Improved Specificity and False Discovery Rates for Multiplex Analysis of Changes in Strain-Specific Anti-Influenza IgG.

Dongmei Li,Martin S Zand,John J Treanor,Jiong Wang

doi:10.1155/2019/3053869

Abstract

We describe a statistical approach to compare absolute antibody concentrations, both within and across subjects, derived from a multidimensional measurement of IgG binding to the influenza surface receptor hemagglutinin (HA). This approach addresses a fundamental problem in the field of vaccine immunology: how to accurately compare the levels of antibodies against multiple influenza strains. The mPlex-Flu assay can simultaneously measure the concentration of IgG antibodies against up to 50 influenza strains with only ≤10 μl of serum. It yields mean fluorescence intensity (MFI) over a 4-log range with low inter- and intrasample variability. While comparison of IgG binding to a single HA between subjects is straightforward, variations in binding behavior across influenza strains, coupled with reagent variations, make quantifying and comparing binding between multiple HA subtypes within subjects challenging. In this paper, we first treat such HA variations as an independent antigen and calculate each subtype antibody concentration using its own standard curve, normalizing variations in HA binding. We applied this method to the analyses of data from an H5 influenza clinical vaccine study. The results demonstrated that there are differences in coefficient estimates and in results of “comparing groups” between those with versus those without consideration of subtype antibody variations. Then, we used simulation studies to show the importance of taking the subtype antibody variations into account in HA strain antibody data analysis. Using a common standard curve for all subtype antibodies resulted in both inflated type I error and lowered specificity when comparing different treatment groups. Our results suggest that using individual standard curves for each influenza HA strain, and independently calculating anti-HA IgG concentrations, allows for adjustment of influenza HA subtype variations in treatment group comparisons in clinical vaccine studies. This method facilitates the direct comparison of serum anti-HA IgG concentrations against different influenza HA subtypes for multiplex assays.

Highlights

Estimating the concentration of antibodies directed against the major influenza viral surface protein hemagglutinin (HA) is critical for studies of antibody-mediated influenza immunity and especially for vaccine development [1]
We sought to determine if accounting for influenza HA strain variation would affect the statistical comparison of vaccine response treatment groups from a clinical influenza vaccine study. e data were generated from an mPLEX-flu assay of samples collected during a previous study of responses to an H5 influenza vaccine (DMID 08-0059) [14]
When the variation in influenza HA subtype differences decreased, we found a concomitant decrease in false discovery rates (FDR) and an increase in specificity (Table 4)

Summary

Introduction

Estimating the concentration of antibodies directed against the major influenza viral surface protein hemagglutinin (HA) is critical for studies of antibody-mediated influenza immunity and especially for vaccine development [1]. Us, the ability to simultaneously measure antibody binding against multiple influenza HA and to accurately compare antibody binding across many influenza strains, especially within and between subject binding distributions, is highly desirable. We have previously described a multiplex-based method that simultaneously measures antibody binding against up to 50 influenza strain hemagglutinin proteins, the mPlex-Flu assay [3, 4]. In mPlex-flu assay, each recombinant influenza strain HA couples to fluorescent microbeads; the mixtures of the HA-coated beads are used to simultaneously detect antibodies binding to multiple influenza strains. In mPlex-flu assay, each recombinant influenza strain HA couples to fluorescent microbeads; the mixtures of the HA-coated beads are used to simultaneously detect antibodies binding to multiple influenza strains. is multidimensional analytic method generates a continuous value for the mean fluorescence intensity (MFI), accurate over a 4-log range, reflecting antibody binding

Methods

Results

Conclusion