Optimization of an enzyme-linked lectin assay suitable for rapid antigenic characterization of the neuraminidase of human influenza A(H3N2) viruses
Introduction
Influenza vaccines reduce the morbidity and mortality associated with annual influenza epidemics. The seasonal influenza vaccine is designed to protect against circulating influenza A H1N1 viruses (A(H1N1)), influenza A H3N2 viruses (A(H3N2)), and influenza B viruses. The influenza virus escapes host immunity through mutations in the major surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). This process is known as antigenic drift (Schild et al., 1974, Webster et al., 1982) and as a result of this drift, the influenza vaccine has to be updated frequently. In the period from 1999 to 2010, the A(H3N2) virus component was updated 6 times (Barr et al., 2010). Recently, it has been shown that only few mutations near the receptor-binding site of HA are responsible for antigenic drift of A(H3N2) viruses circulating between 1968 and 2003 (Koel et al., 2013). For NA, a number of antigenic sites have been described (Air et al., 1985). These antigenic regions surround the enzyme's active site (Colman et al., 1983, Colman et al., 1987) and are highly variable, most likely due to immune pressure (Laver et al., 1982, Luther et al., 1984). Influenza virus surveillance by national influenza centers is done year-round (Russell et al., 2008, Barr et al., 2010). Representatives of the predominant circulating viruses are sent to the World Health Organization (WHO) Collaborating Centers. These centers characterize the viruses by sequencing the HA and NA genes and performing hemagglutination inhibition (HI) assays (Barr et al., 2010). During vaccine strain selection, the main focus is on the genetic and antigenic characterization of HA (Fiore et al., 2010).
Influenza viruses attach to the host cell surface via binding of the HA to sialic acid-containing receptors (Sauter et al., 1989). The enzymatic activity of NA allows virus release from the cell (Palese et al., 1974, Palese and Compans, 1976, Liu et al., 1995) by cleaving the sialic acid residues from the newly formed virus particles and from the host cell (von Itzstein, 2007). Preclinical and clinical studies showed that NA-specific immunity can reduce the severity of disease (Schulman et al., 1968, Murphy et al., 1972, Couch et al., 1974, Kilbourne, 1976, Johansson et al., 1993, Brett and Johansson, 2005). Antibodies directed toward NA inhibit release and spread of newly formed virus particles from infected cells (Compans et al., 1969). The antigenic drift of NA does not closely correspond to that of HA (Schulman and Kilbourne, 1969, Kilbourne et al., 1990, Sandbulte et al., 2011). Considering these findings, investigating options to include routine analysis of NA during vaccine strain selection next to HA seems to be warranted.
Antigenic characterization of NA can be performed using NA inhibition (NI) assays to determine the extent of antibody-mediated interference with enzyme activity (Kilbourne et al., 1968). These assays rely on the enzymatic sialidase activity by measuring cleavage of sialic acid from highly glycosylated proteins such as fetuin. The NI thiobarbituric acid (TBA) assay (Warren, 1959, Webster and Laver, 1967) is based on the detection of free sialic acid. This assay is recommended by the WHO (Cox et al., 2002), but the use of large volumes of hazardous chemicals and laborious procedures has impeded antigenic characterization of NA during influenza virus surveillance. A simplified and miniaturized version of the TBA was developed (Sandbulte et al., 2009), but this assay still remains cumbersome. The recently developed enzyme-linked lectin assay (ELLA) (Lambre et al., 1991, Cate et al., 2010, Couch et al., 2012, Couch et al., 2013, Fritz et al., 2012, Couzens et al., 2014) also relies on the sialidase activity of NA, but instead of measuring free sialic acid, it detects the terminal galactose that becomes exposed after sialic acid cleavage by NA.
A complication of NI assays is the interference of HA-specific antibodies that block NA activity non-specifically when they bind to HA (Schulman and Kilbourne, 1969). Recombinant influenza viruses with a heterologous HA are commonly used for NI assays. Antibodies directed toward the H1 or H3 HA of A(H1N1) and A(H3N2) viruses do not cross-react with a heterologous HA (e.g. H6), and hence only the interaction between NA and NA-specific antibodies is measured (Couzens et al., 2014). However, the generation of recombinant viruses is time-consuming for large numbers of viruses and therefore this method is not suitable for large-scale surveillance of antigenic drift of circulating influenza viruses. For analysis of the antigenic drift of NA, it would be ideal to have the capability of using wild type viruses as antigen in assays that are not impacted by non-specific inhibitors, including antibodies to HA.
In this study, optimized methods to enable rapid antigenic characterization of NA, with wild type viruses as antigen, are described. Since the ELLA is less laborious and shows a good correlation to the miniaturized TBA (Fritz et al., 2012), this assay was selected as a platform. To prevent interference by antibodies directed against HA of wild type A(H3N2) viruses, ferret sera were raised against recombinant influenza A H7N2 viruses (A(H7N2)) viruses that contain the NA of various A(H3N2) viruses. Through this approach it is possible to screen wild type viruses, thus preventing the time-consuming generation of recombinant viruses or proteins for each virus of interest. Reproducibility and sensitivity of the NI assay were highest using virus concentrations that resulted in ∼50% of total NA activity of that virus. Non-specific inhibition of ferret sera was observed for some wild type viruses, especially A(H3N2) viruses, but the critical steps to overcome this non-specific inhibition and obtain reproducible and highly sensitive results are also described.
Section snippets
Cells
Madin–Darby canine kidney (MDCK) cells were cultured in Eagle's minimal essential medium (EMEM, Lonza, Breda, The Netherlands) supplemented with 10% fetal bovine serum (FBS, Sigma–Aldrich, St. Louis, MO, USA), 100 U/ml penicillin (Lonza), 100 U/ml streptomycin (Lonza), 2 mM glutamine (Lonza), 1.5 mg/ml sodium bicarbonate (Lonza), 10 mM HEPES (Lonza), and non-essential amino acids (MP Biomedicals, Europe, Illkirch, France).
293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Lonza)
Principles of the ELLA and NI ELLA
The ELLA measures the enzymatic activity of NA by detecting desialylation of the highly glycosylated fetuin by NA. PNA-HRPO is used to detect the terminal galactoses that become exposed after desialylation of fetuin by NA. The intensity of the signal after addition of the substrate is dependent on the level of desialylation and thus NA activity. In the NI ELLA, binding of NA by specific antibodies will inhibit the enzymatic function of NA and result in a reduction of desialylation and, hence,
Discussion
Monitoring functional antibody responses against NA, in tandem with HA-specific antibody analysis, could enhance the antigenic characterization of influenza viruses during routine surveillance for the purpose of influenza vaccine strain selection. The NI ELLA measures functional inhibition of NA activity by antibodies, and consequently, it has clear relevance to immunity in vivo (Couch et al., 1974, Ogra et al., 1977, Beutner et al., 1979). In this study, the existing NI ELLA was optimized for
Acknowledgements
This work was supported by an NWO VICI (918.96.613) and NIH contract no. HHSN266200700010C. We gratefully thank Geert van Amerongen, Sander Herfst, Eefje Schrauwen, Oanh Vuong, Martin Linster and Ruud van Beek for excellent technical assistance.
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