The Development and Characterisation of a Novel and Modular Virus-like Particle Platform

Abstract

Vaccines are one of the most effective medical interventions to date that have revolutionised the provision of health care against many deadly diseases. As well as protecting against infectious agents, vaccines can be extended for the treatment of chronic inflammatory diseases affecting the skin, the respiratory tract, allergies, rheumatic disorders, and cancer. The idea of a therapeutic vaccine is to treat an ongoing impaired immune disorder, rather than a preventative measure to protect from a pathogen. While there are established biological therapies against some autoimmune diseases, monoclonal therapies are often associated with a high cost, a requirement for high amounts of drug, recurrent dosing, and the potential for anti-drug responses, in addition to class-specific side effects such as neutropenia in some patients. Thus, further research into alternative treatment strategies is crucial. Among delivery platforms for therapeutical vaccines, virus-like particles (VLPs) are the only one having advanced to clinical trial stage. One major limitation in clinical translation of VLP vaccines is the difficulty to manufacture entire proteins (the most effective immunogen) to be presented by the VLP. One way to bypass this limitation is to select specific immunogenic epitopes. Another way is to develop an improved system to allow whole-protein presentation. Both of these strategies were pursued in this thesis, with the emphasis being on a new platform based on the Hepatitis B capsid (HBcAg/HBc₁₄₉), which is well characterised and has been clinically tested. VLPs are self-assembled nanosized structures that resemble the native viral version but lack genomic machinery for infectious replication within the host. The particles tend to be highly resilient to structure manipulation and their underlying repetitive arrangement, geometry and size make them exceptional immunogens in stimulating innate and adaptive responses. The vaccine prototypes assessed in this thesis targeted interleukins 13 and 33 (IL-13 and IL33), with clinical applications in eczema and asthma.

In the early stages, the vaccine prototypes were constructed using Cucumber Mosaic Virus (CMV) VLPs that were designed to present linear epitopes of a helixes A and D of human IL-13. The vaccines were found to be immunogenic and could elicit antibodies for a limited period. The subsequent strategies focused on developing a new VLP platform by using HBc₁₄₉ particles where two high-affinity binding systems of colicin e7 (ColE7)- immunity 7 (Im7) and barstar (Bs)- barnase (Bn) were used as linking agents. This involved insertion of Im7 or Bs in the major immunodominant region (MIR) of HBc₁₄₉ and removal of toxicity by targeted mutations in the cognate partner proteins of ColE7 and Bn that were fused with cytokines IL-13 or IL-33. The proposed concept allows instantaneous decoration of the VLP surface with epitopes that would otherwise be challenging to conjugate under conventional methods. The data presented in this thesis illustrates the successful development and characterisation of Im7 and Bs in-frame-fusions in HBc₁₄₉ capsids displaying fully conformational IL-33 and IL-13 produced in E. coli. The particles mostly displayed T = 3 conformations and exhibited distinctive thickened morphology. This invention aimed to simplify manufacturing processes and deliver a foundation for single-release pharmaceuticals with the ability to present any heterologous epitope on the VLP exterior. Besides therapeutic vaccines, the novel VLPs has vast potential applications due to the capacity of displaying any desired epitope on the surface that can be extended to serological assays, protein purification and personalised medicine.

Date of Award	2022
Original language	English
Supervisor	John Foerster (Supervisor) & Jean-Christophe Bourdon (Supervisor)