Systems Design and Synthetic Construction of Influenza Virus for Flu Vaccine Application

Abstract

Influenza A virus (IAV) is the leading cause of annual flu epidemics, which inflicts about 250,000-500,000 deaths worldwide. The morbidity and mortality rate are much higher when a novel strain of IAV arises, resulting in flu pandemics. Vaccination has been the best prevention strategy for influenza. However, flu viruses constantly evolve and escape the established immunity, thus annual flu vaccination is required. Most current flu vaccine manufacturing platforms use multi-plasmid transfection to rescue seasonal seed viruses, the seed viruses are then used to infect either embryonic chicken eggs or cultured cells to produce viruses. Both production methods have high degrees of variability and produce viruses with a high content of non-infectious particles that reduce vaccine effectiveness. To address the need for more reliable and scalable processes, we applied systems biology and synthetic biology approaches to understand the kinetics of virus replication and to engineer cell lines that can control viral gene expression dynamics. First, we established a new data analysis pipeline using RNA sequencing to study segment-specific kinetics of all IAV RNA molecules. Using the pipeline, InVERT, to study the kinetics of IAV infection, revealed different phases of virus infection, and groups of genes whose kinetics are similar. This was the first-time IAV replication kinetics of all segments is reported. Building on that success, we then developed the second pipeline named InVERT II, which can further differentiate mRNA transcripts made by the viral replication enzyme RdRP from mRNA transcripts synthesized by host cells' RNA Polymerase II, to study the kinetics of virus rescue by transfection. With the understanding gained from the kinetics of virus infection and replication, we engineered the human cell line HEK 293T to express inducible components of IAV that not only have inducible replicative activity but also can package virus particles. This is the first proof of principle to show that mammalian cells can be engineered to produce complex negative-sense RNA viruses. Our integrative approach using both systems biology and synthetic biology has enabled the creation of a platform that could be further optimized for reliable, robust, and scalable flu vaccine manufacturing processes.