Neutralization assays for differential henipavirus serology using Bio-Plex Protein Array Systems

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Abstract

Hendra virus (HeV) and Nipah virus (NiV) are related emerging paramyxoviruses classified in the genus Henipavirus. Both cause fatal disease in animals and humans and are classified as biosafety level 4 pathogens. Here we detail two new multiplexed microsphere assays, one for antibody detection and differentiation and another designed as a surrogate for virus neutralization. Both assays utilize recombinant soluble attachment glycoproteins (sG) whereas the latter incorporates the cellular receptor, recombinant ephrin-B2. Spectrally distinct sGHeV- and sGNiV-coupled microspheres preferentially bound antibodies from HeV- and NiV-seropositive animals, demonstrating a simple procedure to differentiate antibodies to these closely related viruses. Soluble ephrin-B2 bound sG-coupled microspheres in a dose-dependent fashion. Specificity of binding was further evaluated with henipavirus G-specific sera and MAbs. Sera from henipavirus-seropositive animals differentially blocked ephrin-B2 binding, suggesting that detection and differentiation of HeV and NiV neutralizing antibodies can be done simultaneously in the absence of live virus.

Introduction

Nipah virus (NiV) and Hendra virus (HeV) are recently identified paramyxoviruses and are the prototypic members of the new genus Henipavirus. Paramyxoviruses are large, enveloped, negative-sense single stranded RNA viruses, and include members such as Measles virus, Mumps virus, and Respiratory syncytial virus (Lamb and Kolakofsky, 2001). It is a diverse virus family, with various members causing common upper and lower respiratory tract diseases and although less common, other members can cause neurological disease. In contrast, although closely related to each other, NiV and HeV are distinguished from all paramyxoviruses most notably by their broad species tropism and ability to cause fatal disease in animals and humans. HeV first appeared in Australia in 1994 and was transmitted from horses to humans (reviewed Murray et al., 1998); NiV emerged in 1998–1999 in peninsular Malaysia and primarily infected pigs and subsequently humans, however, several other animal species became infected (reviewed Chua, 2003). For both viruses amplification and disease in domestic animals commonly occur prior to transmission of the virus to humans, where infection is manifested as a severe respiratory illness and/or febrile encephalitis (Selvey et al., 1995, Tan and Wong, 2003, Wong et al., 2002). The natural reservoir host of HeV and NiV is believed to be fruit bats in the genus Pteropus (Chua et al., 2002, Halpin et al., 2000). In recent years, both viruses have continued to re-emerge; HeV reappeared in Australia in 1999, 2004, and 2006 with fatal infections in horses and one non-fatal, but sero-converting, human case (Anon., 2004a, McDonald, 2006, Westbury, 2000). In 2004 and 2005, there were three independent NiV outbreaks in Bangladesh (Anon., 2004b, Anon., 2005, Hsu et al., 2004). Significant observations in these outbreaks included a higher incidence of acute respiratory distress syndrome in conjunction with encephalitis, person-to-person transmission, and potentially higher case fatality rates (∼75%). Furthermore, direct transmission of NiV from flying foxes to humans was suggested (Anon., 2004c).

Paramyxoviruses contain two major membrane-anchored envelope glycoproteins that are required for infection of a receptive host cell. All members contain an F glycoprotein which mediates pH-independent membrane fusion between the virus and its host cell, while the second is the attachment glycoprotein which binds the host cell receptor (reviewed in Lamb and Kolakofsky, 2001). Attachment glycoproteins are oligomeric type II membrane glycoproteins, and both dimeric and/or tetrameric (dimer of dimers) configurations exist (Crennell et al., 2000, Morrison, 1988, Russell et al., 1994, Sheehan et al., 1987). For HeV and NiV, the attachment proteins lack hemagglutinin and neuraminidase activities and are designated G. Recently, recombinant, soluble versions of the henipavirus G glycoproteins (sG) were generated which retained several important structural features, such as oligomerization and the ability to bind henipavirus host cell receptors (Bossart et al., 2005). When used as immunogens, homologous and heterologous anti-sG titers were significantly different (Bossart et al., 2005, Mungall et al., 2006), suggesting G may be an ideal antigen for serological differentiation of these two closely related viruses. Additionally, soluble HeV G elicited higher titers of cross-reactive neutralizing antibodies as determined by heterologous serum neutralization titer and may represent a viable vaccine candidate (Mungall et al., 2006).

Recently, ephrin-B2 and ephrin-B3 were identified as receptors employed by HeV and NiV for infection (Bonaparte et al., 2005, Negrete et al., 2005, Negrete et al., 2006). Ephrin-B2 and ephrin-B3 are only the third and fourth host proteins to be identified as paramyxovirus receptors. Both are highly conserved across vertebrate species, expressed in multiple organ systems and are members of a family of receptor tyrosine kinase ligands (Drescher, 2002, Poliakov et al., 2004). Ephrin-B2 and ephrin-B3 have been researched extensively for their role in cancer biology (Castellano et al., 2006, Martiny-Baron et al., 2004, Masood et al., 2005, Nakada et al., 2006) and as a consequence soluble recombinant versions are readily available. When initially discovered as the henipavirus receptor, the affinity of soluble ephrin-B2 for the HeV attachment glycoprotein was demonstrated using Biacore surface plasmon resonance (Bonaparte et al., 2005). More recently, the affinities of ephrin-B2 and ephrin-B3 for the NiV attachment glycoprotein were characterized and when compared, ephrin-B3 had a relatively lower affinity for the attachment glycoprotein but still permitted virus entry (Negrete et al., 2006).

NiV and HeV are classified as biosafety level 4 (BSL4) viruses and as they continue to re-emerge, the ability to diagnose infection becomes critical; however, serological test requiring live virus can be done in only a small number of BSL4 laboratories world wide. Here we combined the use of the soluble attachment glycoproteins, their receptor and a multiplex microsphere platform to develop new assays capable of measuring both virus-specific and neutralizing antibodies. Specifically, we coupled soluble HeV and NiV G (sGHeV and sGNiV) to different microspheres for use on the Bio-Plex Array platform. Henipavirus specific antibodies in sera from seropositive animals were detected and differentiated in a single test. Soluble ephrin-B2 bound both sGHeV- and sGNiV-coupled beads in a dose-dependent fashion. Ephrin-B2 appeared to bind sGNiV more efficiently suggesting that NiV G may have a higher affinity for the host cell receptor. HeV and NiV G-specific sera and monoclonal antibodies (MAbs) were evaluated for their binding to sG and their ability to compete with ephrin-B2 for sG binding. Seropositive sera from different species, including horse and pig field sera, differentially blocked receptor binding to sGHeV and sGNiV, further demonstrating the presence of potentially neutralizing antibodies as well as their specificity. To our knowledge, this is the first report of multiplexed binding and pseudo-neutralization assays that use only recombinant proteins and for HeV and NiV represent a significant advance in serological capability.

Section snippets

Multiplex microsphere assay equipment, software and calibration

Assays were performed on a Bio-Plex Protein Array System integrated with Bio-Plex Manager Software (v 3.0) (Bio-Rad Laboratories, Inc., CA, USA). The high setting was used for the reporter target channel (RP1) and fluorescent identification of microspheres. Reporter conjugate emission wavelengths were maintained using a Bio-Plex Calibration Kit (Bio-Rad, cat. no. 171-203060). Consistent optical alignment, fluidics performance, doublet discrimination and identification of individual bead

Multiplexed antibody detection using sGNiV and sGHeV

Previous studies have demonstrated that HeV and NiV antisera cross neutralize, with each serum being slightly less effective against the heterotypic virus (Berhane et al., 2006, Crameri et al., 2002, Tamin et al., 2002). Additionally, we have demonstrated that antibodies raised against sGHeV were more effective in neutralizing HeV than NiV (Bossart et al., 2005). In more recent immunization studies, sGNiV- and sGHeV-specific cat sera were less effective in SNT against heterotypic virus (Mungall

Discussion

The high level of similarity between HeV and NiV combined with their restriction to high bio-containment laboratories has severely limited the availability of reliable differential serological assays. Although ELISAs have been developed for HeV and NiV, there are technical difficulties in maintaining their reproducibility (Daniels et al., 2001). The antigens are prepared from HeV- or NiV-infected cell cultures and gamma-irradiated for transfer to non-containment laboratories. Consequently, the

Acknowledgments

We thank Gary Crameri, Greer Meehan, Paul Selleck and Chris Morrissy for providing the animal sera used in this study. We are in debt to John White for providing us with his murine MAbs. We thank Lynda Wright for testing all MAbs in the henipavirus SNTs. We are grateful to Christopher C. Broder for critical review of this manuscript prior to its submission and for providing unlimited sG, a critical reagent for these studies. This study was supported by AB-CRC grant 1.013RE to L.W. and Geelong

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