A miniaturized assay for influenza neuraminidase‐inhibiting antibodies utilizing reverse genetics‐derived antigens

Abstract

Background Antibodies to neuraminidase (NA) contribute to protection during influenza virus infection, but NA inhibition (NI) titers are not routinely analyzed in vaccine trials. One reason is the cumbersome nature of the conventional thiobarbituric acid (TBA) NI assay, which uses chemical methods to quantify free sialic acid following incubation of NA with substrate in the presence of serum. In addition, the assay is complicated by the need to use virus of a hemagglutinin (HA) subtype novel to the host to detect NA‐specific antibodies only. Objectives Our primary objectives were to miniaturize the colorimetric NI assay to a format suitable for quantitative analysis of large numbers of samples, and validate the specificity and sensitivity of the miniaturized format with ferret and human sera. An additional aim was to use reverse genetics to construct HA‐mismatched viral reagents bearing NA of recent influenza A vaccine strains and H6 HA. Results Analysis of ferret antisera by the miniaturized assay demonstrated sensitivity and specificity comparable with the conventional assay. Similar increases in the NI titers in sera from vaccinated human volunteers were measured in miniaturized and conventional assays. Inactivated and live‐attenuated vaccines increased NI titers against a given subtype at approximately the same rate. Conclusions The reagents and miniaturized format of the TBA method described here provide a platform for practical serological monitoring of functional antibodies against NA.

Discordant antigenic drift of neuraminidase and hemagglutinin in H1N1 and H3N2 influenza viruses

“…Since NI data had not previously been used to generate an NA antigenic map, we first established that end-point NI titers provided sufficient sensitivity for the analysis. This process was done by comparative analysis of end-point titers (inverse of the serum dilution that inhibits NA activity ≥50%) and the precise 50% inhibition titer (IC50), the inverse of the serum dilution that results in exactly 50% inhibition determined by nonlinear regression analysis (14) of ferret antisera generated in response to H3N2 infection. The maps generated using these datasets were very similar, with an excellent correlation of distances between antigens on the map, r 2 = 0.99 (Fig.…”

“…Antigenic characterization of NA can be performed using NA inhibition (NI) assays to determine the extent of antibody-mediated interference with enzyme activity (13), but the cumbersome nature of the standard NI assay using large volumes of hazardous chemicals has precluded routine analysis of NA. We recently developed a miniaturized format of this assay and confirmed its accuracy and sensitivity for analysis of NI antibody titers in human and animal sera (14). In the present study, we use this assay to characterize the antigenic drift of NA in human H1N1 and H3N2 viruses recommended for United States influenza vaccines over the past ∼15 y (Table S1).…”

Correlates of vaccine protection from influenza and its complications

“…56,57 NA content is not standardized in current inactivated vaccines, and NA-specific immune responses, detected using the neuraminidase inhibition assay, 36,58,59 are rarely measured. Purified HA and NA have been shown to induce a similar magnitude of immune response with similar kinetics, and purified NA is immunogenic in humans.…”

“…The level of memory CTLs correlates with viral clearance from infected hosts, but reactivity of CTLs varies greatly between individuals, making immune correlates based on CTLs difficult to quantitate. 65 A matrix epitope (M1 [58][59][60][61][62][63][64][65][66] ) has historically been used to test human CTL specificity to influenza A viruses. 66 Nucleoprotein (NP) and polymerase basic protein 1 (PB1) epitopes have also been identified and could be utilized as correlates of CMI.…”

Qualitative Differences in T cell responses to Live, Attenuated and Inactivated Influenza Vaccines

“…Neuraminidase inhibition assay: Titration of serum NI antibodies was performed by analyzing NA activity of the HA-mismatched reassortant viruses in a 96-well plate format of the conventional thiobarbituric acid assay [19]. Briefly, serum specimens were serially diluted in PBS across wells of 96-well PCR plates.…”