Profiling of humoral immune responses to influenza viruses by using protein microarray

Abstract

The emergence of pandemic A(H1N1) 2009 influenza showed the importance of rapid assessment of the degree of immunity in the population, the rate of asymptomatic infection, the spread of infection in households, effects of control measures, and ability of candidate vaccines to produce a response in different age groups. A limitation lies in the available assay repertoire: reference standard methods for measuring antibodies to influenza virus are haemagglutination inhibition (HI) assays and virus neutralization tests. Both assays are difficult to standardize and may be too specific to assess possible partial humoral immunity from previous exposures. Here, we describe the use of antigen-microarrays to measure antibodies to HA1 antigens from seven recent and historical seasonal H1, H2 and H3 influenza viruses, the A(H1N1) 2009 pandemic influenza virus, and three avian influenza viruses. We assessed antibody profiles in 18 adult patients infected with A(H1N1) 2009 influenza virus during the recent pandemic, and 21 children sampled before and after the pandemic, against background reactivity observed in 122 persons sampled in 2008, a season dominated by seasonal A(H1N1) influenza virus. We show that subtype-specific and variant-specific antibody responses can be measured, confirming serological responses measured by HI. Comparison of profiles from persons with similar HI response showed that the magnitude and broadness of response to individual influenza subtype antigens differs greatly between individuals. Clinical and vaccination studies, but also exposure studies, should take these findings into consideration, as they may indicate some level of humoral immunity not measured by HI assays.

IgM, IgG, and IgA Antibody Responses to Influenza A(H1N1)pdm09 Hemagglutinin in Infected Persons during the First Wave of the 2009 Pandemic in the United States

“…One approach to reducing the cross-reactivity observed in the HA ELISA is to eliminate the more conserved HA stalk, primarily the HA2 region of the HA, and use the globular head domain HA1 as the antigen. Others have shown the feasibility of this approach for the detection of antibody responses to the A(H1N1)pdm09 virus (7,41).…”

Glycan-Dependent Immunogenicity of Recombinant Soluble Trimeric Hemagglutinin

“…An HA1 antigen microarray was done as described previously (15). Briefly, commercially available HA1 proteins of H1, H2, H3, and H5 influenza A viruses produced in HEK293 cells and purified by His tag purification (Immune Technology, New York, NY) (see Fig.…”

“…After washing, goat anti-mouse IgG (Fc fragment specific) conjugated with Dylight 649 fluorescent dye (Jackson Immuno Research, West Grove, PA) was added. The slides were scanned using a ScanArray Gx Plus microarray scanner (PerkinElmer), and titers were defined as the interpolated serum concentration that provoked a response halfway on a concentration-response curve between the minimum and maximum signals, as described previously (15). Titers lower than the smallest serum dilution (1:20) were set to 20.…”

Influenza virus A(H1N1)2009 antibody-dependent cellular cytotoxicity in young children prior to the H1N1 pandemic

“…To obtain more insights into the role and development of cross-reactive antibodies and their potential role in the observed immune protection against A(H1N1)2009, we used a protein microarray (PA) to dissect antibody reactivity profiles for seasonal and pandemic influenza A strains (Baas et al, 2013;de Bruin et al, 2014;Freidl et al, 2015;Koopmans et al, 2012). We analysed paired serum samples from children aged 9-10 years, collected shortly before and just after the 2009 pandemic.…”