Abstract
BackgroundExisting 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.MethodsWe 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.ResultsUsing 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, q <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, q <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).ConclusionOur 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.
Highlights
Each year, vaccines are generated against the most virulent strains of influenza to minimize global rates of morbidity and mortality associated with influenza infection [1]
Used methods to measure the effectiveness of influenza vaccine on induction of the humoral immune response include the hemagglutination inhibition (HAI) assay and the virus replication neutralization assay, which indirectly measure the effect of vaccination on antibody reactivity [10]
We synthesized 19-mer peptides overlapping by 10 amino acids that span the head region of H1N1 (A/Brisbane/59/2007), H3N2 (A/Brisbane/10/2007), and B (B/Brisbane/60/2008) influenza strains included in the 2008/ 2009 trivalent influenza vaccine (TIV) as well as control peptides containing well-described HA-tag (NH2-YPYDVPDYA-COOH) and FLAG-tag (NH2DYKDDDDK-COOH) sequences [29,30] (Fig. 1c and Table S1)
Summary
Vaccines are generated against the most virulent strains of influenza to minimize global rates of morbidity and mortality associated with influenza infection [1]. Used methods to measure the effectiveness of influenza vaccine on induction of the humoral immune response include the hemagglutination inhibition (HAI) assay and the virus replication neutralization (microneutralization) assay, which indirectly measure the effect of vaccination on antibody reactivity [10] While these assays are currently the gold standard for measuring antibody responses to influenza, and can, to an extent, predict protection from disease, they are limited in that each is specific for only a single antigen, and neither permits broad analysis of more limited epitope determinants recognized by immunoglobulins. Proteomics platforms that can measure a great diversity of antibody binding to influenza antigen targets will be of great use for research and in the clinic for better understanding the antibody response to influenza virus and vaccine To this end, we have developed influenza whole-protein and peptide antigen microarray platforms for the determination of antibody reactivity to conformational and linear epitopes of influenza hemagglutinin (HA). 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
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