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A systematic analysis of Epstein-Barr virus genes and their individual contribution to virus production and composition reveals critical downstream functions
A systematic analysis of Epstein-Barr virus genes and their individual contribution to virus production and composition reveals critical downstream functions
A vaccine to prevent infectious diseases associated with Epstein-Barr virus (EBV) has been put forward decades ago but has not made it to the clinic yet. EBV is a very complex herpes virus and its complexity, uncertainties about viral antigenic targets and technical difficulties to establish and mass-produce viral mutants are major obstacles. To mimic the complexity of the virus which encompasses more than 80 proteins, virus-like particles (VLPs) have a high potential as a vaccine prototype. To explore conditions to optimize and improve virus production, I established and tested an EBV gene library with 78 expression plasmids and a set of designed shRNAs investigating the functions of individual viral genes in the context of virus synthesis. Engineered virus stocks were then systematically characterized with respect to virus titers, bioparticle and physical particle concentration and virus uptake by primary human B cells, EBV’s target cells in vivo. To quantitate virus uptake by these cells, I developed a novel ß-lactamase-based assay that can monitor fusion events of the viral envelope with membranes of recipient cells at the level of single cells by flow cytometry. Together, my results identified several EBV genes such as BALF4, BVLF1 and BKRF4, encoding a viral glycoprotein, a regulator of transcription of late viral genes, and a possible tegument protein, respectively, that improve virus production regarding virus yield, virus composition and quality and virus uptake. My experi¬ments also indicated that EBV does not encode a master gene that governs EBV synthesis dampening virus production, contrary to my initial working hypothesis. Conditional expression of viral genes that improve EBV production as identified in my work will likely enhance and improve yield, assembly and important functional parameters of VLPs (such as their efficient uptake by antigen presenting immune cells, for example) supporting the development of a much improved EB-VLP based vaccine candidate to be used for clinical development and testing.
Epstein-Barr virus, EBV, herpesvirus, mutagenesis, fusion assay, physical particles, infection, virus titer, viral fitness
Chen, Yen-Fu Adam
2021
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Chen, Yen-Fu Adam (2021): A systematic analysis of Epstein-Barr virus genes and their individual contribution to virus production and composition reveals critical downstream functions. Dissertation, LMU München: Faculty of Biology
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Abstract

A vaccine to prevent infectious diseases associated with Epstein-Barr virus (EBV) has been put forward decades ago but has not made it to the clinic yet. EBV is a very complex herpes virus and its complexity, uncertainties about viral antigenic targets and technical difficulties to establish and mass-produce viral mutants are major obstacles. To mimic the complexity of the virus which encompasses more than 80 proteins, virus-like particles (VLPs) have a high potential as a vaccine prototype. To explore conditions to optimize and improve virus production, I established and tested an EBV gene library with 78 expression plasmids and a set of designed shRNAs investigating the functions of individual viral genes in the context of virus synthesis. Engineered virus stocks were then systematically characterized with respect to virus titers, bioparticle and physical particle concentration and virus uptake by primary human B cells, EBV’s target cells in vivo. To quantitate virus uptake by these cells, I developed a novel ß-lactamase-based assay that can monitor fusion events of the viral envelope with membranes of recipient cells at the level of single cells by flow cytometry. Together, my results identified several EBV genes such as BALF4, BVLF1 and BKRF4, encoding a viral glycoprotein, a regulator of transcription of late viral genes, and a possible tegument protein, respectively, that improve virus production regarding virus yield, virus composition and quality and virus uptake. My experi¬ments also indicated that EBV does not encode a master gene that governs EBV synthesis dampening virus production, contrary to my initial working hypothesis. Conditional expression of viral genes that improve EBV production as identified in my work will likely enhance and improve yield, assembly and important functional parameters of VLPs (such as their efficient uptake by antigen presenting immune cells, for example) supporting the development of a much improved EB-VLP based vaccine candidate to be used for clinical development and testing.