Research And Application Of Interferon Response Inhibition On Virus Replication

Vaccines are a type of biological products used to prevent and control the spread of infectious diseases.Vaccines have played an important role in the history of humankind's fight against diseases and are considered one of the greatest achievements in medical science.Depending on the pathogen,vaccines are divided into bacterial or viral vaccines.Cells are almost the only host of viruses,and the culture technology of viruses in cells has become the main way of producing viral vaccines.Cell lines that can be used for vaccine production include primary cell lines:hamster kidney cells,chicken embryo cells;diploid cell lines:human diploid cells,monkey diploid cells;passaged cell lines:Vero cells.Vero cells are interferon(IFN)-deficient cell lines that are susceptible to most viruses.Vero cells produce high-yield and low-cost vaccines,including most commercially available inactivated virus vaccines and live attenuated vaccines such as hepatitis A vaccine,rabies vaccine and polio vaccine.However,as Vero cells are monkey-derived cells,their use in the field of human vaccines has obvious defects,including adverse reactions after vaccination and a higher risk of carcinogenesis owing to the presence of non-human DNA contamination.Currently,the best solution is to replace Vero cells with human diploid cells.For example,the human diploid MRC-5 cell line is widely used in the production of many vaccines and has obvious advantages in terms of safety,especially the removal of non-human DNA contamination from host cells.However,human diploid cells are difficult to culture,not easily infected by most viruses,and have low virus yields.Therefore,vaccines based on MRC-5 cells are expensive.Developing countries in Asia and Africa account for 99%of deaths by rabies worldwide,but more than 90%of people can only be vaccinated with the purified Vero cell rabies vaccine(PVRV).Human diploid cell vaccines(HDCV)are widely used only in developed countries in Europe and America.China is also a large country affected by rabies.The output of HDCV produced in China is one-tenth that of PVRV,and the price of HDCV is10 times higher that of PVRV.The rapid development of the country's economy has increased the demand for the development of a safer HDCV.Therefore,the exploration and development of a new generation of HDCVs have far-reaching strategic significance for upgrading the vaccine industry and promoting vaccine safety.At present,there are still significant obstacles and deficiencies in vaccine production based on human diploid MRC-5 cells.In this study,we compared the differences in the virus replication level between Vero and MRC-5 cells.Based on the regulatory mechanism of the IFN response after the interaction between the rabies virus and cells of the innate immune response,we aimed at improving the replication level of rabies virus in MRC-5 cells.We effectively applied this method to the pilot production of inactivated rabies vaccine,This method provide a reference for the development and application of a new generation of vaccines based on human diploid cells.The main research contents of this study are as follows:1.By comparing the level of viral replication and IFN response in MRC-5 and Vero cells infected with rabies virus,we found that the replication level of rabies viruses in MRC-5 cells was only 1/10 of that in Vero cells,whereas the level of IFN response in MRC-5 cells was significantly higher than that in Vero cells.The relative expression of IFN-?in MRC-5 cells was more than 100 folds higher than that in Vero cells.This finding suggests that the activation of the IFN response caused by the viral infection may be the main reason for the low level of rabies virus replication in human diploid MRC-5 cells.2.By using the IFN response inhibitor and IFN-?/?receptor subunit 1(IFNAR1)monoclonal antibody,effective inhibition of the IFN response of MRC-5 cells after virus infection was achieved.IFN response inhibitors caused an approximately 100 folds reduction in relative expression of IFN-?in MRC-5 cells.The IFN response inhibitor and IFNAR1monoclonal antibody resulted in 10 and 5 folds increase in rabies virus replication in MRC-5cells,respectively,and the level of virus replication in the IFN response inhibitor group reached that in Vero cells.Inhibition of the IFN response was effective in increasing the level of rabies virus replication in MRC-5 cells.3.The rabies virus P protein interacts with IFN response components.Overexpression of the rabies virus P protein in MRC-5 cells demonstrated that the P protein could effectively antagonize the IFN response through its C-terminal domain.This antagonism was enhanced by the pre-expressed P protein,which increased the level of rabies virus replication in MRC-5 cells3 to 4 folds.4.The IFNAR1-silenced MRC-5(MRC-5^IFNAR1-)cell line was successfully established using CRISPR-Cas9 gene-editing technology.The IFNAR1 gene in this cell line produced frameshift mutations,and its expression was reduced by 50%–70%.The levels of IFN responses in the MRC-5^IFNAR1-cell line was significantly reduced by rabies,herpes zoster,and hepatitis A,and rabies virus replication in the MRC-5^IFNAR1-cell line was increased 6 to 7 folds.This study lays the foundation for the application of the MRC-5^IFNAR1-cell line in HDCV production.5.In this study,a 60,000cm² scale pilot production evaluation was performed for the IFN response inhibitor and MRC-5^IFNAR1-cell line.The results showed that the IFN response inhibitor and MRC-5^IFNAR1-cell line increased rabies virus production 10 and 5 folds,respectively.This study lays the foundation for the application of human diploid cells in large-scale production of a rabies inactivated virus vaccine.In this study,the production of an inactivated rabies virus vaccine was used as a case study.At the laboratory and pilot production levels,it was demonstrated that the inhibition of the IFN response could improve the replication level and yield of the virus in human diploid MRC-5cells.An invention patent has been submitted for this method,and the MRC-5^IFNAR1-cell line can be used in large-scale production,providing a new tool that will greatly promote the development and application of HDCVs.