Characterization of Influenza Vaccine Immunogenicity Using Influenza Antigen Microarrays

<div><p>Background</p><p>Existing methods to measure influenza vaccine immunogenicity prohibit detailed analysis of epitope determinants recognized by immunoglobulins. The development of highly multiplex proteomics platforms capable of capturing a high level of antibody binding information will enable researchers and clinicians to generate rapid and meaningful readouts of influenza-specific antibody reactivity.</p><p>Methods</p><p>We developed influenza hemagglutinin (HA) whole-protein and peptide microarrays and validated that the arrays allow detection of specific antibody reactivity across a broad dynamic range using commercially available antibodies targeted to linear and conformational HA epitopes. We derived serum from blood draws taken from 76 young and elderly subjects immediately before and 28±7 days post-vaccination with the 2008/2009 trivalent influenza vaccine and determined the antibody reactivity of these sera to influenza array antigens.</p><p>Results</p><p>Using linear regression and correcting for multiple hypothesis testing by the Benjamini and Hochberg method of permutations over 1000 resamplings, we identified antibody reactivity to influenza whole-protein and peptide array features that correlated significantly with age, H1N1, and B-strain post-vaccine titer as assessed through a standard microneutralization assay (p<0.05, <i>q</i> <0.2). Notably, we identified several peptide epitopes that were inversely correlated with regard to age and seasonal H1N1 and B-strain neutralization titer (p<0.05, <i>q</i> <0.2), implicating reactivity to these epitopes in age-related defects in response to H1N1 influenza. We also employed multivariate linear regression with cross-validation to build models based on age and pre-vaccine peptide reactivity that predicted vaccine-induced neutralization of seasonal H1N1 and H3N2 influenza strains with a high level of accuracy (84.7% and 74.0%, respectively).</p><p>Conclusion</p><p>Our methods provide powerful tools for rapid and accurate measurement of broad antibody-based immune responses to influenza, and may be useful in measuring response to other vaccines and infectious agents.</p></div

Peptide-antigen microarrays on plasmonic gold substrate for profiling antibodies in serum samples of SLE patients and healthy individuals.

<p>A) Box plot of serum IgG antibody reactivity against several peptides and a whole antigen for 20 SLE patients and 20 healthy controls. Acetylated histone H2B peptides were found to be able to differentiate SLE patients and healthy controls (top box plots) together with whole H2B protein (bottom left box plot). While the H3 peptide with the 18th lysine methylated were found not capable of telling SLE patient from healthy control (bottom right box plot). B–D) Heatmaps displaying antibody reactivity to (B) histone peptides only, (C) whole antigens only, and (D) a combination of histone peptides and whole antigens that are identified capable of differentiating SLE patients and healthy controls with false discovery rate (q value) <0.001. The dashed lines are drawn to highlight the separation of SLE and healthy groups identified by using the average linkage Euclidean distance hierarchical clustering method. Color intensity of each grid in the heatmap reflected mean fluorescence intensity of corresponding peptide or antigen spot on the microarray for each SLE patient or healthy individual. In (B) and (C), several SLE patients (labeled in blue and purple color) are misplaced in the healthy group. These patients are grouped in the SLE side in (D) that profiles antibodies against both peptides and whole antigens. However, one healthy individual is mis-placed in the SLE group by this approach, reducing the specificity of this analysis.</p

Plasmonic gold films for peptide microarrays.

<p>A) Scanning electron microscopy (SEM) of an as-made plasmonic gold film containing nano-islands formed on a glass substrate. Inset: an X-ray photoelectron spectrum (XPS) of the film. B) SEM of a plasmonic gold film coated with a layer of avidin proteins (avidin coating caused blurred features of the underlying gold nano-islands). Inset, an XPS spectrum taken on the avidin coated gold substrate showing the presence of nitrogen (in the avidin) over the film. C) A schematic drawing of a multiplexed peptide array (two peptides are shown, red and blue; peptide spots are surrounded by PEG-star coating) on gold for antibody screening based on a 3-layer assay with a NIR fluorophore reporter. D) Intensity of IRDye800 fluorescence (emission ∼ 800 nm) measured on a layer of IRDye800 labeled avidin coated on glass vs. on a plamonic gold film. A ∼100 fold fluorescence enhancement was observed on gold.</p

An integrated peptide-antigen microarray for profiling serum antibodies of 20 patients with systemic lupus erythematosus (SLE).

<p>A) Peptide-antigen microarray layout including modified and unmodified histone H2A, H2B, H3, H4 peptides as well as whole antigens. Ac: acetylated; aa: amino acid; Me1: methylated; Me2: dimethylated; Me3: trimethylated; Ph: phosphorylated; K: Lysine; S: Serine. Number indicates amino acid position from the N-terminus of its corresponding histone proteins. Peptide sequences are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071043#pone.0071043.s008" target="_blank">Table S1</a>. B) Microarray imaging results of a SLE patient serum probed on a plasmonic gold substrate (left) vs. on a commercial streptavidin-glass substrate (right) shown in the same IRDye800 fluorescence intensity scale. C) Dynamic range of fluorescence signal reading among 20 SLE patients measured on each peptide and antigen spots on avidin/gold slide (upper panel) and commercial streptavidin/glass slide (lower panel). Each dot represents a fluorescence intensity measured on a peptide or antigen spot shown along the x-axis in the serum of each of the 20 SLE patients. A dot is drawn only when the fluorescence intensity is above the background by 2 standard deviation of the background signal.</p