Virus-like particle and capsomere vaccines against rotavirus

Abstract

Rotavirus is the major enteric pathogen that is responsible for more than 500,000 deaths of children worldwide annually. The use of live-attenuated oral rotavirus vaccines has significantly reduced child mortality rate in developed countries. However, the situation still remains unacceptably worse in most developing countries due to low efficacy of the vaccines in the region. The vaccines often do not reach the developing countries due to financial and logistic challenges. A high burden of rotavirus disease and the unresolved challenges with the current rotavirus vaccines, particularly in developing countries, have ignited efforts to develop next-generation low-cost, effective and safe vaccine candidates. Virus-like particles (VLPs) have come into focus for their promising application in vaccination because of their unique and attractive set of properties including safety, self-adjuvanticity, developability, economy and the ability to be formulated for stability without a cold chain. Their impact on human health is already evident through commercialization of VLP vaccines against hepatitis B virus infection, human papillomavirus-induced cervical cancer and hepatitis E virus infection. Rotavirus-like particles (RLPs) with proven preclinical immunogenicity and protective efficacy are considered as safe and effective vaccine candidates. While rotavirus-like particles comprising multiple viral proteins can be difficult to process, modular VLPs presenting rotavirus antigenic modules are promising approaches in reducing process complexity and cost. Depending on the physicochemical properties, size and /or surface density of modules, modularization may affect production of stable VLP forming subunits, termed capsomeres, and prevent VLP assembly. This thesis demonstrates a multipronged approach for a low-cost production of stable bacterially-produced modular murine polyomavirus capsomeres and in vitro assembled VLPs presenting a rotavirus highly conserved peptide epitope (RV10) and a conformational and a virus neutralizing 18 kDa antigen (VP8*), separately. The experimental works in this thesis were carried out to address challenges associated with the adverse effects of inserted module’s physicochemical properties, size and density on the production of modular capsomeres and VLPs. Particularly, the expression and purification of stable modular capsomeres for in vitro modular VLP assembly and the enhancement of module-specific immune response were investigated. The outcomes of the work in this thesis are: (i) a rapid and simple high-throughput screening method based on dynamic light scattering measurements was developed to identify additives for enhanced stability of modular capsomeres; (ii) using synthetic biology designs, the hydrophobicity of RV10 modules was engineered for enhanced stability of modular capsomeres; (iii) in vitro assembled modular VLPs displaying RV10 modules were obtained via a module titration approach using Escherichia coli protein co-expression strategy; and (iv) highly stable and immunogenic modular VLPs displaying conformational VP8* were produced using a combination of Escherichia coli protein co-expression, simplified modular capsomere purification steps and cell-free in vitro VLP assembly. The outcomes of this thesis expand the potential and plasticity of the murine polyomavirus VLP platform for presentation of antigenic modules regardless of their adverse physicochemical properties and large size. This work introduces, to the best of our knowledge, the first bacterially-produced capsomeres and in vitro assembled VLPs displaying either hydrophobic RV10 peptide modules or 18 kDa rotavirus VP8* large antigens. The modular capsomeres (CapVP8*) and modular VLPs (VLP-VP8*) induced high levels of VP8*-specific antibodies in mice. The high immunogenicity of CapVP8* and VLP-VP8*likely indicates protective efficacy and makes them a more viable vaccine candidate for further development to prevent rotavirus in the developing world at affordable cost. The strategies developed in this thesis potentially provides a cost-effective production route for modular capsomere and VLPs presenting other antigenic modules from rotavirus and other target pathogens, highly suitable to manufacturing economics for the developing world.