Short communicationQuantification of the total neuraminidase content of recent commercially-available influenza vaccines: Introducing a neuraminidase titration reagent
Introduction
There are two important glycoproteins on the surface of influenza virus particles; hemagglutinin (HA) and neuraminidase (NA). These two proteins play vital roles during the influenza life cycle: HA is involved in the initial attachment of virus to sialic acid receptors on the host cell while NA assists access of the virus to the host cell and hydrolyses surface sialic acids to liberate the nascent virion, allowing it to infect new cells. Currently, annual vaccination is the primary means of controlling influenza, and HA and NA are the major influenza antigens that induce the relevant protective immune responses.
There is approximately four-fold more HA than NA on the surface of the influenza A virus, although the relative reported abundance of these proteins may vary by type/subtype or analytical methods [1]. Overall, the HA is considered immuno-dominant over the NA, with vaccine design and certification standardized on that basis to contain at least 15 μg HA per strain per 0.5 mL dose, higher for the “high dose” vaccine (60 μg per strain per 0.5 mL dose) [2]. By contrast, NA has been the “forgotten antigen” with its content neither monitored nor regulated. Increasing evidence suggests that NA serves important roles in the protection afforded by the influenza vaccine [3], [4], [5], [6], [7], [8], leading to the realization that a better understanding of the NA content of vaccines and its effects are needed. Indeed, a focus group, “NAction!” was recently established to investigate NA-based immunity and design better, broadly protective vaccines [9]. While some methods are available to measure total NA protein, or to assess antibody-responsive levels, no tools are currently available to accurately quantitate active NA content. Access to such information could guide vaccine development.
Several methods have been developed to quantify the NA content of influenza vaccines, most of which are based on an enzyme-linked immunosorbent assay (ELISA) or high-performance liquid chromatography (HPLC) and mass spectrometry [9]. However, these methods are typically time-consuming and do not reflect the content of enzymatically active NA. Enzyme activity measurements are useful to measure relative NA content within an NA type. However, in the absence of reliable specific activity values for each NA they cannot determine absolute amounts of active NA, nor can they be measured in the presence of competing substrates or inhibitors. The “gold standard” for quantitation of active enzyme is an active-site titration agent. Such reagents react stoichiometrically with the enzyme, releasing one equivalent of a readily quantifiable species, typically a fluorophore. Thus, by measuring the total increase in fluorescence the total amount of active enzyme can be determined [10], [11].
Recently, our group reported an active-site titration reagent, TR1, which can quantify influenza NA down to 0.2 nM (about 10 ng/mL) [12]. While TR1 resembles the commonly used NA substrate Mu-NANA, it has a very different mechanism of action: while each molecule of NA hydrolyzes many equivalents of Mu-NANA, NA reacts with just one equivalent of TR1, and releases one equivalent of fluorophore (6,8-difluorocoumarin, F2Mu), upon inactivation (Fig. 1). Concentrations are determined by measurement of total fluorescence change in an “end point” manner. No rates are measured and there is no need for information on specific activity of the specific NA. TR1 reacts with all subtypes of NA, indeed all active forms of the enzyme, with the same response yielding the total, but not component-specific, NA content of the vaccine. Measurements can even be performed in the presence of competing substrates or inhibitors. The TR1 titration approach is fast, high-throughput, operationally simple and its use and accuracy for quantitation of neuraminidases has been demonstrated using a highly pure bacterial neuraminidase that is available in large quantities [12]. Here, we report experience with the TR1 titration approach in quantifying total NA content of six commercially-available vaccines used in North America between 2015/16 and 2017/18, thereby demonstrating the utility of the reagent.
Section snippets
Vaccine samples
Six commercially-available, non-adjuvanted vaccine products per season (2015/16, 2016/17 and 2017/18) were purchased for a total of 18 product-specific NA measurements. Representative vaccines each season included three split standard-dose products – one trivalent (i.e. Fluviral (GlaxoSmithKline (GSK))) and two quadrivalent (i.e. Flulaval (GSK) and Fluzone (Sanofi)); a subunit standard dose vaccine (i.e. Agriflu (Sequiris)); a split high-dose vaccine (i.e. Fluzone High Dose (Sanofi)) and a live
Results
A typical curve obtained upon titration of a commercial vaccine with TR1 is shown in Fig. 2B. The time-dependent increase in fluorescence reflects release of the fluorophore. Under the conditions used here the titration reaction is complete in approximately 10 min. The total change in fluorescence measured from the Y-axis, suitably corrected for background as described in the Methods, is used to calculate the total concentration of fluorophore released, thus of active enzyme, based on the
Discussion
The newly developed active-site titration reagent TR1 reacts stoicheometrically with NA allowing absolute concentrations of active enzyme to be measured by the increase in fluorescence. We use this reagent to show that different brands of vaccine contain different amounts of NA with Fluzone High-Dose vaccine having the highest NA content as would be expected. However the relative NA concentration per brand is reasonably constant for the products and lots we assessed over several seasons. The
Conclusion
The simplicity of this NA-titration reagent makes it a useful tool for the reliable and rapid quantification of total NA content of commercial vaccines as well as the components thereof prior to formulation. The high-throughput, operationally simple nature of the titration reagent renders it amenable to monitoring general trends in NA content as part of routine influenza vaccine manufacturing processes, storage and distribution, as well as in more fundamental studies of influenza virus and
Author contributions
ZG, SGW and DMS conceived the project. ZG and KR performed experimental studies. ZG, SGW and DMS wrote and edited the first draft of the manuscript; KR and GDS edited. GDS and DMS supplied some samples.
Potential conflicts of interest
GDS has received grants for investigator initiated studies unrelated to influenza vaccine from Pfizer and provided paid expert testimony for the Ontario Nurses Association, the Quebec Ministry of Justice and GSK. SGW, ZC, KR and DMS declare no conflicts.
Funding sources
This work was supported by funding from the Canadian Institutes of Health Research with a grant number of FDN-148458.
An account of this work was presented in the form of a poster at the Canadian Immunization Conference, December 2018, Ottawa, Canada.
Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [GDS has received grants for investigator initiated studies unrelated to influenza vaccine from Pfizer and provided paid expert testimony for the Ontario Nurses Association, the Quebec Ministry of Justice and GSK. SGW, ZC, KR and DMS declare no conflicts beyond the submission of a patent application by the University of British Columbia].
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Current address: School of Marine Sciences, Sun Yat-sen University, Zhuhai, Guangdong 518082, China.