The host cell probably plays an important role in neutralization but previous work on influenza virus neutralization used dedifferentiated cultured cells derived from tissues which are not the natural target of the virus. The aim of this investigation was to study the mechanisms of neutralization of influenza virus by different antibody isotypes using fully differentiated, ciliated epithelial cells of tracheal organ cultures. Tracheal organ cultures derived from the mouse were used together with mouse pathogenic strains (A/FPV/Rostock/34 (adpFPV/R) and A/PR/8) and mouse antibodies, so that all the participating components came from the same species. Parallel experiments were performed using cultured BHK cells and erythrocytes to enable comparison between different cell types. As previous work had used only a single saturating amount of antibody I decided to use a range of antibody concentrations with a constant amount of virus. Three potential mechanisms of neutralization were investigated: aggregation of virus, inhibition of virus attachment to cells and inhibition of virus internalization by cells. Each isotype (IgG, IgM and IgA) aggregated influenza virus (by EM determination) when used in sub-saturating amounts. Increasing the amount of antibody reduced aggregation and at higher antibody concentrations, the virus was monodisperse. Sub-saturating amounts of IgG monoclonal antibody partially inhibited the attachment of adpFPV/R to tracheal organ culture cells, BHK cells and erythrocytes. Together, aggregation and inhibition of virus attachment by IgG accounted for more than a 90% loss of infectivity, although this was usually less than the amount of neutralization observed. It is argued that these represent potentially important mechanisms of neutralization. Increasing the IgG concentration increased neutralization and increased attachment of virus to tracheal organ culture and BHK cells but not to erythrocytes. Therefore when there is a high ratio of IgG to virus, neither aggregation or inhibition of virus attachment accounted for any loss of infectivity. Under these conditions IgG did not prevent internalization of virus that had attached to tracheal organ culture or BHK cells and it was concluded that inhibition of an intracellular stage of the virus infectious pathway was the major mechanism of neutralization. Polyclonal IgM partially inhibited attachment of virus to organ culture cells, BHK cells and erythrocytes. Increasing the amount of IgM did not increase virus attachment to any of the cell types. Internalization of IgM-neutralized virus that had attached to tracheal organ culture and BHK cells was blocked, confirming earlier observations when a single saturating concentration of IgM was used. Neutralization of A/PR/8 by sub-saturating amounts of monoclonal polymeric IgA also inhibited virus attachment to BHK cells and erythrocytes. There was no rise in attachment when the amounts of IgA were increased and internalization of attached virus was inhibited. Sub-saturating amounts of IgA also greatly reduced virus attachment to tracheal organ culture cells. However increasing the relative IgA concentration resulted in increased attachment. The highest concentration of IgA used enhanced virus attachment by more than five-fold compared to infectious virus but this was accompanied by no increase in infectivity. Enhanced attachment was neuraminidase-resistant and was possibly mediated via an IgA-specific Fc receptor.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:237807 |
| Date | January 1989 |
| Creators | Outlaw, Mark Charles |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/101997/ |
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