Microbiology & Immunology - Theses
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ItemImproving influenza vaccines( 2013)The World Health Organisation estimates that seasonal influenza is responsible for 3-5 million cases of severe illness and up to half a million deaths annually. It is well established that immunisation is the most cost-effective way to limit the impact of influenza across the community, however vaccine effectiveness is lowest in those most at risk of severe illness including the very young, elderly and the immunocompromised. Studies in this thesis have examined ways in which the production and efficacy of current seasonal influenza vaccines could be improved. We provide proof of principle for vaccines that enhance viral clearance upon co-delivery of a T cell-inducing lipopeptide with the current split influenza virus vaccine. We show the addition of the T cell-inducing lipopeptide to the split virus vaccine led to an improvement in viral clearance irrespective of dose or delivery route compared to the spilt virus vaccine alone, however the degree of viral clearance and immune mediators involved differed. The addition of the T cell-inducing lipopeptide to the split virus vaccine resulted in increased protection either through a boost in antibodies titres, the induction of both antibodies and CD8+ T cells or through the recall of large numbers of vaccine-induced CD8+ T cells alone. The ability to induce CD8+ T cells suggests that this combination vaccine has the potential to protect against seasonal influenza, mismatched strains and novel pandemic viruses, while the observed increase in the antibody titres indicates the potential for dose minimisation, which may be vital when immunising large numbers of people in the event of a pandemic outbreak. Currently the annual production of the split inactivated influenza virus vaccines exploits “classical reassortment” of the seasonal influenza isolate with a highly egg-adapted strain to maximise yields of egg-grown virus. Seed viruses produced in this way are selected based on a high-growth phenotype and the presence of the seasonal haemagglutinin (HA) and neuraminidase (NA) surface antigens. A retrospective analysis of vaccine seed viruses indicated that, unlike other internal proteins that were predominantly derived from the high growth parent, the PB1 gene of the seasonal isolate was selected in greater than 50% of reassortment events analysed. Using the model seasonal H3N2 virus A/Udorn/307/72 (Udorn) and the high-growth A/Puerto Rico/8/34 (PR8) virus we assessed the influence of the source of the PB1 gene on virus growth and vaccine yield. Classical reassortment of these two strains led to the selection of viruses that predominantly had the Udorn PB1 gene mimicking cases where viruses containing the seasonal PB1 dominate the progeny. Using reverse genetics-derived viruses, we showed that the presence of Udorn PB1 resulted in a virus with significantly inferior growth compared to the seed virus with PR8 PB1. Nevertheless, the poorer growing virus had more HA per virion, giving an overall two-fold reduction in the yield of antigen. Analysis of past vaccine viruses also suggested that the inclusion of the seasonal PB1 resulted in higher HA per particle. We postulate that although these seasonal PB1-containing viruses are selected as vaccine seed strains and show higher HA per particle, they may not be the best choice as seed viruses. As the HA vRNA and mRNA levels in infected cells were similar in the presence or absence of the Udorn PB1, we propose that PB1 selectively alters the translation of viral mRNA, affecting the relative protein composition of the virions. To investigate the mechanisms driving the selection of a less fit virus, competitive plasmid transfections were used to show that the Udorn PB1 gene segment co-segregates with the Udorn NA and not HA gene segment. Analysis of chimeric PB1 genes revealed that the co-selection of NA and PB1 segments was not directed through the previously identified terminal packaging sequences but through interactions involving the internal coding region of the PB1 gene. The studies presented in this thesis provide supportive evidence for the use of our combination vaccine to improve protection against influenza infection. We have also added to the knowledge of the “classical reassortment” process used in split influenza virus vaccine production and, by improving our understanding of the drivers of this process have come to better understand some of the limitations to genetic diversity in the creation of novel pandemic strains that arise by reassortment.