Identification of novel influenza A virus exposures by an improved high‐throughput multiplex MAGPIX platform and serum adsorption

Abstract

Background The development of serologic assays that can rapidly assess human exposure to novel influenza viruses remains a public health need. Previously, we developed an 11‐plex magnetic fluorescence microsphere immunoassay (MAGPIX) by using globular head domain recombinant hemagglutinins (rHAs) with serum adsorption using two ectodomain rHAs. Methods We compared sera collected from two cohorts with novel influenza exposures: animal shelter staff during an A(H7N2) outbreak in New York City in 2016‐2017 (n = 119 single sera) and poultry workers from a live bird market in Bangladesh in 2012‐2014 (n = 29 pairs). Sera were analyzed by microneutralization (MN) assay and a 20‐plex MAGPIX assay with rHAs from 19 influenza strains (11 subtypes) combined with serum adsorption using 8 rHAs from A(H1N1) and A(H3N2) viruses. Antibody responses were analyzed to determine the novel influenza virus exposure. Results Among persons with novel influenza virus exposures, the median fluorescence intensity (MFI) against the novel rHA from exposed influenza virus had the highest correlation with MN titers to the same viruses and could be confirmed by removal of cross‐reactivity from seasonal H1/H3 rHAs following serum adsorption. Interestingly, in persons with exposures to novel influenza viruses, age and MFIs against exposed novel HA were negatively correlated, whereas in persons without exposure to novel influenza viruses, age and MFI against novel HAs were positively correlated. Conclusions This 20‐plex high‐throughput assay with serum adsorption will be a useful tool to detect novel influenza virus infections during influenza outbreak investigations and surveillance, especially when well‐paired serum samples are not available.

Antibody Landscape Analysis following Influenza Vaccination and Natural Infection in Humans with a High-Throughput Multiplex Influenza Antibody Detection Assay

“…S1 and S2 in the supplemental material) (16). In our previous studies, we used a serum dilution of 1:40 to detect influenza virus antibody responses using the same assay platform (14,15,17). Here, the linearity analysis suggests that the 1:40 serum dilution falls well within the linear range for most seasonal and novel subtype influenza virus antigens and is suitable to capture the full scope of the antibody responses to most antigens while maintaining the assay throughput.…”

“…To determine seroconversion in the MIADA for paired serum samples, we used a $2-fold rise in MFIs after adjustment of low S1 MFIs to 1,000 (14,15). We found that one A(H1N1)pdm09-infected person and three A(H3N2)-infected persons achieved seroconversion ($2-fold rises in MFIs) against at least one novel subtype rHA (H5, H7, H9, or H13) (Tables S3 and S4), and nine persons showed high MFIs ($2,000) against a novel subtype rHA(s) in S2 and/or S1 sera, although no seroconversions in MFIs were achieved (Table S5 and data not shown).…”

“…In influenza virus serologic studies, a $4-fold rise in HI/MN titers is often considered a positive antibody response (or seroconversion). Here, in the MIADA, we used a $2-fold rise in MFIs (S2/ S1) after adjustment of low MFIs in baseline S1 to 1,000 (to avoid an unreliable fold rise as described previously) as seroconversion (14,15). We also analyzed consistency in non-seroconversion cases measured by HI (,4-fold rise) and MIADA (,2-fold rise).…”

“…We previously developed a Magpix assay for serologic diagnosis of seasonal and novel influenza virus infection (14,15). In the current study, we expanded this platform and developed a high-throughput multiplex influenza antibody detection assay (MIADA) that can measure antibody responses to 42 HA antigens from 10 influenza virus subtypes simultaneously, plus a protein A (PA) control.…”

“…In the current study, we expanded this platform and developed a high-throughput multiplex influenza antibody detection assay (MIADA) that can measure antibody responses to 42 HA antigens from 10 influenza virus subtypes simultaneously, plus a protein A (PA) control. Furthermore, we implemented an antibody adsorption technique using ectodomain (Ecto) HAs from A(H1N1) and A(H3N2) viruses to elucidate the effects of both homosubtypic (within-subtype) and heterosubtypic (cross-subtype) cross-reactivity (14,15).…”