Development of a vaccine platform based on rotavirus-like particle

Abstract

Rotaviruses (RV) are non-enveloped RNA viruses that cause diarrhea, vomiting and gastroenteritis in mammals. In humans, the most susceptible age group for the infection are children under 5 years and overall, RVs are responsible for over 200 000 deaths annually worldwide. The utmost effective method in preventing these deaths are vaccines. The four currently approved oral live-attenuated RV vaccines have had a major impact on decreasing the RV induced disease and death burden during last two decades. However, the problem with the current vaccines is a decreased efficacy in low-income countries when compared to high-income countries, and owing to their live-attenuated nature, they are expensive to produce and have remaining safety issues. Due to this, novel vaccine technologies such as virus-like particles (VLP) are needed in the design of new rotavirus vaccines.

VLPs are non-infectious particles consisting of viral structural proteins, mimicking the symmetric structure of a native virus while lacking the viral genome. They are also able to induce humoral and adaptive immune responses. Rotavirus-like particles (rota-VLPs) consist of one to four different viral proteins (VP2, VP6, VP4 and VP7) which can form single, double, and triple layered particles. Rota-VLPs can be used as vaccines against RVs or utilized as vaccine platforms by engineering the VLPs to display foreign antigens. One interesting approach is utilizing spontaneously covalent bond forming SpyCatcher/SpyTag system. By displaying SpyTag peptides on the surface of rota-VLPs, the particles can be decorated covalently with any SpyCatcher fused vaccine antigens. The aim of this thesis was to produce, purify and characterize wild-type and SpyTag fused rota-VLPs for future vaccine applications.

In this project double layered VP2/VP6 and VP2/VP6-SpyTag, and triple layered VP2/VP6/VP4 and VP2/VP6/VP4-SpyTag rota-VLPs, were produce utilizing insect cell – baculovirus expression vector system. The production parameters, including multiplicity of infection and harvesting time, were optimized for each rota-VLP. Different harvesting methods and purification workflows, consisting of varying combinations of tangential flow filtration (TFF), sucrose gradient purification, ultracentrifugation, and size exclusion chromatography (SEC), were tested. SDS-PAGE and Western Blot analysis as well as dynamic light scattering and transmission electron microscopy were used, respectively, to assess the success of the production and purification steps, and to characterize the produced rota-VLPs.

As a result, VP2/VP6 particles were successfully produced and purified utilizing TFF, ultracentrifugation and SEC resulting in highly homogenous and intact double layered rota-VLPs. Similarly, relatively pure VP2/VP6/VP4 and VP2/VP6/VP4-SpyTag particles were obtained with same purification methods although in lower concentration. Despite promising results, the intactness of the triple layered particles could not be validated. The purification workflow tested with VP2/VP6-SpyTag particles was unsuccessful and resulted in sample loss. Nevertheless, the results showed that the Spy-Tag was inserted successfully to VP6, and the recombinant protein was producible. Solubility issues were encountered with all the rota-VLPs during the whole process.

In conclusion, the attempt of producing four distinct rota-VLPs was partially successful. Further optimization of the buffer composition and purification methods are needed before proceeding into the immunogenicity analyses of the produced rota-VLPs in animal models. Altogether, this thesis provides insights to the future development of rota-VLP based vaccines.