Influenza is an infectious disease caused by Orthomyxoviridae viruses that infect 3-5 million people worldwide during seasonal epidemics. Influenza vaccines must be produced and administred annually to respond to the high rate of mutations and possible exchange of genomic segments between different strains. The vaccines are produced by a well established prodedure in inoculated embryonated eggs. Influenza virions are purified by sucrose gradient and then split by detergent filtered and inactivated by formaldehyde. Samples of split virions contain mainly HA and NP viral proteins.
To monitor quality of produced vaccines, quantitative information about the oligomerization state and molecular assembly of antigens in produced lots is desirable. We have developed a new approach to determine the size distribution of particles in split-viron influenza vaccine samples by electron microscopy and could thus interpret elution profiles from the asymmetrical flow field-flow fractionation (A4F) method. Samples were imaged by negative-stain electron microscopy using organo-tungstate stain (nanoW, pH 8.0) at 19,000x magnification (5.59 Å/pix) and 4-μm defocus (Figure 1). About 40-60 micrographs were initially filtered to remove variations in background and improve signal-to-noise ratio . Individual particles were then segmented by a watershed algorithm . Areas of the segmented 2D projections were determined in ImageJ  and plotted as histograms of the size distribution. (Figure 2). This approach is able to distinguish among different oligomeric states of HA and NP subunits in the sample, however all larger assemblies and aggregates are not resolved.
In parallel, we analyzed the same samples by A4F. Elution profiles resolved three peaks of small particles (diameter 200 nm). While the first three peaks correspond to the HA and NP oligomers resolved in the size distribution resolved in the analysis by electron microscopy, the latter peaks reflect presence of large lipid vesicles with embedded HA proteins. Interpretation of the A4F elution profiles allows routine screening and monitoring of produced split-virion influenza vaccines.
This study has been funded by Sanofi Pasteur.
 Tang, G. et al. (2007) J. Struct. Biol., 157, 38-46.
 Heymann, J.B. (2001) J. Struct. Biol.,133, 156-169.
 Schneider, C.A. et al. (2012) Nature Methods, 9, 671-675.
To cite this abstract:Marie-Claire Nicolai, Aurelie Deliot, Frederic Ronzon, Jean-François Cotte, Catherine Manin, Daniel Nemecek; Quantification of particles in samples of a split-virion influenza vaccine. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/quantification-of-particles-in-samples-of-a-split-virion-influenza-vaccine/. Accessed: July 13, 2020
EMC Abstracts - https://emc-proceedings.com/abstract/quantification-of-particles-in-samples-of-a-split-virion-influenza-vaccine/