Bead-based suspension array for simultaneous detection of antibodies against the Rift Valley fever virus nucleocapsid and Gn glycoprotein

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Abstract

A multiplex bead-based suspension array was developed that can be used for the simultaneous detection of antibodies against the surface glycoprotein Gn and the nucleocapsid protein N of Rift Valley fever virus (RVFV) in various animal species. The N protein and the purified ectodomain of the Gn protein were covalently linked to paramagnetic Luminex beads. The performance of the resulting multiplex immunoassay was evaluated by testing a comprehensive and well-characterized panel of sera from sheep, cattle and humans. The suitability of this multiplex immunoassay to differentiate infected from vaccinated animals (DIVA) was investigated by testing sera from lambs vaccinated with a paramyxovirus vaccine vector expressing the RVFV surface glycoproteins Gn and Gc. The results suggest that the bead-based suspension array can be used as a DIVA assay to accompany several recently developed experimental vaccines that are based on RVFV glycoproteins, and are devoid of the N protein.

Highlights

► A bead-based suspension array was developed for serology of Rift Valley fever virus. ► It can detect antibodies against the nucleocapsid and glycoprotein Gn simultaneously. ► The assay is capable of detecting IgG antibodies in sheep, cattle and human sera. ► The assay may be applied as DIVA test with vaccines based on surface glycoproteins.

Introduction

Rift Valley fever virus (RVFV) is a mosquito borne virus that belongs to the Phlebovirus genus of the Bunyaviridae family (Elliott, 1996). The virus was first isolated in 1930 (Daubney et al., 1931) near Lake Naivasha in Kenya and has since caused devastating outbreaks throughout Africa (Bird et al., 2009, Gerdes, 2004).

The RVFV RNA genome comprises a small (S), medium (M) and large (L) segment. The S genome segment encodes the nucleocapsid (N) protein in genomic-sense orientation and a non-structural protein (NSs) in the antigenomic-sense orientation (Elliott, 1996). The NSs protein suppresses host innate immune responses and was shown to be the major virulence determinant of the virus (Billecocq et al., 2004, Bouloy et al., 2001, Habjan et al., 2009, Ikegami et al., 2009). The M segment encodes the two structural glycoproteins Gn and Gc and two non-structural proteins, a 78-kDa protein and the NSm protein, of which the latter was shown to have an anti-apoptotic function (Won et al., 2007). The L segment encodes the RNA-dependent RNA polymerase protein.

The first commercially available ELISAs used inactivated whole virus as the antigen (Paweska et al., 2003, Paweska et al., 2005a, Paweska et al., 2005b). Although these ELISAs in general perform very well, the use of recombinant proteins can provide advantages with respect to safety, stability and cost-effectiveness. Several ELISAs based on recombinant antigen have been developed and commercialized, but all these assays detect antibodies against the N protein (Jansen van Vuren and Paweska, 2009, Jansen van Vuren et al., 2007, Paweska et al., 2007, Paweska et al., 2008). Preferably, a serological confirmation test should detect antibodies against other immunogenic proteins of the virus. The virus neutralization test is based on the detection of antibodies against the structural glycoproteins, and can therefore be used for such serological confirmation (Gerdes, 2008). However, the classical virus neutralization test requires live virus to be handled under appropriate biosafety containment facilities, and takes up to seven days for completion. A less laborious, glycoprotein-based serological assay would therefore be very advantageous.

Recently, an efficient method to produce milligram amounts of the Gn ectodomain in a highly pure, soluble form was developed (de Boer et al., 2010). In the work described in the present paper, the production of this protein was improved, and both the recombinant Gn ectodomain and purified N protein were used to develop a Luminex assay for the simultaneous detection of antibodies against both proteins.

Luminex technology uses variously coloured polystyrene beads, the surface of which is carboxylated to allow covalent coupling of antigens. Conjugated beads can be incubated with serum to capture specific antibodies, after which a fluorescent secondary antibody is added to bind to the captured serum antibodies. The Luminex system uses a red laser to determine the colour of the bead and a green laser to detect bound secondary antibodies (Krishhan et al., 2009). With this system, multiple serological components can be tested simultaneously with a single sample (Perkins et al., 2006). The work described in this paper demonstrated that a newly developed Luminex assay can detect antibodies against both the RVFV Gn and N proteins simultaneously. Moreover, the results suggest that this assay may be used to differentiate infected from vaccinated animals (DIVA) when combined with new experimental vaccines based on RVFV glycoproteins.

Section snippets

Production of the N protein

Production of recombinant RVFV nucleoprotein N has been described previously (Martin-Folgar et al., 2010). Briefly summarized, the gene encoding the N protein of RVFV strain MP-12 (GenBank accession number DQ380154.1, protein ID ABD38739.1), flanked by SalI and XhoI sites, was cloned into plasmid pET32a (Novagen, Merck KGaA, Darmstadt, Germany) to generate a fusion protein with the 109 amino acid thioredoxin protein (Trx) containing a 6× His-tag for purification purposes. The resulting plasmid,

Production of Trx-N and the Gn ectodomain

The thioredoxin fusion system was initially chosen to increase the fraction of soluble recombinant N protein in E. coli cells. After induction of expression, most of the recombinant protein was found in the lysozyme fraction, which is indicative of the accumulation in the periplasmic space. Large amounts of this protein were easily produced, facilitating further downstream nickel resin-based purification procedures. The analysis of the expression by Coomassie staining showed the presence of a

Discussion

This study reports the development of a Luminex assay that can be used for the simultaneous detection of antibodies against the RVFV N protein and the structural glycoprotein Gn. The assay was shown to be capable of detecting IgG antibodies in sheep, cattle and human sera and proof-of-concept of IgM detection was provided. Based on the limited number of sera analysed, the assay was shown to be comparable in terms of sensitivity and specificity with commercially available RVFV ELISAs. This

Acknowledgments

The authors would like to thank Dr. Catherine Cêtre-Sossah (Centre International de Recherche Agronomique pour le Développement, Montpellier, France), Dr. Philippe Marianneau and Dr. Michel Pépin (Agence Nationale de Sécurité Sanitaire, France), Dr. Martin Eiden (Friedrich Loeffler Institute, Germany) and Dr. Francesc Xavier Abad Morejón de Girón (Centre de Recerca en Sanitat Animal, Spain) for providing the ring trial sera, Dr. Christiaan A. Potgieter and Shirley J. Smith (ARC-OVI) for

References (26)

  • R. Williams et al.

    Validation of an IgM antibody capture ELISA based on a recombinant nucleoprotein for identification of domestic ruminants infected with Rift Valley fever virus

    J. Virol. Methods

    (2011)
  • A. Billecocq et al.

    NSs protein of Rift Valley fever virus blocks interferon production by inhibiting host gene transcription

    J. Virol.

    (2004)
  • B.H. Bird et al.

    Rift Valley fever virus

    J. Am. Vet. Med. Assoc.

    (2009)
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    1

    Present address: Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, R3E 3R2 Winnipeg, Canada.

    2

    Present address: National Institute for Public Health (RIVM), Centre for Infectious Disease Control, Laboratory for Zoonoses and Environmental Microbiology, P.O. Box 1, 3720 BA Bilthoven, The Netherlands.

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