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

Wang J, Hilchey SP, Hyrien O, Huertas N, Perry S, Ramanunninair M, Bucher D, Zand MS - PLoS ONE (2015)

Bottom Line: Pre- and post-intramuscular trivalent influenza vaccination levels of virus specific Ig were consistent with HAI titer and ELISA measurements.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

The mean fluorescence intensities (MFIs) generated from the binding of eight unique rHA-coupled beads (octoplex assay) with serially diluted human serum for determination of HA specific IgG, IgA, and IgM antibody amounts.A two-step Luminex assay was performed with recombinant hemagglutinins (rHA) of influenza vaccine related subtype strains: A/California/07/2009 (A/Cal09), A/Perth/16/2009 (A/Perth09), A/Victoria/210/2009 (A/Vic09), A/Victoria/361/2011 (A/Vic11), B/Brisbane/60/2008 (B/Bri08), B/Wisconsin/1/2010 (B/Wis10), B/Massachusetts/2/2012 (B/Mass12), A/Port Chalmers/1/1973 (A/P.C 73), A/Hong Kong/1/1968 (A/HK68) as described (see Methods). (a) Detection of human rHA specific IgG (seven 3-fold serial dilutions starting from 1:1000). (b) Detection of human rHA specific IgA (seven 3-fold serial dilutions starting from 1:50), and (c) Detection of human rHA specific IgM (seven 3-fold serial dilutions starting from 1:10). Serum dilutions for the different Ig isotypes were based on relative concentrations needed to remain within the 4-Log10 linear range of the mPlex-Flu assay.
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pone.0129858.g001: The mean fluorescence intensities (MFIs) generated from the binding of eight unique rHA-coupled beads (octoplex assay) with serially diluted human serum for determination of HA specific IgG, IgA, and IgM antibody amounts.A two-step Luminex assay was performed with recombinant hemagglutinins (rHA) of influenza vaccine related subtype strains: A/California/07/2009 (A/Cal09), A/Perth/16/2009 (A/Perth09), A/Victoria/210/2009 (A/Vic09), A/Victoria/361/2011 (A/Vic11), B/Brisbane/60/2008 (B/Bri08), B/Wisconsin/1/2010 (B/Wis10), B/Massachusetts/2/2012 (B/Mass12), A/Port Chalmers/1/1973 (A/P.C 73), A/Hong Kong/1/1968 (A/HK68) as described (see Methods). (a) Detection of human rHA specific IgG (seven 3-fold serial dilutions starting from 1:1000). (b) Detection of human rHA specific IgA (seven 3-fold serial dilutions starting from 1:50), and (c) Detection of human rHA specific IgM (seven 3-fold serial dilutions starting from 1:10). Serum dilutions for the different Ig isotypes were based on relative concentrations needed to remain within the 4-Log10 linear range of the mPlex-Flu assay.

Mentions: The mPlex-flu assay presents an array of HAs to a small amount of serum, giving rise to the possibility that Ig species which cross-react to conserved epitopes across multiple HAs may bind to multiple beads. If a widely cross-reactive anti-HA antibody was present in small amounts, this may decrease the mPLEX-flu sensitivity for individual HA proteins. To assess if competition for antibodies with cross-reactive specificities would occur, we assayed antibody binding against individual beads (monoplex) and a combined mixture of 8 coupled bead sets (octoplex) by incubation with 3-fold dilutions of standard serum (Fig 1). The MFIs of the monoplex beads were nearly identical to those generated using the octoplex bead set (see S6 Fig) This result is consistent with minimal detectable interference when assaying multiple beads sets together simultaneously, compared to assaying the bead sets separately. This result is identical to similar studies of Luminex assays reported by others [32, 35].


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

Wang J, Hilchey SP, Hyrien O, Huertas N, Perry S, Ramanunninair M, Bucher D, Zand MS - PLoS ONE (2015)

The mean fluorescence intensities (MFIs) generated from the binding of eight unique rHA-coupled beads (octoplex assay) with serially diluted human serum for determination of HA specific IgG, IgA, and IgM antibody amounts.A two-step Luminex assay was performed with recombinant hemagglutinins (rHA) of influenza vaccine related subtype strains: A/California/07/2009 (A/Cal09), A/Perth/16/2009 (A/Perth09), A/Victoria/210/2009 (A/Vic09), A/Victoria/361/2011 (A/Vic11), B/Brisbane/60/2008 (B/Bri08), B/Wisconsin/1/2010 (B/Wis10), B/Massachusetts/2/2012 (B/Mass12), A/Port Chalmers/1/1973 (A/P.C 73), A/Hong Kong/1/1968 (A/HK68) as described (see Methods). (a) Detection of human rHA specific IgG (seven 3-fold serial dilutions starting from 1:1000). (b) Detection of human rHA specific IgA (seven 3-fold serial dilutions starting from 1:50), and (c) Detection of human rHA specific IgM (seven 3-fold serial dilutions starting from 1:10). Serum dilutions for the different Ig isotypes were based on relative concentrations needed to remain within the 4-Log10 linear range of the mPlex-Flu assay.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4478018&req=5

pone.0129858.g001: The mean fluorescence intensities (MFIs) generated from the binding of eight unique rHA-coupled beads (octoplex assay) with serially diluted human serum for determination of HA specific IgG, IgA, and IgM antibody amounts.A two-step Luminex assay was performed with recombinant hemagglutinins (rHA) of influenza vaccine related subtype strains: A/California/07/2009 (A/Cal09), A/Perth/16/2009 (A/Perth09), A/Victoria/210/2009 (A/Vic09), A/Victoria/361/2011 (A/Vic11), B/Brisbane/60/2008 (B/Bri08), B/Wisconsin/1/2010 (B/Wis10), B/Massachusetts/2/2012 (B/Mass12), A/Port Chalmers/1/1973 (A/P.C 73), A/Hong Kong/1/1968 (A/HK68) as described (see Methods). (a) Detection of human rHA specific IgG (seven 3-fold serial dilutions starting from 1:1000). (b) Detection of human rHA specific IgA (seven 3-fold serial dilutions starting from 1:50), and (c) Detection of human rHA specific IgM (seven 3-fold serial dilutions starting from 1:10). Serum dilutions for the different Ig isotypes were based on relative concentrations needed to remain within the 4-Log10 linear range of the mPlex-Flu assay.
Mentions: The mPlex-flu assay presents an array of HAs to a small amount of serum, giving rise to the possibility that Ig species which cross-react to conserved epitopes across multiple HAs may bind to multiple beads. If a widely cross-reactive anti-HA antibody was present in small amounts, this may decrease the mPLEX-flu sensitivity for individual HA proteins. To assess if competition for antibodies with cross-reactive specificities would occur, we assayed antibody binding against individual beads (monoplex) and a combined mixture of 8 coupled bead sets (octoplex) by incubation with 3-fold dilutions of standard serum (Fig 1). The MFIs of the monoplex beads were nearly identical to those generated using the octoplex bead set (see S6 Fig) This result is consistent with minimal detectable interference when assaying multiple beads sets together simultaneously, compared to assaying the bead sets separately. This result is identical to similar studies of Luminex assays reported by others [32, 35].

Bottom Line: Pre- and post-intramuscular trivalent influenza vaccination levels of virus specific Ig were consistent with HAI titer and ELISA measurements.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus