Development of an influenza virus protein microarray to measure the humoral response to influenza virus infection in mallards

Abstract

Avian influenza viruses pose a serious zoonotic threat, in part because current seasonal influenza virus vaccines only offer strain-specific protection, instead of heterosubtypic or universal protection against influenza virus infection. Understanding the humoral response to vaccination and natural infection in the broadest context possible is important to developing defenses against influenza zoonosis. Protein microarrays are a novel platform well suited to assaying the humoral immune response broadly and efficiently. We developed an influenza virus protein microarray (IVPM) that could be used to assay sera from many species, including humans. Waterfowl such as mallard ducks are natural reservoirs for many influenza A viruses, but their humoral immune response to infection is poorly understood. To establish this technology, we assayed sera from mallards experimentally infected with two low-pathogenic common avian influenza viruses (H3N8 and H4N5) for reactivity to influenza virus hemagglutinin (HA) by IVPM. The IVPM results correlated well with results from an established enzyme-linked immunosorbent assay, supporting the validity of the IVPM as a serological assay in influenza virus research. Interestingly, successive infection with H3N8 followed by H4N5 virus in mallard ducks induced antibodies that were broadly reactive against group 2 hemagglutinins. We also analyzed sera from wild mallards and observed serological evidence for infection in those sera. With serological information, it may be possible to infer infection history of wild avian species and gain a better understanding of the infection dynamics of influenza viruses in their natural reservoir. This might ultimately lead to interventions that enhance our pandemic preparedness.

Influenza Virus Infection Induces a Narrow Antibody Response in Children but a Broad Recall Response in Adults

“…This method was chosen because it is more quantitative than assaying one serum dilution only. We have also shown in the past (13) and here that the method correlates well with ELISA results, can detect antibodies with high specificity, and can detect broadly neutralizing antibodies that bind to fragile and conformational epitopes (Fig. S2).…”

“…However, performing individual ELISAs against recombinant HAs of many different virus strains and subtypes is tedious and timeconsuming and can be sample intensive. Recently, we therefore developed influenza virus protein microarrays (IVPMs) (13). For this technology, we print a library of recombinant HA proteins, including all HA subtypes, on microarrays which are then probed with serial dilutions of serum (see Fig.…”

Meeting report and review: Immunological assays and correlates of protection for next‐generation influenza vaccines

“…14 Cross reactive CD4+ and CD8+ cells have also been identified as correlates of protection in human challenge and cohort studies. [4][5][6][7] Other immunological markers, including antibody effector functions as measured in antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cell-mediated phagocytosis (ADCP) assays, 25-28 complement activation, mucosal antibody levels, entry inhibition titers as measured by pseudotype particle entry inhibition assays, 29 antibodies to the ectodomain of the matrix 2 ion channel (M2e), antibodies to matrix protein 1 (M1) and nucleoprotein (NP), 30 influenza virus protein arrays (IVPM), [31][32][33][34] and many others are currently being investigated to assess whether they correlate with protection. Importantly, systems immunology approaches are being used to identify new immunological markers that could then be tested for their potential to predict whether protective immunity was induced through vaccination.…”

“…Cell-based ELISA or flow cytometry 30 Influenza virus proteins Influenza virus protein arrays (IVPM) [31][32][33][34] Immune markers Systems immunology 35,36 validation.). Standardization of sampling is also useful in this context.…”

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

“…Building on extensive previous work by multiple contributors in the field 15,16,17,18,19 , an influenza protein microarray was recently developed that contains over 250 purified hemagglutinin (HA) antigenic variants with representation of all 18 subtypes 12,20 . Using this methodology, a natural influenza infection was demonstrated to generate broadly reactive IgG and IgA antibodies against phylogenetically related HA subtypes, while an intramuscular influenza vaccination generated only subtype-specific IgG antibodies 21 .…”