Influenza virus : Protection and adaptation

University dissertation from Stockholm : Karolinska Institutet, Microbiology and Tumor Biology Center (MTC)

Abstract: Influenza is an acute respiratory disease caused by influenza type A and B viruses. Human influenza viruses may infect up to 15% of the total population during the seasonal epidemics, causing many cases of severe illness. Each year, approximately 350 million doses of influenza vaccine are produced for protection of those at risk of severe disease. The thesis focuses on the protection against influenza virus infection and disease (Paper I and II) as well as the analysis of the antigen variations found in primarily the hemagglutinin (HA) gene of the virus (Paper III and IV). The time required for the production of influenza vaccines is 6-8 months. A more effective and rapid method of production is desirable, both for the annual epidemics and in case of an influenza pandemic. A DNA-based vaccine could be a useful alternative. In paper I, the immunological response in ferrets after intramuscular immunisation with a plasmid construct expressing chimeric influenza HA proteins was evaluated. Strain specific antibodies were elicited, but none of the ferrets immunised with a DNA or subunit vaccine were protected from infection when challenged with an influenza A/H3N2 virus homologous or heterologous to the vaccine. Considerable enhancement of the immune response induced by DNA immunization will be needed before the approach can be a realistic alternative for vaccination of humans. Hem agglutination inhibition (HAI) is the standard method for determination of protective levels of influenza antibodies. The method is not efficient for all virus subtypes and strains, and alternative methods suitable for large-scale examinations are desirable. In paper II, an in situ neutralisation test (NT) for the measurement of influenza antibodies was created and evaluated in two human cell-lines, human fibroblasts (HS27) cells and human salivary gland epithelial duct (HSG) cells, and in Madin-Darby canine kidney (MDCK) cells. The HS27 cell line gave stable results and was most suitable for antigen detection with enzyme-linked immunosorbent assay. It was therefore chosen for the analysis of the humoral response after an influenza A infection in patients treated or not treated with the antiviral drug zanamivir. No titre differences between the groups could be verified at 28 days after onset. The NT using HS27 cells revealed heterologous NT-titre rises after the influenza infection. The antigenic drift occurring in human influenza viruses is mainly affecting the cell receptor binding glycoprotein, HA. It is important to identify antigenic changes in the HA occurring in vitro since they may be mistaken for drift occurring in vivo. In paper III, we characterised the variable region of the HA gene from nine recent human influenza A/H3N2 viruses after up to 11 passages in both HSG cells and MDCK cells. Ten amino acid alterations were identified in both MDCK-propagated and HSG-propagated strains. All altered residues were either close to the receptor-binding site or within it. We conclude that these alterations most likely represent an adaptation to growth in vitro. To further study antigenic drift in vitro we have passaged a human influenza A virus in MDCK cells in the absence or presence of three polyclonal neutralising sera (Paper IV) and sequenced the HA gene from selected passages. One mutation causing the amino acid change I140M was found after 20 and retained after 28 passages in the presence of serum. Our analysis of the HA gene from different passages showed that during propagation in vitro in the presence of specific sera escape mutations in HA are not easily obtained. The study confirms the relative genetic stability of the HA gene of influenza A/H3N2 viruses in cell culture.

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