Elucidating primary human nasal epithelial cell responses to LAIV/IAV infection in 2-dimensional culture systems

Proudlove, Christopher James (2023) Elucidating primary human nasal epithelial cell responses to LAIV/IAV infection in 2-dimensional culture systems. PhD thesis, University of Glasgow.

Abstract

Influenza continues to represent a global health challenge, causing an estimated 650,000 deaths annually. The ability of Influenza viruses to undergo genetic drift and shift, leading to evasion of vaccination efforts, requires annual global surveillance of circulating influenza strains to best match vaccines to prevalent viral targets. Influenza vaccines can be split broadly in to two categories, Inactivated Influenza Vaccines (IIVs) and Live Attenuated Influenza Vaccines (LAIV). LAIV induces an attenuated active infection in the nasal tract, leading to specific and lasting adaptive immunity to influenza infection. Previous vaccination seasons with LAIV have found variable vaccine effectiveness (VE) with H1N1 subtypes, leading to continued studies to identify potential causes of variable VE. Madin-Darby Canine Kidney (MDCK) cells are widely used during LAIV research and development. However previous studies have found MDCK cells to be significantly more permissive to IAV than human airway cells. Here we elucidate the potential of using 2D primary human nasal epithelial (hAECn) cells in place of MDCK cells for LAIV medium-throughput assays. Using focus-forming unit (FFU)-style assay, we show that MDCK cells are significantly more permissive to LAIV than hAECn during single-cycle infection. We show that this is not a result of attachment/entry defects in hAECn cells and that LAIV A/Slovenia/2903/2015 has a higher attachment/entry efficiency than LAIV A/Bolivia/559/2013 in hAECn cells by quantifying intracellular virions 2hpi using absolute quantification digital-droplet PCR. We show by RT-qPCR that in hAECn cells, LAIV A/Slovenia/2903/2015 induces greater Myxoma resistance 1 (MX1) expression than LAIV A/Bolivia/559/2013. We show by 2-step RT-qPCR, that there are donor-dependent differences in MX1 expression levels, and by pooling donors, the interferon stimulated gene (ISG) response falls within the range of the constitutive individual donors. We used open-access transcriptomic data from GTEX/Human Protein Atlas to show that constitutive expression of lung ISGs in 578 individuals is dependent on both sex and age, with males showing a higher level of differential ISG expression as a result of changing age compared to females, only Complement Factor H (CFH) showed significant difference as a result of advanced age in both females and males. Collectively, these data show hAECn cells may provide a more physiologically representative system for 2-Dimensional (2D) medium-throughput IAV/LAIV assays than MDCK cells, however, optimisation of immortalisation techniques is required to make them a cost-effective alternative to MDCK cells. Our data suggests that LAIV H1N1 strains induce different expression levels of ISG (MX1), we suggest this may contribute to differences in strain-specific adaptive responses and subsequent variable VE. We show that there is donor specific ISG responses (MX1) to LAIV, however analysis of constitutive lung ISG expression within the population show high levels of variation which may account for this. More work is required to understand the feasibility of using 2D hAECn cells to replace MDCK cells in IAV/LAIV research and development assays.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QR Microbiology > QR355 Virology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
• Supervisor's Name: Boutell, Dr. Chris
• Date of Award: 2023
• Depositing User: Theses Team
• Unique ID: glathesis:2023-83974
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 06 Dec 2023 10:11
• Last Modified: 06 Dec 2023 10:12
• Thesis DOI: 10.5525/gla.thesis.83974
• URI: https://theses.gla.ac.uk/id/eprint/83974