Profiling of humoral immune responses to influenza viruses by using protein microarray  M. Koopmans, E. de Bruin, G.-J. Godeke, I. Friesema, R. van Gageldonk, M. Schipper, A. Meijer, R. van Binnendijk, G.F. Rimmelzwaan, M. D. de Jong, A. Buisman, J. van Beek, D. van de Vijver, J. Reimerink  Clinical Microbiology and Infection  Volume 18, Issue 8, Pages 797-807 (August 2012) DOI: 10.1111/j.1469-0691.2011.03701.x Copyright © 2012 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 1 Set-up of antigen microarrays, containing triplicate wells of HA1 peptides of the viruses indicated, and a typical result from testing of serum from a 9-year-old child sampled before and after the first pandemic wave. Titres are calculated from the inflection point of a dilution series, as described in the methods section. X axes denote serum dilutions, and Y axes the median spot fluorescence intensity as measured by ScanArray. Clinical Microbiology and Infection 2012 18, 797-807DOI: (10.1111/j.1469-0691.2011.03701.x) Copyright © 2012 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 2 Comparison of kinetics of antibody response to homologous antigen following infection with pandemic influenza virus A(H1N1)2009 measured by protein array and HI test in 16 cases with virologically confirmed influenza A(H1N1) 2009, and two contacts, all without a history of vaccination. Time points on the X axis denote first, second, third and fourth sampling time point (Materials and methods). (a) Kinetics of response measured by HI assay per person; (b) kinetics of response measured by protein array per person; (c) proportion of persons with titre ≥40 by time point; (d) proportion of persons with titre ≥40, cumulative. Clinical Microbiology and Infection 2012 18, 797-807DOI: (10.1111/j.1469-0691.2011.03701.x) Copyright © 2012 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 3 Geometric mean titres of antibodies to different HAI antigens (bars) as measured by microarray, for persons sampled during 2008 for different age groups (X-axis) Figures show GMTs measured in the population pre-pandemic for (a) (top left) recent seasonal influenza viruses of subtype HI, (b) (top right) historical influenza viruses of subtype HI and the 2009 pandemic virus; (c) (bottom left) recent seasonal influenza viruses of subtype H3; and (d) influenza virus strains that are not currently circulating (i.e. H2, and avian H5, H7 and H9). The dotted line shows the cut-off of 40 used to calculate the fold increase in antibody titre from the 18 adult patients. Clinical Microbiology and Infection 2012 18, 797-807DOI: (10.1111/j.1469-0691.2011.03701.x) Copyright © 2012 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 4 Individual reactivity to a range of seasonal, pandemic and non-human influenza viruses in serum samples from persons sampled during 2008 in the Netherlands. Left column indicates year of birth of individuals. - denotes titre for respective antigen was below 40. + indicates titres of 40 or more, measured by protein microarray. *Year of birth. Blue bar indicates pandemic years. Clinical Microbiology and Infection 2012 18, 797-807DOI: (10.1111/j.1469-0691.2011.03701.x) Copyright © 2012 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 5 Changes in antibody titres to different influenza HA1 proteins, expressed as fold difference. Minimum titres were set arbitrarily at 40 to reduce effect of small changes on titres in the low range. SD denotes standard deviation. (a) Fold response in children sampled before and after the pandemic wave; (b) fold response in 16 patients with virologically confirmed influenza and two infected controls. Dotted lines indicate onefold (left) and two-fold (right) change in average GMT. Note differences in scale. Clinical Microbiology and Infection 2012 18, 797-807DOI: (10.1111/j.1469-0691.2011.03701.x) Copyright © 2012 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 6 Antibodies to H1 2009, HI 2007 and H3 2007 HAI antigens as measured by protein microarray in paired serum samples from 21 children sampled before (March 2009) and after (March 2010) the pandemic wave in the Netherlands. Clinical Microbiology and Infection 2012 18, 797-807DOI: (10.1111/j.1469-0691.2011.03701.x) Copyright © 2012 European Society of Clinical Infectious Diseases Terms and Conditions

FIG. 7 Microarray-based profile of antibody responses in patients with virologically confirmed influenza. Patients were grouped according to their responses in HI titre as low (left panel), moderate (middle panel) or high (right panel). Headings indicate year of birth of the patients. For calculation of fold changes a minimum titre of 40 was used as cut-off. Clinical Microbiology and Infection 2012 18, 797-807DOI: (10.1111/j.1469-0691.2011.03701.x) Copyright © 2012 European Society of Clinical Infectious Diseases Terms and Conditions