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Description
Despite massive vaccination programmes, Hepatitis B Virus (HBV) remains the major cause of liver diseases. Therefore, more effective but at the same time economically feasible and commonly available preventive vaccines and CHB therapeutic vaccines, are particularly welcome. Commonly used, yeast-produced 2nd-generation preventive vaccines are based on the small surface antigen (S-HBsAg). More effective are the 3rd-generation vaccines containing also the medium and/or large surface antigens (M/L-HBsAg). Yet, they are produced in costly mammalian cells, thus their use is limited. The main component of tested CHB therapeutic vaccines is the core antigen (HBcAg), inducing strong Th1 response - mostly cellular type, which can be supplemented with HBsAgs or their immunodominant domains - inducers of Th2 response - humoral type. Virus-Like Particles (VLPs) VLPs based on HBcAg are well- characterized potential vaccine templates. Antigens or their fragments (epitopes) loaded on the surface of VLPs are therefore presented in a highly ordered structure, thus effectively for the immune system. VLP-assembled plant-derived antigens are considered alternative vaccines, due to low cost, biosafety, bioactivity, and possibility of oral immunisation. Immunogenicity of plant-produced S-HBsAg or HBcAg has been already proved. Both antigens, when used in injection immunization or that comprising injection priming and oral boosting, induced significant response of proper polarization. These results indicate that key epitopes of both antigens, combined in one type of chimeric VLPs, can induce the immune response of mixed Th1/Th2 polarization, required for CHB therapy. The main goal of this project is the development and the determination of immunogenicity of novel types of chimeric VLPs - formed by plant-produced HBcAg displaying key HBsAg epitopes: aSHBs and ΔpreS1 as well as TransLocation Motif (TLM) – enhancing permeability and immunogenicity of VLPs.
Summary of project results
The main achieved objective of the project is the construction of the panel of 10 plant expression vectors coding chimeric proteins composed of HBcAg as a carrier and an epitope (ΔpreS1, ΔpreS2, a-SHBs) and HBcAg or S-HBsAg as reference proteins, both protein types
with/no TLM (translocation motif), to finally assembly into chimeric or reference VLPs (Virus-Like Particles). The vectors panel provides actual basis for further actual research, including VLPs production in transient expression system, then research on VLPs preparation
and finally immunogenicity studies in animal model. However these objectives were not achieved due to resignation of the Principal investigator. Nevertheless, prepared vectors, as key research tools makes possible either partial continuation of the project under condition of employing of suitably qualified researcher or project reactivation under condition of adequate funds.
Construction of the panel of expression vectors is associated with an additional objective achieved, which is preparation of vectors coding chimeric protein (TLM)-HBcAg- ΔpreS2. These vectors were not originally planned in the project. However, these were constructed,
considering the role of preS2 in the infection of hepatocytes during HBV infection, as well as immune response induced and consequently – the role for efficacy of potential vaccines. The panel of VLPs displaying ΔpreS1, ΔpreS2 and a-SHBs would make possible to manipulate
composition of a vaccine preparations on purpose to evoke immune response specifically for a case. Next additional objective achieved is modeling of proteins TLM-HBcAg-ΔpreS1 and TLMHBcAg- ΔpreS2. These results are preliminary and predicted structure would require
experimental confirmation. However, if so, it would open possibilities to develop tools for modeling other analogous chimeric proteins of the type HBcAg MIR-epitope.
The most important project deliverables:
- Construction of the panel of 10 plant expression vectors coding chimeric proteins composed of HBcAg as a carrier and an epitope (ΔpreS1, ΔpreS2, a-SHBs) and HBcAg or S-HBsAg as reference proteins, both protein types with/no TLM (translocation motif), to finally assembly into chimeric or reference VLPs (Virus-Like Particles).
- Preliminary agroinfiltration with obtained vectors.
- Preliminary results from the modeling of proteins TLM-HBcAg-ΔpreS1 and TLMHBcAg-ΔpreS2.
The achieved objective of the project, i.e. construction of the panel of 10 plant expression vectors for VLPs production is necessary for further research, which only then it will open the possibility for actual implementation. However the project results can be considered as the foundation for research on plant-derived therapeutic vaccine(s) against chronic hepatitis B. Moreover, as planned vaccine would be of VLP type, the projects results can be considered important for the field of production of chimeric VLPs. Consequently, if research continued, the project results are important for plant biotechnology, especially biopharming, nanotechnology, immunology and medicine.
The project implementation achieved the stage of completion of the first phase - construction of the panel of 10 plant expression vectors for VLPs production. This makes possible continuation of research, which then and after dissemination of assumed results, will have
social implications due to importance for biotechnology, nanotechnology, immunology and medicine.
As the positive social impact of the project can be considered two review papers, which concerns important and timely aspects of plant biotechnology, including the use of HBcAg VLPs as a scaffold for heterologous epitopes – analogous for the use of HBcAg for displaying
HBsAg epitopes: ΔpreS1, ΔpreS2, a-SHBs.