Improving Modern Influenza Virus Propagation by Engineering Glycan Expression

There are currently 4 strains of influenza circulating in humans causing seasonal epidemics: 2 subtypes of influenza A virus (A/H1N1 and A/H3N2) and 2 influenza B viruses (B/Yamagata) and B/Victoria). Influenza A viruses are named depending on their main surface proteins; hemagglutinin (HA) and neuraminidase (NA). HA facilitates attachment to cells via its interaction with sialic acid (Sia) terminating glycans present on the cell surface, whilst NA allows release of virions from cells by cleaving Sia residues. Sia can be linked to glycans via alpha2-3 or alpha2-6 glycosidic bonds. Human influenza viruses preferentially bind alpha2-6 linked Sia, which is the main configuration present on receptors in the upper respiratory tract (URT) in humans. Conversely, avian viruses preferentially bind to alpha2-3 linked Sia, which is mainly present in birds as well as the deep lung of humans.
Virus propagation is an essential step in evaluating influenza virus strains. Traditionally, MDCK cell lines are used, but modern human A/H3N2 viruses grow poorly in MDCK cells as the majority of Sia present on the cells are alpha2-3 linked. MDCK SIAT cells, which express human alpha2-6 sialyltransferase, have a higher proportion of alpha2-6 linked Sia, however, viruses grown in these cells develop mutations in their HA and NA gene segments making them difficult to characterize. Recently, a cell line which expresses an even higher proportion of alpha2-6 linked Sia, termed hCK cells, was developed. Virus propagation was improved in this cell line, yet mutations have still been seen, such as D151G in the viral NA, which allows NA to compensate for poor HA binding. Research suggests that A/H3N2 virus HA has evolved to preferentially bind bi-antennary complex N-glycans containing extended N-acetlylactosamine (LacNAc) chains terminating in alpha2-6 linked Sia. This project will explore this by engineering glycan biosynthetic pathways to express these types of glycans in cells. We aim to create a stable cell line with the ability to effectively propagate modern A/H3N2 viruses which do not acquire mutations.
Glycomic workflows paired with mass spectrometry will be used to characterize the glycans present on cloned cells, in order to ensure the correct structures are present. In conjunction with this, cloned cells will be tested to assess their susceptibility to modern A/H3N2 viruses. The genetic stability of viruses propagated in these cells will also be assessed.