Evolution of Recent Human H3N2 viruses

Influenza A virus (IAV) epidemics impose a large economic and health burden. Seasonal recurrence of IAVs is driven by antigenic drift of its surface glycoproteins hemagglutinin and neuraminidase, and urges frequent changes in IAV vaccine composition. Updating of vaccine strains on basis of antigenic characterization is not self-evident and recently further hampered by changes in receptor-binding properties that interfere with antigenic characterization techniques and with strain propagation in eggs and cell culture. This research plan describes the development of assays, applying biolayer interferometry technology (BLI), by which antigenic drift and evolution of receptor-binding properties can be studied in concert. Research will in particular focus on recent H3N2 strains with the aim to improve selection of well-matched vaccine strains. It should also result in directing the generation of cell lines that are adapted for efficient production of contemporary H3N2 viruses while minimizing the acquisition of cell-culture-adaptations that may affect their antigenicity.

Aims and Approach
I) Obtain insight into the evolution of receptor-binding of recent H3N2 viruses.
We will further develop our BLI assays for the analysis of virus-receptor interaction dynamics and the analysis of the HA-NA-receptor balance of  H3N2 viruses. We will screen for receptors to which recent H3N2 viruses preferably bind by using synthetic glycans as well as tailor-made (e.g. by applying a range of glycosyl transferases) recombinant glycoproteins (produced in 293T cells) that better mimic the IAV receptors encountered in vivo.
II) Development of cell lines for production of H3N2 viruses lacking cell-culture-adaptations affecting their antigenicity.
Tailor-made glycoproteins allowing enhanced H3N2 virion-binding as identified during I) will first be transiently expressed in 293T cells and subsequently in MDCK cells, which are routinely used to generate high titre IAV stocks. We expect that increasing the number of sialoglycans containing branched and long polylactosamine chains at the cell surface exposed glycoproteins will increase binding and infection by recent H3N2 viruses. After providing proof on concept, stable cell lines will be generated and we will analyse whether the presence of an optimized receptor repertoire  on these stable cell lines will prevent the acquisition of mutations in HA and NA by recent H3N2 viruses.
III) Development of alternatives to the hemagglutination-inhibition (HAI) assay .
BLI in combination with the tailor-made glycoproteins or synthetic glycans will be used to develop an alternative HAI assay that circumvents the current requirement for HA-dependent hemagglutination of erythrocytes. A BLI based assay will enable to quantify the ability of antibodies/antisera to interfere with virus-receptor interactions by (1) inhibition of virus-association to optimal receptors or (2) by induction of virus dissociation from receptors. To validate the method we will generate and compare antigenic maps (based on titers corresponding to 50% inhibition of virus binding or dissociation) using viruses and anti-sera that have been used previously for creating antigenic maps by HAI for H3N2 viruses isolated between 1968 and 2002. Subsequently, we will use these assays for antigenic characterization of recent H3N2 viruses using receptors to which these viruses bind well.  

IAVs cause a huge health and economic burden. The estimated annual total economic burden to the healthcare system and society runs in billions of euros, dollars, yuans. These costs will explode in case of an influenza pandemic. Research, such as proposed here, will help to better understand the evolution of human IAVs. This hopefully increases the  possibilities to predict its impact on antigenic change thus allowing the generation of better matched vaccines in a timely manner.

Mengying Liu