Optimization of an enzyme-linked lectin assay suitable for rapid antigenic characterization of the neuraminidase of human influenza A(H3N2) viruses

https://doi.org/10.1016/j.jviromet.2015.02.014Get rights and content

Highlights

  • An enzyme-linked lectin assay (ELLA) was optimized to monitor antigenic drift of NA.

  • Non-specific inhibition of NA activity by ferret sera was overcome by RDE-treatment.

  • Sera elicited against recombinant viruses allow use of wild-type viruses as antigen.

  • The optimized ELLA permits screening of many viruses with a limited number of sera.

  • The optimized ELLA is rapid, highly sensitive and reproducible.

Abstract

Antibodies to neuraminidase (NA), the second most abundant surface protein of the influenza virus, contribute to protection against influenza virus infection. Although traditional and miniaturized thiobarbituric acid (TBA) neuraminidase inhibition (NI) assays have been successfully used to characterize the antigenic properties of NA, these methods are cumbersome and not easily amendable to rapid screening. An additional difficulty of the NI assay is the interference by hemagglutinin (HA)-specific antibodies. To prevent interference of HA-specific antibodies, most NI assays are performed with recombinant viruses containing a mismatched HA. However, generation of these viruses is time consuming and unsuitable for large-scale surveillance. The feasibility of using the recently developed enzyme-linked lectin assay (ELLA) to evaluate the antigenic relatedness of NA of wild type A(H3N2) viruses was assessed. Rather than using recombinant viruses, wild type A(H3N2) viruses were used as antigen with ferret sera elicited against recombinant viruses with a mismatched HA. In this study, details of the critical steps that are needed to modify and optimize the NI ELLA in a format that is reproducible, highly sensitive, and useful for influenza virus surveillance to monitor antigenic drift of NA are provided.

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.

References (61)

  • P. Palese et al.

    Characterization of temperature sensitive influenza virus mutants defective in neuraminidase

    Virology

    (1974)
  • T.J. Pritchett et al.

    Basis for the potent inhibition of influenza virus infection by equine and guinea pig alpha 2-macroglobulin

    J. Biol. Chem.

    (1989)
  • C.A. Russell et al.

    Influenza vaccine strain selection and recent studies on the global migration of seasonal influenza viruses

    Vaccine

    (2008)
  • K.A. Ryan-Poirier et al.

    Alpha 2-macroglobulin is the major neutralizing inhibitor of influenza A virus in pig serum

    Virology

    (1993)
  • L. Warren

    The thiobarbituric acid assay of sialic acids

    J. Biol. Chem.

    (1959)
  • E.M. Anders et al.

    Bovine and mouse serum beta inhibitors of influenza A viruses are mannose-binding lectins

    Proc. Natl. Acad. Sci. U. S. A.

    (1990)
  • S.F. Baker et al.

    Influenza A and B virus intertypic reassortment through compatible viral packaging signals

    J. Virol.

    (2014)
  • K.R. Beutner et al.

    Evaluation of a neuraminidase-specific influenza A virus vaccine in children: antibody responses and effects on two successive outbreaks of natural infection

    J Infect Dis

    (1979)
  • F.M. Burnet et al.

    The receptor-destroying enzyme of V. cholerae

    Aust. J. Exp. Biol. Med. Sci.

    (1947)
  • S. Chutinimitkul et al.

    In vitro assessment of attachment pattern and replication efficiency of H5N1 influenza A viruses with altered receptor specificity

    J. Virol.

    (2010)
  • P.M. Colman et al.

    Structure of the catalytic and antigenic sites in influenza virus neuraminidase

    Nature

    (1983)
  • P.M. Colman et al.

    Three-dimensional structure of a complex of antibody with influenza virus neuraminidase

    Nature

    (1987)
  • R.W. Compans et al.

    Effect of antibody to neuraminidase on the maturation and hemagglutinating activity of an influenza A2 virus

    J. Virol.

    (1969)
  • R.B. Couch et al.

    Induction of partial immunity to influenza by a neuraminidase-specific vaccine

    J. Infect. Dis.

    (1974)
  • R.B. Couch et al.

    Antibody correlates and predictors of immunity to naturally occurring influenza in humans and the importance of antibody to the neuraminidase

    J. Infect. Dis.

    (2013)
  • N. Cox et al.

    WHO Manual on Animal Influenza Diagnosis and Surveillance

    (2002)
  • E. de Wit et al.

    Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments

    Virus Res.

    (2004)
  • E. de Wit et al.

    Molecular determinants of adaptation of highly pathogenic avian influenza H7N7 viruses to efficient replication in the human host

    J. Virol.

    (2010)
  • A.E. Fiore et al.

    Prevention and control of influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices (ACIP)

    MMWR. Recommendations and reports: Morbidity and mortality weekly report. Recommendations and reports/Centers for Disease Control

    (2010)
  • A. Flandorfer et al.

    Chimeric influenza A viruses with a functional influenza B virus neuraminidase or hemagglutinin

    J. Virol.

    (2003)
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