Multi-Dimensional Measurement of Antibody-Mediated Heterosubtypic Immunity to Influenza

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.

Identification of novel influenza A virus exposures by an improved high‐throughput multiplex MAGPIX platform and serum adsorption

“…The platform of this 20‐plex MAGPIX, test flow, and serum adsorption is described in Figure . The high‐throughput MAGPIX platform can generate an antibody profile to 19 influenza antigens simultaneously by using very small volume of serum samples (<10 µL), and the similar platform showed a 4 ‐ log10 linear range of sensitivity …”

“…Seroconversion with a ≥ 4‐fold rise in HI or MN antibody titers is indicative of recent influenza virus infection or vaccination . In recent years, magnetic multiplex fluorescence microsphere immunoassays (MAGPIX) have been developed to investigate antibody profiles following influenza vaccination/infection and aid in the diagnosis of influenza . Previously, we developed a high‐throughput MAGPIX assay with serum adsorption using two ectodomain recombinant HAs (rHAs) from A(H1N1)pdm09 (A/California/7/2009, CA/09) and A/Perth/16/2009 (Perth/09, A(H3N2)) to identify novel influenza exposures when well‐paired human serum samples were available .…”

Novel multiplex assay platforms to detect influenza A hemagglutinin subtype‐specific antibody responses for high‐throughput and in‐field applications

“…Results from DPP can be obtained in a non‐laboratory setting in less than 30 minutes, making it an appealing choice for in‐field applications. MAGPIX is cost‐effective and high throughput, requires limited sample volume and staff time, and reduces aliquot errors . Both platforms are multiplex assays with several influenza subtype antigens, ideal for serum specimen collected from locations where multiple subtypes of influenza viruses cocirculate.…”

“…A multiplex protein microarray using globular head domain HA1 (GH HA1) was developed to investigate antibody profiling, and high sensitivity and specificity were achieved by bivariate model of microarray using HAs from pH1N1 and A/South Carolina/1918 (H1N1) viruses . Recently, a mPLEX‐Flu assay using full‐length HA was also used to evaluate HA profiles and demonstrated good correlation between the median fluorescence intensity (MFI) and anti‐HA antibodies in ELISA …”

“…4,7,8 Recently, a mPLEX-Flu assay using full-length HA was also used to evaluate HA profiles and demonstrated good correlation between the median fluorescence intensity (MFI) and anti-HA antibodies in ELISA. 9 Detections of antibody responses to influenza in humans are often complicated by the complex antibody profiles generated from past exposure through infection or vaccination with multiple influenza viruses. Novel influenza viruses may share common epitopes with seasonal viruses that an individual may have been exposed to previously, causing both within-subtype (homosubtypic) or cross-subtype (heterosubtypic) cross-reactivity.…”