Abstract
Neuraminidase (NA) immunity leads to decreased viral shedding and reduced severity of influenza disease; however, NA content in influenza vaccines is currently not regulated, resulting in inconsistent quality and quantity of NA that can vary from manufacturer to manufacturer, from year to year, and from lot to lot. To address this problem, we have developed an assay for NA quantification that could be used by the industry to move toward developing influenza vaccines that induce a predictable immune response to NA. The VaxArray Influenza Seasonal NA Potency Assay (VXI-sNA) is a multiplexed sandwich immunoassay that relies on six subtype-specific monoclonal antibodies printed in microarray format and a suite of fluor-conjugated “label” antibodies. The performance of the assay as applied to a wide range of influenza vaccines is described herein. The assay demonstrated high NA subtype specificity and high sensitivity, with quantification limits ranging from 1 to 60 ng/mL and linear dynamic ranges of 24–500-fold. When compared to an enzymatic activity assay for samples exposed to thermal degradation conditions, the assay was able to track changes in protein stability over time and exhibited good correlation with enzyme activity. The assay also demonstrated excellent analytical precision with relative error ranging from 6 to 12% over day-to-day, user-to-user, and lot-to-lot variation. The high sensitivity and reproducibility of the assay enabled robust detection and quantification of NA in crude in-process samples and low-dose, adjuvanted vaccines with an accuracy of 100 ± 10%.
Highlights
There is increasing scientific evidence that neuraminidase (NA) within influenza vaccines leads to NA immunity, decreased viral shedding, and reduced severity of influenza disease.[1,2,3,4,5,6,7,8,9,10,11,12] In a recent clinical trial, anti-NA immunity correlated more significantly with the reduction of all tested disease severity measures and had a stronger effect on prognosis than anti-hemagglutinin (HA) immunity.[11]
The assay is available as an off-the-shelf kit that relies on a panel of monoclonal antibody (mAb) that were selected for broad coverage of each seasonal subtype and high specificity, allowing for multiplexed analysis of multivalent influenza vaccines
The selection of multiple broadly reactive mAbs increases the chance that the antibodies will detect new vaccine strains as they emerge since, if
Summary
There is increasing scientific evidence that neuraminidase (NA) within influenza vaccines leads to NA immunity, decreased viral shedding, and reduced severity of influenza disease.[1,2,3,4,5,6,7,8,9,10,11,12] In a recent clinical trial, anti-NA immunity correlated more significantly with the reduction of all tested disease severity measures and had a stronger effect on prognosis than anti-hemagglutinin (HA) immunity.[11]. We previously reported that the VXI-sNA is predictive of NA immunity for an N2 subtype within an H3N2 monovalent vaccine.[31] we summarize the development and overall performance of the multiplexed NA potency assay for all NA subtypes (N1, N2, and B-NA) in seasonal influenza vaccines produced by a wide range of manufacturing methods. Very little monoclonal antibody (mAb) is required for each assay, allowing a typical antibody production run to produce enough antibody for the quantification of over 500,000 samples. This reagent sparing approach overcomes the issues around reagent scarcity outlined in the recent NAction!
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