Virus production for vaccines still is a challenging issue, particularly with slow-growing viruses. Many vaccines are produced in embryonated hen’s eggs or continuous avian cell lines; however, it is well established that the rate determining step in the manufacture of numerous vaccines is the induction of antiviral immune responses within the host cell that inhibit the replication of vaccine viruses. Furthermore, millions of eggs are required to meet vaccine requirements. Relatively little data is available on the immune capacity of the chicken embryo, and the innate response to vaccine inoculation and growth.
Type I interferons protect cells from virus infection through the induction of a group of genes collectively named interferon-stimulated genes (ISGs). Among these interferon-stimulated proteins, are the IFITMs (interferon-inducible transmembrane) which have been shown to restrict the replication of several highly pathogenic viruses, including severe acute respiratory syndrome (SARS) coronavirus, Ebola virus, influenza A viruses, and flaviviruses (dengue virus). Through analysis of conserved synteny between the IFITM locus in the human genome and a region of the chicken genome, combined with targeted PacBio sequencing of the chicken IFITM locus, we have established that IFITM1, 2, 3, and 5 are present in the chicken genome, and are induced in response to avian viruses. We aim to generate chIFITM knock-out cell lines using cutting edge gene editing techniques such as CRISPR/Cas9, which will directly target the chIFITM locus. We believe that this approach will overcome the bottleneck in vaccine production, resulting in increased vaccine yields and reducing the time in which vaccines can be manufactured.
Using a combination of synthetic siRNAs and CRISPR/Cas9 gene editing techniques, we have successfully ablated the transcriptional expression of each gene in vitro, and determined the effect of the loss of these genes on antiviral immunity and viral titre.