Hepatitis B (HBV) and C viruses (HCV) are important human pathogens resulting in more than 500 million people being currently carriers. Sadly, Romania has the highest prevalence of HBV/HCV infections among the EU countries (up to 7% of the population). Chronically infected patients of HBV and HCV are at high risk to develop severe liver diseases, like fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Current therapies against HBV/HCV, based on replication inhibitors and immune system activators are associated with severe side effects. Moreover, both viruses are prone to the development of resistance to antiviral inhibitors. The objective of this project is to contribute to sustainable improvements in health of vulnerable social groups in Romania through bilateral collaboration of Romania and Norway with international partners participation. The outcomes are (i) Plant-produced HCV and HBV vaccines; (ii) Production of oral HCV and HBV vaccines in lettuce to further reduce the cost of vaccine production; and (iii) PhD education for capacity building in Romania and the Romania-Norway plant production platform, which will facilitate our future collaboration. The Norwegian Institute for Agricultural & Environmental Research (Bioforsk) will be responsible with the expression of HBV/HCV vaccine candidates in tobacco and lettuce plants as well as with the cost estimation of the production of the new vaccines and the social impact of the project results.
Summary of project results
It is estimated that over 6% of the world’s population is infected with the Hepatitis B virus (HBV) or Hepatitis C virus (HCV) and at risk of serious hepatic diseases such as fibrosis, cirrhosis or cancer. The current treatment of HBV/HCV infections consists in replication inhibitors and/or immune system activators which may have serious side-effects, determine the emergence of resistant mutations, do not completely eliminate the virus (HBV) or are extremely costly (HCV). Prophylaxis remains the most efficient way to address HBV/HCV infections, yet, there are issues associated with the HBV vaccine available, such as production costs and failure in almost 10% of vaccinated adults, while no commercial vaccine against HCV exists. In this context, the GreenVac project aimed to design new HBV/HCV antigens with superior immunologic properties and produce them in plants for potential vaccine development at reduced costs. 9 viral antigens were obtained: 3 HBV and 6 HCV. These were characterized biochemically and functionally in mammalian cells HEK 293T and plants Lactuca Sativa and Nicotiana Benthamiana. A selection of antigens with best properties was further made (2 HBV and 3 HCV) for immunology studies. BALB/c mice were vaccinated with these antigens using 12 different immunization schemes. The results of the immunization studies clearly indicated that the newly designed HBV antigen, HBV-S/preS121-47, has significantly better immunogenic properties than HBV-S, the protein component of the standard vaccine produced in yeast. The elicited antibodies have the ability to neutralize the HBV infection in tissue culture. These remarkable properties recommend the HBV-Ins antigen as a possible alternative for immunization of people who do not respond to the current vaccine and the plants as a viable and economical technological platform to produce this type of chimeric viral antigens. It was demonstrated for the first time the successful production of the E1E2 dimer in plants and its ability to trigger production of neutralizing antibodies by oral administration. The N-glycosylation process appears to be important for the antigenic properties of the E1E2 dimer and the quality of the immune response. A Romanian-Norwegian plant vaccine production platform, reuniting complementary expertise and technologies was developed. Results are published in high-impact factor journals (7,4 and 4,2, 3), disseminated in conferences (11) and under preparation for publication (3).
Summary of bilateral results
One of the most important results of the GreenVac project is validation of plants as viable technological platform for production of HBV/HCV antigens at low cost. By combining our expertise, we successfully cloned, characterized and produced a series of 9 novel HBV and HCV antigens in mammalian cells and Lactuca Sativa and Nicotiana Benthamiana plants. The antigens with best biochemical properties were short listed, purified at large scale and used for immunization experiments in animal models. Following a detailed analysis of the humoral and cellular immune responses obtained, it was concluded that a newly designed HBV chimeric protein has significantly better immunogenic properties than the current HBV vaccine antigen. The project has also demonstrated in premiere that complex HCV viral proteins can be correctly processed in plant cells and are able to trigger production of virus neutralizing antibodies in vaccinated mice. To fulfill the complex, transdisciplinary objectives of the GreenVac project, the consortium teams have shared technical expertise, scientific knowledge and specific reagents, adding great value and synergism to the results produced. The partnership established in this project is the first Romania-Norway bilateral collaboration in vaccine development, an important domain of public health. This fruitful collaboration has already led to submission of two applications for European funding of other projects (the EraNet Program) thus strengthening the current bilateral partnership and international visibility. In addition to the important amount of data in the field of HBV/HCV prophylaxis by vaccination, generated in this project, the most important achievement of this collaboration is the development of a functional partnership, which will last far beyond the project life-span. The project’s objectives would not have been possible to fulfill without the joint expertise of the Norwegian partner and the Romanian promoter and partners, with complementary knowledge in plant science (absent in Romania) and HBV/HCV fundamental virology and vaccine development and immunology, respectively (absent in Norway). Importantly, the project strongly benefited from the involvement of a highly reputable, internationally recognized academic institution in the HBV/HCV field from Lyon, France (CIIL), with whom PP had established a successful collaboration long before the start of this project, which has now extended at the level of the entire consortium.