Abstract
The human immune response to influenza vaccination depends in part on preexisting cross-reactive (heterosubtypic) immunity from previous infection by, and/or vaccination with, influenza strains that share antigenic determinants with the vaccine strains. However, current methods for assessing heterosubtypic antibody responses against influenza, including the hemagglutination-inhibition (HAI) assay and ELISA, are time and labor intensive, and require moderate amounts of serum and reagents. To address these issues we have developed a fluorescent multiplex assay, mPlex-Flu, that rapidly and simultaneously measures strain specific IgG, IgA, and IgM antibodies against influenza hemagglutinin (HA) from multiple viral strains. We cloned, expressed and purified HA proteins from 12 influenza strains, and coupled them to multiplex beads. Assay validation showed that minimal sample volumes (<5 μl of serum) were needed, and the assay had a linear response over a four Log10 range. The assay detected nanogram levels of anti-influenza specific antibodies, had high accuracy and reproducibility, with an average percentage coefficient of variation (%CV) of 9.06 for intra-assay and 12.94 for inter-assay variability. Pre- and post-intramuscular trivalent influenza vaccination levels of virus specific Ig were consistent with HAI titer and ELISA measurements. A significant advantage of the mPLEX-Flu assay over the HAI assay is the ability to perform antigenic cartography, determining the antigenic distances between influenza HA’s, without mathematical correction for HAI data issues. For validation we performed antigenic cartography on 14 different post-influenza infection ferret sera assayed against 12 different influenza HA’s. Results were in good agreement with a phylogenetic tree generated from hierarchical clustering of the genomic HA sequences. This is the first report of the use of a multiplex method for antigenic cartography using ferret sera. Overall, the mPlex-Flu assay provides a powerful tool to rapidly assess the influenza antibody repertoire in large populations and to study heterosubtypic immunity induced by influenza vaccination.
Highlights
Annual immunization against influenza infection is one of the largest coordinated international public health efforts [1, 2]
Recombinant hemagglutinin protein (HA) proteins prepared from different expression systems, such as human cell-lines and insect cells, have been shown to differ in their biological activity and ability to bind anti-HA antibodies due to differences in glycosylation patterns and protein folding from natively isolated virus [30]
It has been reported that Recombinant HA (rHA) expressed by insect cells appeared to preserve both the antigenic properties and biological activity of HA proteins [31]
Summary
Annual immunization against influenza infection is one of the largest coordinated international public health efforts [1, 2]. Antibodies directed against the influenza surface hemagglutinin protein (HA) are a major source of protective immunity, blocking viral binding of the HA1 subunit to sialic acid expressed on the surface of target epithelial cells, and preventing viral entry [3]. Influenza virus infection and vaccination both induce antibodies that bind to molecularly similar influenza subtypes, a phenomenon termed heterosubtypic immunity (HSI), and a major reason for the success of seasonal influenza vaccination [4, 5]. Vaccines containing HAs with small antigenic changes from the prior years’ influenza strains have a high probability of inducing memory B cells to classswitch and secrete IgG anti-HA antibodies. Pandemic strains are defined by low antigenic homology to prior vaccines and previously circulating influenza strains, and evade the human adaptive immune response [6,7,8,9]
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