title: 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.
authors.affiliation: Division of Nephrology, Department of Medicine and the Rochester Center for Biodefense Immune Modeling, University of Rochester Medical Center, Rochester, New York, United States of America.
authors.forename: Jiong
authors.initials: J
authors.lastname: Wang
authors.affiliation: Division of Nephrology, Department of Medicine and the Rochester Center for Biodefense Immune Modeling, University of Rochester Medical Center, Rochester, New York, United States of America.
authors.forename: Shannon P
authors.initials: SP
authors.lastname: Hilchey
authors.affiliation: Department of Biostatistics and Computational Biology, University of Rochester Medical Center, Rochester, New York, United States of America.
authors.forename: Ollivier
authors.initials: O
authors.lastname: Hyrien
authors.affiliation: Division of Nephrology, Department of Medicine and the Rochester Center for Biodefense Immune Modeling, University of Rochester Medical Center, Rochester, New York, United States of America.
authors.forename: Nelson
authors.initials: N
authors.lastname: Huertas
authors.affiliation: Division of Nephrology, Department of Medicine and the Rochester Center for Biodefense Immune Modeling, University of Rochester Medical Center, Rochester, New York, United States of America.
authors.forename: Sheldon
authors.initials: S
authors.lastname: Perry
authors.affiliation: Department of Microbiology and Immunology, New York Medical College, Valhalla, New York, United States of America.
authors.forename: Manojkumar
authors.initials: M
authors.lastname: Ramanunninair
authors.affiliation: Department of Microbiology and Immunology, New York Medical College, Valhalla, New York, United States of America.
authors.forename: Doris
authors.initials: D
authors.lastname: Bucher
authors.affiliation: Division of Nephrology, Department of Medicine and the Rochester Center for Biodefense Immune Modeling, University of Rochester Medical Center, Rochester, New York, United States of America; Rochester Center for Health Informatics, University of Rochester Medical Center, Rochester, New York, United States of America.
authors.forename: Martin S
authors.initials: MS
authors.lastname: Zand
mesh_terms: D000914:Antibodies, Viral / Q000276:immunology*; D000956:Antigens, Viral / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology; D006801:Humans; D007136:Immunoglobulins / Q000097:blood; D007252:Influenza Vaccines / Q000097:blood / Q000276:immunology; D007251:Influenza, Human / Q000276:immunology*; D010802:Phylogeny
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007136:Immunoglobulins; D007252:Influenza Vaccines