) was expressed in E. coli and the assembly was confirmed by electron microscopy. As a proof of principle, two ‘model’ antigens- ovalbumin (OVA) and N meningitidis NadA- were fused to the Fc fragment of mouse IgG2a and produced separately. The binding functionality of HBc-PA-DD to IgG and the Fc- antigen fusions (Fc-OVA or Fc-NadA) was confirmed by Bio-Layer Interferometry assay. An in vitro mouse bone marrow-derived dendritic cells were observed to take up non-covalently linked Fc-OVA or Fc-NadA-HBc-PA-DD complex by FACS. Uptake of the Fc-OVA-HBc-PA-DD complex does not show any significant difference as compared to Fc-OVA alone, whereas, uptake of Fc-NadA was significantly higher than Fc-NadA-HBc-PA-DD complex. However, both Fc-OVA-HBc-PA-DD and Fc- NadA-HBc-PA-DD showed high expression of MHC-II, CD40 and CD80 activation markers compared to Fc-OVA and Fc-NadA alone. Uptake studies were also carried out to determine whether this approach would also be suitable for receptor- mediated delivery of Fc-OVA-HBc-PA-DD by targeting the CD205 receptor. The results showed enhanced uptake of the Fc-OVA or Fc-NadA-HBc-PA-DD complexes, when the complex was targeted using an anti-mouse CD205-monoclonal antibody, as compared to the same complex without targeting antibody. Confocal imaging confirmed the complete internalization of both FITC-labelled Fc-antigens when complexed with HBc-PA-DD. In addition, CD4+ T cells from OT-II mice specific for the OVA epitope were co-cultured with antigen stimulated DCs. These results showed that the HBc-PA-DD VLP platform can successfully deliver Fc-OVA to DCs for efficient processing and presentation by MHC class-II molecules and is capable of inducing OVA-specific T-cell clonal proliferation. In addition, CD205 receptor-mediated targeting of Fc-OVA-HBc-PA-DD complex showed a higher proliferation, activation and differential cytokine profiles of CD4+ T cells as compared to the same complex without targeting antibody. This simple and flexible way of attaching antigens onto the VLP surface could help in accelerating vaccine development, as well as finding uses in other nanoparticle applications.

Date of Award	1 Jun 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Jeremy Derrick (Main Supervisor)