In vitro adaptation of Influenza A PR8 virus to the murine lung adenoma cell line LA-4 and development of mRNA vaccine platform expressing the PR8 hemagglutinin subunit HA1

This thesis investigates Influenza A virus using the Puerto Rico/8/1934 (H1N1) [PR8] strain as a model to improve strategies for studying pathogenicity and developing immunization approaches. Influenza A viruses remain a major global health threat, responsible for over 250,000 deaths annually. This significant impact is primarily attributed to their broad host range and high mutation rate, thereby necessitating continuous research efforts to address this ongoing concern. Chapter 2 describes the in vitro adaptation of PR8 to the murine lung adenoma cell line LA-4, aiming to eliminate the need for repeated in vivo mouse passages, which are normally required to increase virulence in in vivo pathogenicity and immunization studies. After an initial expansion in the MDCK cell line, the standard propagation system used by ATCC, PR8 was subjected to 45 blind passages in the murine cell line. Viral infections were monitored by cytopathic effect observation and confirmed by fluorescence microscopy. Virus titration was performed using hemagglutination and TDIC50 assays. Replication efficiency assessed at virus passages 5, 15, 25, 35, and 45 indicated an increase in viral growth efficiency with the progression of passages. Whole-genome sequencing, performed on the same passages using amplicon-based Nanopore sequencing, revealed a progressive accumulation of single nucleotide polymorphisms (SNPs) over serial passages, resulting in nine amino acid substitutions across seven of the eight genomic segments. Additionally, a growth kinetics assay comparing the replication profiles of the PR8 wild-type virus and the LA-4-adapted variant at passage 45 showed higher viral titres starting from 24 hours post-infection for the PR8 variant compared to the wild type. These findings highlight that studying viral dynamics in murine cell models is a crucial, cost-effective, and ethical step to understand viral adaptation and pathogenicity, reducing reliance on animal models before in vivo experimentation. Following, the in vitro adaptation of PR8, Chapter 3 describes the development of a synthetic mRNA vaccine targeting the HA1 subunit of the PR8 hemagglutinin protein. In recent years, mRNA vaccines have gained prominence for their rapid production and potential to provide broad protection. In this study, a DNA construct, containing all necessary regulatory elements for mRNA synthesis, was designed and used as a template for in vitro transcription. Post-transcriptional modifications included the addition of a 5′ cap and a 3′ poly(A) tail to enhance stability and translation efficiency. The purified mRNA was delivered into human HeLa cells using cationic lipids (Lipofectamine™ 3000) as transfection agent. Expression of the HA1 protein was confirmed by Western blot analysis as early as 3 hours post-transfection. This study successfully developed and transfected an mRNA encoding the HA1 protein in vitro, laying the groundwork for a novel influenza vaccine platform with promising potential for future in vivo research.