Cold-adapted (ca) live attenuated influenza vaccines (LAIVs) have been introduced as alternatives to existing inactivated influenza vaccines. The influenza A components of the FDA-approved ca LAIVs (Flumist®; Medimmune) have common internal genes derived from the donor strain A/Ann Arbor/6/60 ca and surface genes derived from current wild-type (wt) epidemic strains. The aim of this thesis was to investigate determinants of immunogenicity for reassortants of A/Ann Arbor/6/60 ca, using a range of immunological assays, including recently developed MHC tetramer techniques. From the study, the extent of viral replication in the respiratory tract of mice, the primary site of inoculation, was a key factor in determining ca vaccine immunogenicity. Replication was shown to be influenced by both viral surface Ags and the host MHC. The H3 ca reassortants CR6, CR18, CR29 and CR6-35* exhibited greater replication efficiency (as determined by their PFU:HAU ratios) than the H1 ca reassortants CR35 and CR6-35. The H3 ca reassortant CR6 caused a 3.79% loss in body weight but no losses were observed for the H1 ca reassortant CR35 and the ca H2N2 donor strain A/Ann Arbor/6/60 ca. Higher HI responses were detected after 3 weeks in groups infected with the H3 ca reassortant CR6 (GMT 80) than with the H1 reassortant CR35 (GMT 10) and the H2 ca donor strain A/Ann Arbor/6/60 ca (GMT 13). Recently developed techniques were used to evaluate specific T-cell response to ca LAIVs. Fluorescent-labelled tetramer is the key reagent for use in tetramer-based flow cytometry assays. The NP366-374 peptide of influenza A viruses comprises an immunodominant epitope that is highly conserved between subtypes. Tetramers developed for A/PR/8/34 (H1N1) were able to detect NP-specific cytotoxic T lymphocytes (CTLs) induced by A/Ann Arbor /6/60 ca (H2N2). An attempt to prepare the A/Ann Arbor/6/60 ca-specific-NP-tetramer is described. H-2Db monomers were successfully refolded with the peptide, but only 20% were able to form tetramers through biotin-streptavidin linkage, resulting in a poor capacity to stain. By contrast, an IFN-γ ICC assay developed in parallel demonstrated that peptide NP366-374 was able to restimulate A/Ann Arbor/6/60 NP ca-specific CTLs and secrete IFN-γ when tested in vitro. Specific-B and T cell responses induced in the lungs in response to infection by ca reassortants exhibited great variability that was determined by the growth characteristics of different viruses. Type I (CTL) responses were induced by low yielding ca reassortants, such as CR35 (H1N1). Viruses with enhanced growth characteristics, such as CR6 (H3N2), produced higher Type II (HA-specific Ab) responses. In addition, host factors, such as MHC type, were found to play an important role in responses to the same viruses. Susceptible mouse strains, such as C57BL/6, showed higher CTL but lower serum Ab responses than more resistant strains, such as BALB/c. Throughout this PhD project, a fine balance between the humoral and CMI, local and systemic immune responses induced by ca LAIVs was demonstrated. The need to assess local immune responses, in addition to serum antibody levels, for the evaluation of vaccine efficacy was an important conclusion of the thesis.
| Identifer | oai:union.ndltd.org:ADTP/246528 |
| Date | January 2009 |
| Creators | Xue, Lumin, Lumin.Xue@csl.com.au |
| Publisher | RMIT University. Applied Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.rmit.edu.au/help/disclaimer, Copyright Lumin Xue |
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