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BackgroundDetections of influenza A subtype‐specific antibody responses are often complicated by the presence of cross‐reactive antibodies. We developed two novel multiplex platforms for antibody detection. The multiplexed magnetic fluorescence microsphere immunoassay (MAGPIX) is a high‐throughput laboratory‐based assay. Chembio Dual Path Platform (DPP) is a portable and rapid test that could be used in the field.MethodsTwelve recombinant globular head domain hemagglutinin (GH HA1) antigens from A(H1N1)pdm09 (pH1N1), A(H2N2), A(H3N2), A(H5N1), A(H7N9), A(H9N2), A(H13N9), B/Victoria lineage, B/Yamagata lineage viruses, and protein A control were used. Human sera from U.S. residents either vaccinated (with H5N1 or pH1N1) or infected with pH1N1 influenza viruses and sera from live bird market workers in Bangladesh (BDPW) were evaluated. GH HA1 antigens and serum adsorption using full ectodomain recombinant hemagglutinins from A(pH1N1) and A(H3N2) were introduced into the platforms to reduce cross‐reactivity.ResultsSerum adsorption reduced cross‐reactivity to novel subtype HAs. Compared to traditional hemagglutination inhibition or microneutralization assays, when serum adsorption and the highest fold rise in signals were used to determine positivity, the correct subtype‐specific responses were identified in 86%‐100% of U.S. residents exposed to influenza antigens through vaccination or infection (N=49). For detection of H5N1‐specific antibodies in sera collected from BDPW, H5 sensitivity was 100% (six of six) for MAGPIX, 83% (five of six) for DPP, H5 specificity was 100% (15/15), and cross‐reactivity against other subtype was 0% (zero of six) for both platforms.ConclusionMAGPIX and DPP platforms can be utilized for high‐throughput and in‐field detection of novel influenza virus infections.

Background.-In 2016, an influenza A(H7N2) virus outbreak occurred in cats in New York City's municipal animal shelters. One human infection was initially detected.Methods.-We conducted a serological survey using a novel approach to rule out cross-reactive antibodies to other seasonal influenza viruses to determine whether additional A(H7N2) human infections had occurred and to assess exposure risk.Results.-Of 121 shelter workers, one had serological evidence of A(H7N2) infection, corresponding to a seroprevalence of 0.8% (95% confidence interval, .02%-4.5%). Five persons exhibited low positive titers to A(H7N2) virus, indicating possible infection; however, we could not exclude cross-reactive antibody responses to seasonal influenza viruses. The remaining 115 persons were seronegative. The seropositive person reported multiple direct cat exposures without using personal protective equipment and mild illness with subjective fever, runny nose, and sore throat.

Annual vaccination is routinely used in organ transplant recipients for immunization against seasonal influenza. However, detailed analysis of the kinetics of vaccine-induced immune responses in this population is lacking. In this study, we investigated the kinetics of vaccine strains-specific antibody responses to trivalent influenza vaccine in a group of renal transplant recipients and a control group. First, we found that the geometric mean hemagglutination inhibition titer against all 3 vaccine strains in the transplant cohort was significantly low when compared to control subjects. Next, whereas the control group sera showed significantly higher HA-specific IgG and isotype IgG1 antibodies at all four time points, a similar increase in the transplant group was delayed until day 28. Interestingly, within the transplant group, subjects receiving belatacept/MMF/prednisone-based regimen had significantly lower levels of total IgG and HA-specific IgG when compared to tacrolimus/MMF/prednisone-based regimen. Even though IgG-ASC response in both cohorts peaked at day 7 post-vaccination, the frequency of IgG-ASC was significantly low in the transplant group. Taken together, our studies show delayed kinetics and lower levels of influenza vaccine-specific antibody responses in renal transplant recipients and, more importantly, indicate the need to probe and improve current vaccination strategies in renal transplant recipients.

We used an improved ADCC assay to investigate HA-ADCC antibody responses in human sera following either seasonal or avian influenza vaccination. Our results suggest that detection of both neutralizing and non-neutralizing antibodies may better reflect protective capacity of HA-specific antibodies induced by avian influenza vaccines.

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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.

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