WO2007005244A1

WO2007005244A1 - Henipavirus receptor and uses thereof

Info

Publication number: WO2007005244A1
Application number: PCT/US2006/023618
Authority: WO
Inventors: Benhur Lee; Oscar A. Negrete
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2005-07-05
Filing date: 2006-06-16
Publication date: 2007-01-11
Anticipated expiration: 2008-01-05

Abstract

The present invention provides compositions and methods of preventing or treating viruses in the Henipavirus genus. The Henipavirus can be a Nipah (NiV) virus or a Hendra (HeV) virus. In particular, the invention provides inhibitors and modulators of NiV viral attachment to the cell through interaction of the Hendravirus attachment (G) glycoprotein with EphrinB2 or EphrinB3.

Description

HENIPAVIRUS RECEPTOR AND USES THEREOF

Statement of Rights to Inventions Made Under Federally Sponsored Research

[0001] This invention is supported by Grant No. AI59051 and AI61824 of the National Institutes of Health. The United States government may have certain rights in this invention.

Technical Field

[0002] The present invention relates to the fields of virology and immunology. In particular, the present invention relates to Nipah virus (NiV) or Hendra virus (HeV) infection and the role of their cellular receptors, EphrinB2 and EphrinB3, in the identification of antiviral agents and candidates for viral vaccines.

Background Art

[0003] Emerging viral pathogens present a critical threat to global health and economy. Nipah (NiV) and Hendra (HeV) viruses are members of the newly defined Henipavirus genus of the Paramyxoviridae (Chua, et al, (2000) Science 288, 1432-5; Harcourt, et al. (2000) Virology 271, 334-49), and are designated a priority pathogen in the NIAID Biodefense Research Agenda. Since 1999, NiV outbreaks have occurred in Malaysia, Singapore and Bangladesh (Hsu, et al, (2004) Emerg Infect Dis 10, 2082-7; Parashar, et al, (2000) J Infect Dis 181, 1755-9). Zoonotic diseases such as those caused by NiV have become an increasing threat in several parts of the world (Daszak, et al, (2004) Ann NY Acad Sd 1026, 1-11)]. NiV, in particular, could be a devastating agent of economic or agrobioterrorism if used against the pig farming industry (Lam, (2003) Antiviral Res 57, 113-9).

[0004] The Nipah virus exhibits an unusually broad host range including humans, pigs, dogs, cats, horses, guinea pigs, hamsters and fruit bats (it's presumptive natural host) (Chua, et al, (2000) Science 288, 1432-5; Field, et al, (2001) Microbes Infect 3, 307-14; Wong, et al, (2003) Am J Pathol 163, 2127-37). Such broad host tropism is rare amongst extant paramyxoviruses. With the possible exception of fruit bats, disease mortality of all other hosts has been shown during natural or experimental infection (Wong, et al, (2003) Am J Pathol 163, 2127-37; Hooper, et al, (2001) Microbes Infect 3, 315-22). However, the mortality rate in pigs is less than 5% even though the transmission rate approaches 100% (Chua, et al, (2000) Science 288, 1432-5; Lam, (2003) Antiviral Res 57, 113-9), suggesting that zoonotic transmission to humans has increased the pathogenicity of the virus.

[0005] The habitat of the pteropid fruit bat, considered as the natural reservoir host, spans from the east coast of Africa across southern and Southeast Asia, east to the Philippines and Pacific islands, and south to Australia (Field, et ah, (2001) Microbes Infect 3, 307-314). Although NiV outbreaks have only occurred in Malaysia, Bangladesh, and Singapore, increased surveillance in other geographical regions of the pteropid habitat found bats to harbor NiV (Reynes, et al, (2005) Cambodia. Emerg Infect Dis 11, 1042-1047).

[0006] Therefore, NiV continues to remain a potential threat to both human and animal populations. This underscores the need for the development of antiviral therapeutics. A complete understanding of Nipah viral entry at the level of receptor engagement may help in these efforts, since, to date, the NiV and HeV receptors have not been identified, limiting the ability to develop targeted compounds that prevent viral entry into cells or elicit protective immunity.

Summary of the Invention

[0007] The present invention provides compositions and methods of preventing or treating viruses in the Henipavirus genus. The Henipavirus can be the NiV or HeV. hi particular, the invention provides inhibitors and modulators of NiV or HeV viral attachment to the cell through interaction of the NiV or HeV G attachment with EphrinB2 or EphrinB3.

[0008] In one aspect, provided herein is a pharmaceutical composition comprising a polypeptide comprising a soluble EphrinB2, EphrinB3, ephB4, or a fragment thereof, and pharmaceutically acceptable excipient. Binding of the soluble EphrinB2 or fragment thereof to a Henipavirus attachment (G) glycoprotein, binding of the soluble EphrinB3 or fragment thereof to a Henipavirus G glycoprotein, or binding of the soluble ephB4 or fragment thereof to a cell-bound EphrinB2, prevents or inhibits binding of the Henipavirus attachment (G) glycoprotein to a cell-bound EphrinB2 or EphrinB3 polypeptide. The EphrinB2 or EphrinB3 fragment can be a Henipavirus attachment (G) glycoprotein-binding fragment. The soluble EphrinB2, EphrinB3, ephB4, or fragment thereof, can further comprises a heterologous domain such as an Fc domain. The polypeptides of the present composition can be human or pig in origin. Sometimes, the soluble EphrinB2, EphrinB3 or ephB4, or fragment thereof, comprises a synthetic or recombinant polypeptide or a peptidomimetic. [0009] In another aspect, provided herein is a pharmaceutical composition comprising a polypeptide comprising a soluble Henipavirus attachment (G) glycoprotein or fragment thereof, and pharmaceutically acceptable excipient, wherein binding of the soluble Henipavirus attachment (G) glycoprotein or fragment thereof to a cell-bound EphrinB2 or EphrinB3 polypeptide prevents or inhibits downstream signaling. In one embodiment, the downstream signaling is necessary for entry of the Henipavirus into the cell.

[0010] In another aspect, provided herein is a pharmaceutical composition comprising a polypeptide comprising a soluble Henipavirus attachment (G) glycoprotein-binding protein, and pharmaceutically acceptable excipient, wherein binding of the soluble Henipavirus attachment (G) glycoprotein-binding protein to the Henipavirus attachment (G) glycoprotein prevents or inhibits binding of the Henipavirus attachment (G) glycoprotein to an EphrinB2 or EphrinB3 polypeptide. In one embodiment, the soluble Henipavirus attachment (G) glycoprotein-binding protein comprises an antibody that specifically binds to the Henipavirus attachment (G) glycoprotein. The antibody can be a recombinant, synthetic or humanized antibody.

[0011] In yet another aspect, provided herein is a method for inhibiting or preventing binding of a Henipavirus to a cell, wherein the method comprises contacting the cell with a composition as set forth above, thereby inhibiting or preventing binding of the Henipavirus to a cell. The cell can be a human or a pig cell. The cell can be contacted with the pharmaceutical composition in vivo, in vitro or ex vivo.

[0012] Further provided herein is a method for inhibiting or preventing binding of a Henipavirus to a cell, wherein the method comprises contacting the cell with a composition comprising a polypeptide comprising a soluble EphrinB2, EphrinB3, ephB4, or a fragment thereof, thereby inhibiting or preventing binding of the Henipavirus to a cell.

[0013] Also provided herein is a method for inhibiting or preventing infection of a cell by a Henipavirus, wherein the method comprises contacting the cell with a composition comprising a polypeptide comprising a soluble EphrinB2, EphrinB3, ephB4, or a fragment thereof, thereby inhibiting or preventing binding of the Henipavirus to the cell and inhibiting or preventing infection of the cell by the Henipavirus.

[0014] In one aspect, provided herein is a method for inhibiting or preventing binding of a Henipavirus to a cell, wherein the method comprises contacting the cell with a composition comprising a Henipavirus attachment (G) glycoprotein-binding antibody or binding fragment thereof, thereby inhibiting or preventing binding of the Henipavirus to a cell. [0015] In another aspect, provided herein is a soluble EphrinB2, EphrinB3, ephB4, or a fragment thereof, for use as a medicament.

[0016] Also provided is a soluble Henipavirus attachment (G) glycoprotein or fragment thereof, for use as a medicament.

[0017] In another aspect, provided herein is a soluble EphrinB2, EphrinB3, ephB4, or a fragment thereof, for use as a Henipavirus antiviral agent.

[0018] Further provided herein is a use of a polypeptide comprising a soluble EphrinB2, EphrinB3, ephB4, or a fragment thereof for the manufacture of a medicament for the treatment or prevention of a Henipavirus infection.

[0019] In one aspect, provided herein is a method for identifying an inhibitor of a Henipavirus-induced cell-cell fusion, which comprises: providing a test agent, Henipavirus, and cells comprising EphrinB2 or EphrinB3 polypeptides, biologically active fragments capable of binding to the Henipavirus attachment (G) glycoprotein; contacting one set of cells with the test agent and the Henipavirus, wherein the cells are contacted with the test agent and the Henipavirus simultaneously, or contacted with the test agent before the Henipavirus, and contacting one set of cells with only the Henipavirus; and detecting the presence or absence of cell fusion, whereby the test agent is identified as an inhibitor of Henipavirus-induced cell-cell fusion when the cell fusion produced in the system contacted with the test agent is different than the cell fusion produced in a system not contacted by the test agent.

[0020] In another aspect, provided herein is a method for identifying an inhibitor of a Henipavirus infection, which comprises: providing a test agent; contacting a system comprising a first cell and a second cell with the test agent, wherein the first cell comprises a Henipavirus attachment (G) glycoprotein, the second cell comprises EphrinB2, EphrinB3, or a biologically active fragment thereof capable of binding to the Henipavirus attachment (G) glycoprotein of the first cell; and detecting the presence or absence of cell fusion, whereby the test agent is identified as an inhibitor of Henipavirus viral infection when the cell fusion produced in the system contacted with the test agent is different than the cell fusion produced in a system not contacted by the test agent.

[0021] For these methods, the cells can be human cells or pig cells. The viral envelope protein can be exogenously expressed. The viral envelope protein receptor can be exogenously or endogenously expressed. In some embodiments, the cell fusion is detected by the presence or absence of a signal produced by the functional reporter molecule. In one embodiment, the functional reporter molecule is β-galactosidase. The test agent can be a polypeptide, a carbohydrate or a small molecule.

[0022] Further provided herein is a Nipah viral infection inhibitor molecule identified by the methods provided herein.

[0023] In yet another aspect, provided herein is a method for identifying an antiviral agent, which comprises providing a test agent; contacting a Henipavirus attachment (G) glycoprotein with an EphrinB2 polypeptide, a EphrinB3 polypeptide, or a Henipavirus attachment (G) glycoprotein-binding fragment thereof, in the presence or absence of a test agent; and detecting attachment between the Henipavirus attachment (G) glycoprotein with the EphrinB2 polypeptide, with the EphrinB3 polypeptide, or with the Henipavirus attachment (G) glycoprotein-binding fragment of EphrinB2 or EphrinB3. The test agent is identified as an antiviral agent when the attachment in the system contacted with the test agent is different than the attachment produced in a system not contacted by the test agent. The Henipavirus attachment (G) glycoprotein can be present in a Henipavirus or in a pseudo-type virus. The EphrinB2 polypeptide, EphrinB3 polypeptide, or Henipavirus attachment (G) glycoprotein- binding fragment thereof can be present in a host cell. In one embodiment, a decrease in attachment of the Henipavirus attachment (G) glycoprotein to the EphrinB2 polypeptide, EphrinB3 polypeptide, or Henipavirus attachment (G) glycoprotein-protein binding fragment of EphrinB2 or EphrinB3 in the presence of the test agent corresponds to a decrease in entry of a Henipavirus into the host cell. The test agent can be a polypeptide, a carbohydrate or a small molecule. In some embodiments, the test agent is a soluble EphrinB2 receptor or a fragment thereof, an antibody specific for Henipavirus attachment (G) glycoprotein, EphrinB2 or EphrinB3, or siRNA. In some embodiments, the test agent is a tyrosine kinase inhibitor or an ion channel inhibitor.

[0024] Also provided herein is a method for reducing the infectivity by Henipavirus of a host cell susceptible to infection by Henipavirus, comprising administering to a subject in need thereof an antiviral agent that inhibits the ability of Henipavirus attachment (G) glycoprotein to attach to EphrinB2 or EphrinB3 and thereby reducing infectivity of the virus. In some embodiments, the method is performed in vivo. The subject can be a human or a pig.

[0025] In one aspect, provided herein is a method for eliciting a protective immune response against Henipavirus, which comprises administering a Henipavirus attachment (G) glycoprotein or a fragment thereof to a subject in need thereof. [0026] In yet another aspect, provided herein is a method for selectively identifying peptides to elicit a protective immune response for Henipavirus, which comprises obtaining a peptide library of Henipavirus attachment (G) glycoprotein; and identifying the peptide required for Henipavirus attachment (G) glycoprotein attachment to EphrinB2 or EphrinB3, whereby the peptide that mediates Henipavirus attachment (G) glycoprotein attachment to EphrinB2 or EphrinB3 is a peptide that will elicit a protective immune response for Henipavirus.

Brief Description of the Drawings

[0027] Figure 1 illustrates how soluble NiV-G binds to a 48 kDa membrane protein. Figure Ia shows how equal amounts of the NiV-G-Fc (thick line) or Fc-only (filled histogram) were incubated with permissive 293T, Vero, and HeLa cells or non-permissive CHO-pgsA745, PKl 3, or human Raji B cells. Cell surface binding was detected by a PE-conjugated anti- human IgG secondary antibody. Figure Ib illustrates how cell surface proteins from permissive 293T and Vero cells or non-permissive CHO-pgsA745 cells were biotinylated, immunoprecipitated by NiV-G-Fc or Δ28 NiV-G-Fc, ran on a non-denaturing SDS-PAGE gel and detected by western blotting with HRP-conjugated streptavidin (SAV) or anti-human Fc (α-Fc).

[0028] Figure 2 illustrates how the ectodomain of NiV-G binds specifically to EphrinB2. Figure 2a shows the soluble HA-tagged ectodomain of NiV-G (sNiV-G-HA) bound to EphrinB2-Fc but not EphrinBl-Fc in an ELISA based assay (see Methods). Δ28 sNiV-G-HA, with an identical deletion as in Δ28 NiV-G-Fc, did not bind to EphrinB2-Fc or EphrinBl-Fc. One representative experiment out of three is shown. Data are averages of triplicates +/- S.D. Figure 2b illustrates how 10 μg/ml of EphrinB2-Fc but not EphrinBl-Fc was able to block sNiV-G-HA binding to permissive 293T cells. sNiV-G-HA binding was detected by a mouse monoclonal anti-HA antibody followed by a PE-conjugated anti-mouse IgG secondary antibody. Figure 2c shows NiV-G-Fc bound to EphrinB2-transfected but not to pcDNA3- transfected CHO-pgsA745 and human Raji B cells. Cell surface binding was detected as in Fig. Ia.

[0029] Figure 3 illustrates how EphrinB2 is necessary for NiV fusion. Figure 3a shows how NiV-F/G expressing "effector" PKl 3 cells were placed on permissive (293T or Vero cells) or non-permissive (PKl 3 or human Raji B) "target" cells and fusion quantified as described in Methods. Figure 3b illustrates how a Fusion assay was performed as in (a) for 293T and Vero cells except that EphrinB2-Fc or EphrinBl-Fc (10 μg/ml) was added to the target cells 30 minutes prior to addition of NiV envelope expressing effector cells. Figure 3c shows a fusion assay performed with transfected Raji B target cells and PKl 3 effector cells. Figure 3d illustrates inhibition studies on Raji B cells were performed as in (b) (ephB4-Fc: 100 μg/ml). Fusion in each case was normalized to that obtained in the absence of any blocking reagent. Figure 3e shows fusion assay between microvascular endothelial target cells and NiV envelope expressing PKl 3 effector cells as described. Inhibition studies were performed as in (d). Data are shown as averages +/- S.D. from at least 2 independent experiments.

[0030] Figure 4 illustrates how EphrinB2 mediates entry of NiV-F/G pseudotyped viruses. Figure 4a shows how VSV-G or NiV-F/G mediated entry into 293T cells was neutralized specifically by their respective anti-sera. Matched phase contrast and fluorescent images are shown. Figure 4b illustrates how NiV-F/G or VSV-G pseudotyped viruses were used to infect Vero cells in the presence or absence of EphrinBl-Fc or EphrinB2-Fc (10 μg/ml). RFP production was analyzed by FACS. Figure 4c shows how human EphrinB2 or pcDNA3- transfected CHO-ρgsA745 cells were infected with NiV-F/G pseudotyped VSV-RFP and FACS-analyzed for RFP production. Figure 4d illustrates how NiV-F/G pseudotyped VSV- RFP viruses were used to infect cortical rat neurons in the presence or absence of EphrinBl-Fc or EphrinB2-Fc (10 μg/ml). Representative matched phased contrast and fluorescent images are shown. Figure 4e shows how additional inhibition studies were performed with microvascular endothelial cells (EphrinBl/B2-Fc, lOμg/ml; ephB2/B4-Fc, lOOμg/ml).

[0031] Figure 5 shows the protein sequence of EρhrinB2 (SEQ ID NO: 1). The two peptides identified by LC-MS/MS (tandem MS) peptide sequencing are capitalized, bold, and underlined. The results from LC-MS/MS (tandem MS) are based on independent peptide sequencing. Therefore, all proteins detected are actual proteins present in a sample.

[0032] Figure 6 shows the construction of NiV-G-Fc and sNiV-G-HA. NiV-F and NiV-G were codon optimized and synthesized by Geneart (Germany) using an in-house proprietary software that addresses codon-usage, elimination of cryptic splice-sites, as well as the stability of DNA/RNA secondary structures (GenBank Accession numbers AY816748 and AY 816745, respectively). The ectodomain of NiV-G (amino acid residues 71-602) was fused to the Fc constant region (CH2-CH3) of human IgGl (NiV-G-Fc) or placed in frame with a hemagglutinin tag (sNiV-G-HA) and placed under a CMV promoter. This latter construct also has a myc and 6X-his epitope tag. Since NiV-G is a type II transmembrane protein, the kappa light-chain signal sequence was placed at the N-terminus of both NiV-G-Fc and sNiV-G-HA to promote secretion of soluble protein into the supernatant.

[0033] Figure 7 illustrates that soluble NiV-G binds to EphrinB3 with lower affinity than EphrinB2. In Figure 7a, 1.0 μg/ml, 0.1 μg/ml, and 0.01 μg/ml of the indicated ephrin-Fc fusion proteins were allowed to bind to soluble NiV-G-coated plates in an ELISA format (see Materials and Methods). The amount of ligand bound was detected colorimetrically using an antihuman Fc antibody conjugated to horseradish peroxidase. One representative experiment out of three is shown. Data are averages of triplicates ± standard error (SE). In Figure 7b, EphrinB2 and B3 stably transfected CHO-pgsA745 cells (CHO-B2 and CHO-B3, respectively) were used to measure NiV-G-Fc cell surface binding. Increasing concentrations of NiV-G-Fc were added to either CHO-B2 cells (dashed line with squares) or CHO-B 3 cells (solid line with triangles), and binding was assessed by flow cytometry using R-phycoerythrin-conjugated anti- Fc antibodies. Regression curves were generated as described in Materials and Methods. Each data point is an average ± SE from three experiments. Figure 7c illustrates how surface plasmon resonance (BIAcore 3000) measured the binding kinetics of NiV-G-Fc to both EphrinB2-Fc and EphrinB3-Fc in response units (RU). NiV-G-Fc was immobilized to a CM5 sensor chip via an amide coupling procedure, and increasing concentrations of EphrinB2-Fc and EphrinB3-Fc were flowed as analyte over the sensor chip. One representative experiment out of two is shown. Figure 7d illustrates the K_d, K_0n (association-rate), and K_of_f (dissociation- rate) determined by fitting the binding chromatogram data from (C) with BIAcore evaluation software (version 3.1) using the 1:1 Langmuir binding model.

[0034] Figure 8 illustrates that pseudotyped and live NiV use EphrinB2 and B3 for cellular entry. Figure 8a illustrates ephrin expression measured by flow cytometry on CHO-pgsA745 parental cells (CHO) and CHO-ρgsA745 cells stably expressing EphrinBl, B2, and B3 (CHO- Bl, CHO-B2, and CHO-B3). To bind the CHO cell lines, 10 μg/ml of EphA2, 10 μg/ml EphB3-Fc, and 1 nM of NiV-G-Fc were used, and the amount of binding was detected by flow cytometry as in Figure 7b. Data are representative of three experiments. Figure 8b shows NiV-F and G glycoproteins pseudotyped onto a VSV-ΔG-Luc core virus (NiV- VSV-ΔG-Luc) and used to infect parental CHO-pgsA745 (CHO), CHOBl, CHO-B2, and CHO-B3 cells. Entry of the indicated dilutions of NiV- VSV-ΔG-Luc viruses was measured by quantifying Renilla Luc activity according to manufacturer's directions. Relative light units (RLU) were acquired and quantified on a Veritas luminometer. Data are shown as averages of triplicates 6 standard deviation of a representative experiment. In three independent experiments, viral entry into CHO-B3 cells was reduced by 21%, 28%, and 46%, respectively, compared to CHO- B2 cells (p = 0.05, paired t-test). In Figure 8c, the listed MOIs of live NiV were used to infect the indicated cell lines (105 cells per infection). Foci of syncytia were observed 24 h postinfection. Vero E6 cells are fully permissive for NiV infection and were used as positive control cells. Note the larger number of syncytia seen on CHO-B2 versus CHO-B3 cells.

[0035] Figure 9 illustrates that EphrinB2 and B3 bind NiV-G at an overlapping site. NiV- VSV-ΔG-Luc pseudotyped viruses were used to infect CHO-B2 (Figure 9a) and CHO-B3 (Figure 9b) cells in the presence of the indicated amounts of EphrinBl, B2, and B3-Fc fusion proteins (Bl-Fc, B2-Fc, and B3-Fc, respectively). Entry was measured as in Figure 8a. Data are the average of triplicates ± standard deviation, and one representative experiment of three is shown.

[0036] Figure 10 illustrates that the Leu-Trp residues present in the G-H loop of EphrinB2 and B3 are the critical determinants of NiV-G binding. Figure 10a shows the sequence alignment of human (hu) EphrinBl (SEQ ID NO: 2), mouse (ms) EphrinBl (SEQ ID NO: 3), rat (rt) EphrinBl (SEQ ID NO: 4); human (hu) EphrinB2 (SEQ ID NO: 5), mouse (ms) EphrinB2 (SEQ ID NO: 6), rat (rt) EphrinB2 (SEQ ID NO: 7); human (hu) EρhrinB3 (SEQ ID NO: 8) and mouse (ms) EphrinB3 (SEQ ID NO: 9) ectodomains using the Jotun Hein algorithm (DNAstar Megalign software). Six residues in the ephrin B-class ectodomain reveal solvent-exposed amino acids (Himanen, et ai, (2001) Nature 414, 933-938) that contain identical residues in both EphrinB2 and B3 but different residues in EphrinBl (open box). Examination of the ephrin binding loop (G-H loop) indicates the L-W residues in ephrin B2 and B3 are replaced by Y-M residues in EphrinBl (filled box). Figure 10b shows ephrin-Fc mutants created by substituting the L-W residues present in EphrinB2 and B3 with Y-M residues using site-directed mutagenesis (B2_YM-FC and B3_YM-FC). Conversely, the Y-M residues in EphrinBl were exchanged for the L-W residues (BI_LW-FC); 10 nM, 1 nM, and 0.1 nM of both wild-type (Bl, B2, and B3) and mutant (Bl_Lw, B2_YM, and B3_YM) ephrin-Fc proteins were tested for their ability to bind NiV-G-HA in an ELISA. The amount of binding was measured the same as in Figure 7a. The data are averages of three experiments done in triplicates ± standard error.

[0037] Figure 11 shows that the Leu-Trp residues in G-H loop of EphrinB3 are necessary for pseudotyped NiV entry. Figure 11a shows the percentage of ephrin cell surface expression (CSE) measured by flow cytometry on CHO-pgsA745 parental cells (CHO) and CHO-pgs745 cells stably expressing both full-length wild-type ephrins (Bl, B2, and B3) and mutant ephrms (BI_LW, B2γ_M, and B3_YM); 10 μg/ml of EphB3-Fc (solid bar) and 1 iiM of NiV-G-Fc (open bar) were used to bind the CHO cell lines, and the amount of binding was detected the same as in Figure 7b. The data are an average of triplicates ± standard deviation (SD). Figure lib shows the same CHO cell lines used above seeded at 10⁵ cells per well and infected with pseudotyped NiV- VSV-ΔG- Luc virus. The amount of entry was detected as in Figure 8a. One representative experiment of three is shown, and data are an average of triplicates ± SD. In three independent experiments, the viral entry into B2_YM cells was reduced by 45%, 68%, and 85%, respectively, compared to wild-type B2 cells (p < 0.03, paired t-test).

[0038] Figure 12 illustrates that soluble NiV-G and HeV-G bind EphrinB2 with similar affinities. Figure 12a and 12b show surface plasmon resonance (BIAcore 3000) measurements of the binding kinetics of NiV-G-FC (a) and HeV-G-Fc (b) to ephrin B2-Fc in response units (RU). Soluble G-Fc was immobilized to a CM5 sensor chip via an amide coupling procedure and increasing nM concentrations of EphrinB2-Fc were flowed as an analyte over the sensor chip. One representative experiment out of two is shown. Figure 12c shows K_0n, K_off (association- rate), and Ka (dissociation-rate) determined by fitting the binding chromatogram data from (A) with BIAcore evaluation software (version 3.1) using the 1 :1 Langmuir binding model.

[0039] Figure 13 illustrates a comparison of NiV-G and HeV-G Fc fusion construct binding on cell surface expressed EphrinB2 and B3. Ephrin B2 (Figure 13a) and B3 (Figure 13b) stably transfected CHOpgsA745 cells (CHO-B2 and CHO-B3, respectively) were used to measure NiV-G-HA (squares) and HeV-G-HA (triangles) cell surface binding. Increasing concentrations of soluble G-Fc (sG-Fc) were added to either CHO-B2 cells or CHO-B3 cells and binding was assessed by flow cytometry using R-phycoerythrin-conjugated anti-Fc antibodies. Regression curves were generated.

Modes of Carrying Out the Invention

[0040] The invention provides compositions and methods for preventing or treating infection with viruses of Henipavirus genus. The Henipavirus can be a Nipah (NiV) virus or a Hendra (HeV) virus. Endothelial cells are major cellular targets for the Nipah virus such that syncytial or multinucleated giant endothelial cells in blood vessels are considered a pathognomonic feature of Nipah viral disease (Wong, et ah, (2002) Am J Pathol 161, 2153- 67). The fusion (F) and attachment (G) proteins of NiV mediate syncytia formation, and many cell lines from numerous animal species are permissive for NiV envelope-mediated fusion (Bossart, et al, (2002) J Virol 76, 11186-98; Tamin, et al, (2002) Virology 296, 190-200), suggesting that the receptor for NiV is highly conserved. The present invention lies in the identification of EphrinB2 and EphrinB3 as receptors for NiV and HeV. The attachment of NiV to EphrinB2 or EphrinB3 is mediated through the NiV attachment (G) protein and allows the virus to enter the cell through a fusion event. Similarly, the attachment of HeV to EphrinB2 or EphrinB3 is mediated through the HeV attachment (G) protein and allows the virus to enter the cell through a fusion event. Thus, the disruption of the NiV-G:EphrinB2 interaction, the NiV-G:EphrinB3 interaction, the HeV-G:EphrinB2 interaction, the HeV- G:EphrinB3 interaction, or subsequently mediated fusion events reduces NiV or HeV infectivity and represents a method to prevent or treat viral infection and related symptoms. [0041] For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections that follow.

A. Definitions

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, published patent applications and other publications and sequences from GenBank and other databases referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in patents, published patent applications and other publications and sequences from GenBank and other data bases that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

[0043] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, MOLECULAR CLONING: A LABORATORY MANUAL, 2ND ED. (Sambrook et al, 1989); OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait, ed., 1984); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al, eds., 1987); and CURRENT PROTOCOLS IN IMMUNOLOGY (J. E. Coligan et al, eds., 1991).

[0044] As used herein, "a" or "an" means "at least one" or "one or more."

[0045] As used herein, the term "antibody" refers to an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an antibody is any form of antibody or fragment thereof that exhibits the desired biological activity, e.g., binding the specific target antigen. Thus, it is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, nanobodies, diabodies, multispecific antibodies (e.g., bispeciflc antibodies), and antibody fragments including but not limited to scFv, Fab, and Fab₂, so long as they exhibit the desired biological activity.

[0046] As used herein, the term "EphrinB2" refers to a receptor tyrosine kinase ligand that binds to an EphB receptor, specifically, the EphB4 receptor. Other synonyms of EphrinB2 include EFNB2, eph-related receptor tyrosine kinase ligand 5, EPLG5, ligand of eph-related kinase 5, LERK5, LERK-5, HTK ligand, HTKL, and HTK-L. The EphrinB2 proteins and nucleic acids of the present methods are not limited to a particular source or species. Thus, the proteins and nucleic acids can be isolated or recombinant, including but not limited to the sequences disclosed in Bennett et al., Proc. Natl Acad. Sd. U.S. A. 92, 1866-70 (1995); Cerretti et al, MoI. Immunol. 32, 1197-1205 (1995)); U.S. Pat. No. 6,303,769; U.S. Patent Publication No. 2004/0110150; and at NIH Database Accession Number NM__004093 (human).

[0047] As used herein, the term "EphrinB3" refers to a receptor tyrosine kinase ligand that binds to an EphB receptor, specifically, the EphA4, EphBl, EphB2, and EphB3 receptors Flanagan & Vanderhaegen, (1998) Annu. Rev. Neurosci., 21, 309-345; Pasquale, (1997) Curr. Opin. Cell Biol., 9, 608-615). Other synonyms of EρhrinB3 include EFNB3, eph-related receptor tyrosine kinase ligand 8, EPH-related receptor transmembrane ligand, EFL-6, ELF-3, ELK-L3, EPLG8, ligand of eph-related kinase 8, NLERK-2, LERK8, and LERK-8. The EphrinB3 proteins and nucleic acids of the present methods are not limited to a particular source or species. Thus, the proteins and nucleic acids can be isolated or recombinant, including but not limited to the sequences disclosed in Tang, et al., (1997) Genomics 41, 17- 24; Gale, et al, (1996) Oncogene 13,1343-1352; Strausberg, et al., (2002) Proc. Natl. Acad. ScL U.S.A. 99,16899-16903 and at NIH Database Accession Number NMJ)01406 (human).

[0048] The term "EphB4" refers to the endogenous receptor for EphrinB2 ligand. EphB4 is typically a membrane bound receptor that must be oligomerized to be active. The EphrinB2 proteins and nucleic acids of the present methods are not limited to a particular source or species. Thus, the proteins and nucleic acids can be isolated or recombinant, including but not limited to the sequences disclosed in Accession No. NM_00444 (nucleic acid sequence); NP_004435; Sakamoto et al, Biochem. Biophys. Res. Commun. 321: 681-87 (2004).

[0049] As used herein, a "fragment thereof refers to a fragment that still substantially retains at least one function of the full length polypeptide. Normally, the derivative or fragment retains at least 50% of its binding activity to NiV, HeV, EphB4, EphrinB2 or EphrinB3, as appropriate. Preferably, the derivative or fragment retains at least 60%, 70%, 80%, 90%, 95%, 99% and 100% of its binding activity. In one embodiment, the fragment of the EphrinB2 or EphrinB3 binds to a Henipavirus attachment (G) glycoprotein, which can be a NiV-attachment (G) glycoprotein or a Hendra Virus (HeV) attachment (G) glycoprotein.

[0050] As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See e.g., REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY 20th Ed. (Lippincott, Williams & Wilkins 2003). Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.

[0051] As used herein, the terms "small interfering RNA" ("siRNA") or "short interfering RNAs") refer to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNA interference.

[0052] As used herein the term "subject" refers to mammalian subjects. Exemplary subjects include, but are not limited to humans, bats, monkeys, dogs, cats, mice, rats, guinea pigs, hamsters, cows, horses, pigs, goats and sheep, hi some embodiments, the subject has cancer and can be treated with the agent of the present invention as described below.

[0053] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of a compound that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject is effective to prevent or ameliorate the disease condition or the progression of the disease. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. In one embodiment, an effective amount is an amount that inhibits or reduces viral entry into a cell. In a further embodiment, an effective amount is an amount that inhibits or reduces EphrinB2 or B3 signaling, preferably by inhibiting or reducing interaction between the ephrin and its endogenous ligand.

B. Inhibitors of viral attachment and methods of identifying such inhibitors

[0054] In one aspect, provided herein is a pharmaceutical composition comprising a polypeptide comprising a soluble polypeptide selected from the group consisting of ephB4 or a fragment thereof, EphrinB2, EphrinB3, and a Henipavirus attachment (G) glycoprotein binding fragment of EphrinB2 or EphrinB3, and pharmaceutically acceptable excipient, for preventing or inhibiting binding of the Henipavirus attachment (G) glycoprotein to a cell-bound EphrinB2 or EphrinB3 polypeptide. Binding of the soluble EphrinB2 or fragment thereof to a Henipavirus attachment (G) glycoprotein, binding of the soluble EphrinB3 or fragment thereof to a Henipavirus G glycoprotein, or binding of the soluble ephB4 or fragment thereof to a cell- bound Ephrin B2, prevents or inhibits binding of the Henipavirus attachment (G) glycoprotein to a cell-bound EphrinB2 or EphrinB3 polypeptide. The EphrinB2 or EphrinB3 fragment can be a Henipavirus attachment (G) glycoprotein-binding fragment. The soluble EphrinB2, EphrinB3, ephB4, or fragment thereof, can further comprises a heterologous domain such as an Fc domain. The polypeptides of the present composition can be human or pig in origin. Sometimes, the soluble EphrinB2, EphrinB3 or ephB4, or fragment thereof, comprises a synthetic or recombinant polypeptide or a peptidomimetic.

[0055] In another aspect, provided herein is a pharmaceutical composition comprising a polypeptide comprising a soluble Henipavirus attachment (G) glycoprotein or fragment thereof, and pharmaceutically acceptable excipient, wherein binding of the soluble Henipavirus attachment (G) glycoprotein or fragment thereof to a cell-bound EphrinB2 or EphrinB3 polypeptide prevents or inhibits downstream signaling. In one embodiment, the downstream signaling is necessary for entry of the Henipavirus into the cell.

[0056] In another aspect, provided herein is a pharmaceutical composition comprising a polypeptide comprising a soluble Henipavirus attachment (G) glycoprotein-binding protein, and pharmaceutically acceptable excipient, wherein binding of the soluble Henipavirus attachment (G) glycoprotein-binding protein to the Henipavirus attachment (G) glycoprotein prevents or inhibits binding of the Henipavirus attachment (G) glycoprotein to an EphrinB2 or EphrinB3 polypeptide. In one embodiment, the soluble Henipavirus attachment (G) glycoprotein-binding protein comprises an antibody that specifically binds to the Henipavirus attachment (G) glycoprotein. The antibody can be a recombinant, synthetic or humanized antibody.

[0057] In yet another aspect, provided herein is a method for inhibiting or preventing binding of a Henipavirus to a cell, wherein the method comprises contacting the cell with a composition as set forth above, thereby inhibiting or preventing binding of the Henipavirus to a cell. The cell can be a human or a pig cell. The cell can be contacted with the pharmaceutical composition in vivo, in vitro or ex vivo.

[0058] Further provided herein is a method for inhibiting or preventing binding of a Henipavirus to a cell, wherein the method comprises contacting the cell with a composition comprising a polypeptide comprising a soluble EphrinB2, EphrinB3, ephB4, or a fragment thereof, thereby inhibiting or preventing binding of the Henipavirus to a cell.

[0059] Also provided herein is a method for inhibiting or preventing infection of a cell by a Henipavirus, wherein the method comprises contacting the cell with a composition comprising a polypeptide comprising a soluble EphrinB2, EphrinB3, ephB4, or a fragment thereof, thereby inhibiting or preventing binding of the Henipavirus to the cell and inhibiting or preventing infection of the cell by the Henipavirus.

[0060] In one aspect, provided herein is a method for inhibiting or preventing binding of a Henipavirus to a cell, wherein the method comprises contacting the cell with a composition comprising a Henipavirus attachment (G) glycoprotein-binding antibody or binding fragment thereof, thereby inhibiting or preventing binding of the Henipavirus to a cell.

[0061] In another aspect, provided herein is a soluble EphrinB2, EphrinB3, ephB4, or a fragment thereof, for use as a medicament.

[0062] Also provided is a soluble Henipavirus attachment (G) glycoprotein or fragment thereof, for use as a medicament.

[0063] In another aspect, provided herein is a soluble EphrinB2, EphrinB3, ephB4, or a fragment thereof, for use as a Henipavirus antiviral agent.

[0064] Further provided herein is a use of a polypeptide comprising a soluble EphrinB2, EphrinB3, ephB4,, or a fragment thereof for the manufacture of a medicament for the treatment or prevention of a Henipavirus infection.

[0065] In one aspect, provided herein is a method for identifying an inhibitor of a Henipavirus-induced cell-cell fusion, which comprises: providing a test agent, Henipavirus, and cells comprising EphrinB2 or EphrinB3 polypeptides, biologically active fragments capable of binding to the Henipavirus attachment (G) glycoprotein; contacting one set of cells with the test agent and the Henipavirus, wherein the cells are contacted with the test agent and the Henipavirus simultaneously, or contacted with the test agent before the Henipavirus, and contacting one set of cells with only the Henipavirus; and detecting the presence or absence of cell fusion, whereby the test agent is identified as an inhibitor of Henipavirus-induced cell-cell fusion when the cell fusion produced in the system contacted with the test agent is different than the cell fusion produced in a system not contacted by the test agent.

[0066] In another aspect, provided herein is a method for identifying an inhibitor of a Henipavirus infection, which comprises: providing a test agent; contacting a system comprising a first cell and a second cell with the test agent, wherein the first cell comprises a Henipavirus attachment (G) glycoprotein, the second cell comprises EphrinB2, EphrinB3, or a biologically active fragment thereof capable of binding to the Henipavirus attachment (G) glycoprotein of the first cell; and detecting the presence or absence of cell fusion, whereby the test agent is identified as an inhibitor of Henipavirus viral infection when the cell fusion produced in the system contacted with the test agent is different than the cell fusion produced in a system not contacted by the test agent.

[0067] For these methods, the cells can be human cells or pig cells. The viral envelope protein can be exogenously expressed. The viral envelope protein receptor can be exogenously or endogenously expressed. In some embodiments, the cell fusion is detected by the presence or absence of a signal produced by the functional reporter molecule. In one embodiment, the functional reporter molecule is β-galactosidase. The test agent can be a polypeptide, a carbohydrate or a small molecule.

[0068] Further provided herein is a Nipah viral infection inhibitor molecule identified by the methods provided herein.

[0069] In yet another aspect, provided herein is a method for identifying an antiviral agent, which comprises providing a test agent; contacting a Henipavirus attachment (G) glycoprotein with an EphrinB2 polypeptide, a EphrinB3 polypeptide, or a Henipavirus attachment (G) glycoprotein-binding fragment thereof, in the presence or absence of a test agent; and detecting attachment between the Henipavirus attachment (G) glycoprotein with the EphrinB2 polypeptide, with the EphrinB3 polypeptide, or with the Henipavirus attachment (G) glycoprotein-binding fragment of EphrinB2 or EphrinB3, whereby the test agent is identified as an antiviral agent when the attachment in the system contacted with the test agent is different than the attachment produced in a system not contacted by the test agent. The Henipavirus attachment (G) glycoprotein can be present in a Henipavirus or in a pseudo-type virus. The EphrinB2 polypeptide, EphrinB3 polypeptide, or Henipavirus attachment (G) glycoprotein-binding fragment thereof can be present in a host cell. In one embodiment, a decrease in attachment of the Henipavirus attachment (G) glycoprotein to the EphrinB2 polypeptide, EphrinB3 polypeptide, or Henipavirus attachment (G) glycoprotein-protein binding fragment thereof in the presence of the test agent corresponds to a decrease in entry of a Henipavirus into the host cell. The test agent can be a polypeptide, a carbohydrate or a small molecule. In some embodiments, the test agent is a soluble EphrinB2 receptor or a fragment thereof, an antibody specific for Henipavirus attachment (G) glycoprotein, EphrinB2 or EphrinB3, or siRNA. In some embodiments, the test agent is a tyrosine kinase inhibitor or an ion channel inhibitor.

[0070] Also provided herein is a method for reducing the infectivity by Henipavirus of a host cell susceptible to infection by Henipavirus, comprising administering to a subject in need thereof an antiviral agent that inhibits the ability of Henipavirus attachment (G) glycoprotein to attach to EphrinB2 or EphrinB3 and thereby reducing infectivity of the virus. In some embodiments, the method is performed in vivo. The subject can be a human or a pig.

[0071] In one aspect, provided herein is a method for eliciting a protective immune response against Henipavirus, which comprises administering a Henipavirus attachment (G) glycoprotein or a fragment thereof to a subject in need thereof.

[0072] In yet another aspect, provided herein is a method for selectively identifying peptides to elicit a protective immune response for Henipavirus, which comprises obtaining a peptide library of Henipavirus attachment (G) glycoprotein; and identifying the peptide required for Henipavirus attachment (G) glycoprotein attachment to EphrinB2 or EphrinB3, whereby the peptide that mediates Henipavirus attachment (G) glycoprotein attachment to EphrinB2 or EphrinB3 is a peptide that will elicit a protective immune response for Henipavirus.

[0073] For these methods, the cells are from any suitable source including, but not limited to mammalian and avian cells. In some embodiments, the cells are of human or pig origin. The cell can be freshly isolated {i.e., primary) or derived from a short term- or long term- established cell line. Exemplary biological cell lines include pig kidney fibroblast (PKl 3), Raji B cell CEM, HeLa epithelial carcinoma, Chinese hamster ovary (CHO) cell, Vero cell and 293T cell. Such cell lines are described, for example, in the Cell Line Catalog of the American Type Culture Collection (ATCC, Rockville, MD). [0074] The viral envelope protein can be endogenously or exogenously expressed. Likewise, the viral envelope protein receptor can be exogenously or endogenously expressed. Endogenous expression by a cell as provided herein can result from constitutive or induced expression of endogenous genes.

[0075] Exogenous expression by a cell as provided herein can result from the introduction of the nucleic acid sequences encoding a viral envelope protein receptor or viral envelope protein. In some cases, use of a nucleic acid sequence for a viral envelope protein co-receptor may also be employed. Transformation may be achieved using viral vectors, calcium phosphate, DEAE-dextran, electroporation, cationic lipid reagents, or any other convenient technique known in the art. The manner of transformation useful in the present invention are conventional and are exemplified in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, F.M., et al., eds. 2000). Exogenous expression of the viral envelope protein, its receptor, and, when applicable, its co-receptor can be transient, stable, or some combination thereof. Exogenous expression can be enhanced or maximized by co-expression with one or more additional proteins, e.g., NiV-F or HeV-F. Exogenous expression can be achieved using constitutive promoters, e.g., SV40, CMV, and the like, and inducible promoters known in the art. Suitable promoters are those which will function in the cell of interest.

[0076] Also provided herein are vectors or plasmids containing a nucleic acid that encodes for a viral envelope protein receptor, or viral envelope protein. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by a skilled artisan. Additional vectors can also be found, for example, in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, F.M., et al., eds. 2000) and Sambrook et al, "Molecular Cloning: A Laboratory Manual," 2nd ED. (1989).

[0077] The level of expression of the viral envelope protein, its receptor, and when applicable, its co-receptor is that required to mediate the cell fusion event. One of ordinary skill in the art can determine the required level of expression for fusogenic activity using assays routinely employed in the art.

[0078] In some embodiments, the cell fusion is detected by the presence or absence of a signal produced by the functional reporter molecule. In one embodiment, the functional reporter molecule is β-galactosidase or luciferase. The test agent can be a polypeptide, a carbohydrate or a small molecule.

[0079] Any fusogenic event can be detected using suitable methods. See, e.g., Holland et al., Virology 319:343-52 (2004). In one embodiment, fusion between Ephrin-B2 or EphrinB3 expressing cells and viral protein expressing cells is measured using a reporter gene assay in which the cytoplasm of one cell population contains vaccinia virus-encoded T7 RNA polymerase and the cytoplasm of the other contained E.coli LacZ gene linked to the T7 promoter, β-galactosidase is then synthesized and detected only in fused cells. See, e.g., Bossart et al, J. Virol. 76:11186-98 (2002). Any suitable signal generating molecule can be employed including, but not limited to enzymatic, catalytic, and the like, where such signal generating activity can be measured in at least a semi-quantitative fashion. Exemplary molecules include, but are not limited to ribonuclease, staphylcoccal nuclease, DHFR, β- lactamase, ubiquitin, ras-based recruitment systems (RRS and SOS), G-protein signaling, green fluorescent protein (GFP), fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), fusion-protein based systems such as yeast two hybrid method, and the like. See, e.g., Remy, et al, Science 283:990-93 (1999); Remy, et al, Proc. Natl. Acad. ScL USA 96:5394-99 (1999); U.S. Patent Nos.: 6,270,964, 6,294,330 and 6,428,951; Wehrman, et al, Proc. Natl. Acad. ScL USA 99:3469-74 (2002); U.S. Patent Appl'n. No. 20030175836; Johnsson, et al, Proc. Natl. Acad. ScL USA 91 :10340-44 (1994); U.S. Patent Nos.: 5,503,977 and 5,585,245; Aronheim, Methods MoI. Biol. 250:251-62 (2004); Maroun et al, Nucleic Acids Res. 27:e4 (1999); Aronheim, Nucleic Acids Res. 25:3373-74 (1997); Ehrhard et al, Nature Biotechnol. 18:1075-79 (2000); Remy, et al, Methods 32:381- 88 (2004); Pollok et al, Trends Cell Biol 9:57-60 (1999); Adams, et al, Nature 349:694-97 (1991); Fields, et al, Nature 340:245-46 (1989); Ray P. et al, Proc. Natl. Acad. ScL USA 99:3105-10 (1999); Xu et al, Proc. Natl. Acad. Sd USA 96:151-56 (1999); Ayoub etal, J. Biol. Chem. 277:21522-28 (2002); Paulmurugan et al, Cancer Res. 64:2113-19 (2004).

[0080] In one embodiment, the functional reporter molecule is an enzyme whose activity can be monitored by the appearance of a product of the enzymatically catalyzed reaction or by disappearance of the enzyme substrate. In another embodiment, the functional reporter molecule can be detected without addition of exogenous substrate by measurement of some endogenous property {e.g., luminescence, chemiluminescence).

[0081] In embodiments where the functional reporter molecule is an enzyme that converts a substrate to a detectable product, the detection step typically first requires contacting the cell lysate with a substrate for the reporter enzyme. The substrate may be contacted with the lysate using any convenient protocol, e.g., by placing the lysate into a container having the substrate, by introducing the substrate into the lysate, etc. The nature of the particular substrate necessarily depends on the nature of the reporter enzyme which is present in the two fragments. For example, the substrate can be one that is converted by the reporter enzyme into a chromo genie product. Of interest in certain embodiments are substrates that are converted by the enzyme into a fluorescent product. The amount of substrate that is contacted with the lysate may vary, but typically ranges from about 1 femtomolar to 10 millimolar.

[0082] The substrate conversion can be evaluated in whole cells or in lysate depending on the nature of the substrate and the final detectable product as is known in the art.

[0083] In one embodiment, the lysate is evaluated for the presence or absence of detectable product following a predetermined incubation period, where this incubation period typically ranges from about 1 minute to about 2 hours. The particular detection protocol employed varies depending on the nature of the detectable product. For example, where the detectable product is a fluorescent product, the detection protocol employs the use of a fluorescent light detection means, e.g., a fluorescent light scanner, which can scan the lysate for the presence of fluorescent signal. The presence or absence of detectable signal from the signal producing system, e.g., detectable product in the lysate, is then used to derive information as to whether cell fusion occurred. The presence of a signal in the lysate is indicative of cell fusion. The signal can be correlated to the cell fusion event in a qualitative or quantitative manner. One also can employ a threshold value, whereby any signal above the threshold value represents insufficient activity and any signal below the threshold value represents sufficient activity. One also can evaluate the signal in a quantitative or a semi-quantitative manner, in which the amount of signal detected is used as a direct indication of the level of cell fusion events. The amount of signal detected may be linear or non-linear relative to the amount of cell fusion depending on the sensitivity of the reporter molecule and substrate employed. In one embodiment, a larger amount of signal indicates a greater amount of cell fusion, such that the amount of signal has a direct relationship with the amount of cell fusion.

[0084] The above signal evaluation may be accomplished using any convenient means. Thus, the signal maybe subjectively evaluated by comparing the signal to a set of control signals. The evaluation may be done manually or using a computing or data processing means that compares the detected signal with a set of control values to automatically provide a value for the cell fusion activity. Quantified interactions can be expressed in terms of a concentration of signal molecule, test inhibitor molecule (as described in the section below), or protein component required for emission of a signal that is 50% of the maximum signal (IC₅o). Also, quantified interactions can be expressed as a dissociation constant (K_d or Kj) using kinetic methods known in the art. [0085] Test agents or inhibitors can also be identified by assessing binding characteristics. Such assays are well known in the art, e.g., BIAcore analysis. The test agent-protein complex, free substance or non-complexed proteins may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof. To facilitate the assay of the components, antibody against the Ephrin or Eph receptor or the substance, or labeled Ephrin or Eph receptor, or a labeled substance may be utilized. The antibodies, proteins, or test agents may be labeled with a detectable substance using conventional methods.

[0086] Further provided herein is a Henipaviral infection inhibitor molecule identified by the methods provided herein.

[0087] Inhibitors are those molecules that reduce or eliminate the viral-cell fusion event, cell-cell fusion events, or otherwise inhibit infectivity of the Henipavirus. Such inhibition can occur through direct binding of one or more critical binding residues of a viral envelope protein, its receptor, or when applicable, its co-receptor or through indirect interference including steric hindrance, enzymatic alteration of the fusogenic proteins {i.e., viral envelope protein and its complementary receptors and co-receptors (if applicable)), and the like. As used herein, the term "inhibitor" or "'antiviral agent" includes both protein and non-protein moieties. In one embodiment, the inhibitor or agent is a small molecule. In another embodiment, the inhibitor or agent is a protein.

[0088] A variety of different test agents may be identified using the method as provided herein. Test agents can encompass numerous chemical classes. In certain embodiments, they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Test cell agents can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and may include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The test agents can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Test agents also include biomolecules like peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Test cell fusion inhibitory molecules of interest also can include peptide and protein agents, such as antibodies or binding fragments or mimetics thereof, e.g., Fv, F(ab')₂ and Fab. [0089] Test agents can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological, agents maybe subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidifϊcation, etc. to produce structural analogs.

[0090] Exemplary compounds useful in the present invention include, but are not limited to the compounds of soluble EphrinB2, EphrinB3, ephB4, NiV-G, HeV-G or fragments thereof, antibodies specific for NiV-G, HeV-G, EphrinB3 or EphrinB2, antisense nucleic acids, siRNA, or small molecule inhibitors of attachment or cell signaling cascades. Other exemplary inhibitory compounds include 5-{[5-(4-nitrophenyl)-2-furyl]methylene}-3-(2-phenylethyl)-2- thioxo-l,3-thiazolidin-4-one (C22 H16 N2 O4 S2), and 3-allyl-5-{[5-(3-bromophenyl)-2- furyl]methylene}-2-thioxo-l,3-thiazolidin-4-one (C17 H12 Br N 02 S2).

[0091] In one embodiment, the test agents or inhibitors are soluble Ephrin and Eph proteins as well as soluble NiV-G or HeV-G. Soluble proteins can be prepared any suitable methods including, but not limited to conventional methodologies such as heterologous fusion proteins GST fusion proteins, MBP, His, ThioHis, Fc, Myc tag, HA tag, or other epitopes or domains that allow for purification procedures to be utilized while retaining the biological activity of the purified protein. In some cases, the fusion domains can be removed by the inclusion of a proteolytic cleavage site between the fusion partner and the soluble polypeptide.

[0092] In another embodiment, the test agent or inhibitor is an antibody or a biologically active fragment thereof. The antibody may be one that modulates the biological activity in a way that reduces or inhibits entry of the virus into the cell. While not being limited to any particular theory of action, the antibody may block NiV-G or HeV-G attachment to EphrinB2 or EphrinB3 or interfere with the downstream signaling that elicits the fusion event. Conventional methods can be used to prepare the antibodies. The antibodies can be generated in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes. Therefore, the antibody useful in the present methods is typically a mammalian antibody. Phage techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are routine and well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, a recombinant antibody can be produced by transfecting a host cell with a vector comprising a DNA sequence encoding the antibody. One or more vectors can be used to transfect the DNA sequence expressing at least one V_L and one V_H region in the host cell. Exemplary descriptions of recombinant means of antibody generation and production include Delves, ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997); Shephard, et al, MONOCLONAL ANTIBODIES (Oxford University Press, 2000); and Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (Academic Press, 1993).

[0093] In another embodiment, the inhibitory compound is an siRNA molecule. RNA interference or "RNAi" refers to a selective intracellular degradation of RNA. RNA interference is a post-transcriptional, targeted gene-silencing technique that uses double- stranded RNA (dsRNA) to degrade messenger RNA (mRNA) containing the same sequence as the dsRNA. The process occurs when an endogenous ribonuclease cleaves the longer dsRNA into shorter, 21- or 22-nucleotide-long RNAs, termed small interfering RNAs or siRNAs. The smaller RNA segments then mediate the degradation of the target mRNA. Kits for synthesis of RNAi are commercially available. See, e.g., U.S. Application No. 20040203145. Thus, RNAi directed to the expression of EphrinB2, EphrinB3 or any critical upstream or downstream effector for EphrinB2 or B3 expression or function, particularly those related to fusion events are contemplated.

[0094] In some embodiments, the inhibitory compound is a peptide or peptidomemtic. Exemplary peptides include soluble extracelluar domains of EphrinB2, EphrinB3, or ephB4, or soluble Henipavirus-G. Methods of making peptidomimetics based upon a known sequence are known in the art. See, e.g. U.S. Patent Nos. 5,631,280; 5,612,895; and 5,579,250. Use of peptidomimetics can involve the incorporation of a non-amino acid residue with non-amide linkages at a given position. One embodiment of the present invention is a peptidomimetic wherein the compound has a bond, a peptide backbone or an amino acid component replaced with a suitable mimic. Examples of unnatural amino acids which may be suitable amino acid mimics include but are not limited to β-alanine, L-α-amino butyric acid, L-γ-amino butyric acid, L-α-amino isobutyric acid, L-ε-amino caproic acid, 7-amino heptanoic acid, L-aspartic acid, L-glutamic acid, N-ε-Boc-N-α-CBZ-L-lysine, N-ε -Boc-N-α-Fmoc-L-lysine, L- methionine sulfone, L-norleucine, L-norvaline, N-α-Boc-N-δ-CBZ-L-ornithine, N-δ-Boc-N-α- CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, and Boc-L-thioproline.

[0095] Sometimes the inhibitor compounds are antisense nucleic acids. The term "antisense oligonucleotide" as used herein means a nucleotide sequence that is complementary to its target. For example, EphrinB2 or EphrinB3 antisense can be created and introduced into a cell using routine methods and as disclosed herein. See, e.g., Lichtenstein et ah, ANTISENSE TECHNOLOGY: A PRACTICAL APPROACH (Oxford University Press 1998). The term "oligonucleotide" refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted oligomers comprising non-naturally occurring monomers or portions thereof, which function similarly. Such modified or substituted oligonucleotides may be preferred over naturally occurring forms because of properties such as enhanced cellular uptake, or increased stability in the presence of nucleases. The term also includes chimeric oligonucleotides which contain two or more chemically distinct regions. For example, chimeric oligonucleotides may contain at least one region of modified nucleotides that confer beneficial properties (e.g. increased nuclease resistance, increased uptake into cells), or two or more oligonucleotides of the invention may be joined to form a chimeric oligonucleotide.

[0096] The antisense oligonucleotides of the present invention may be ribonucleic or deoxyribonucleic acids and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The oligonucleotides may also contain modified bases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4- thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5- trifiuoro cytosine.

[0097] Other antisense oligonucleotides of the invention may contain modified phosphorous, oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. For example, the antisense oligonucleotides may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates. In an embodiment of the invention there are phosphorothioate bonds links between the four to six 3 '-terminus bases. In another embodiment phosphorothioate bonds link all the nucleotides.

[0098] The antisense oligonucleotides of the invention may also comprise nucleotide analogs that may be better suited as therapeutic or experimental reagents. An example of an oligonucleotide analogue is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides. See, e.g., Nielsen, et ah, Science 254:1497 (1991). Other oligonucleotides may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones. For example, the nucleotides may have morpholino backbone structures. See, e.g., U.S. Pat. No. 5,034,506. Oligonucleotides may also contain groups such as reporter groups, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an antisense oligonucleotide. Antisense oligonucleotides may also have sugar mimetics.

[0099] The antisense oligonucleotides may be introduced into tissues or cells using techniques in the art including vectors (retroviral vectors, adenoviral vectors and DNA virus vectors) or physical techniques such as microinjection. The antisense oligonucleotides may be directly administered in vivo or may be used to transfect cells in vitro which are then administered in vivo. In one embodiment, the antisense oligonucleotide may be delivered to macrophages and/or endothelial cells in a liposome formulation.

[0100] The polypeptides and peptides of the invention, and polypeptides and peptides used in the compositions and methods of the invention, can comprise "mimetic" and "peptidomimetic" forms. The terms "mimetic" and "peptidomimetic" refer to a synthetic chemical compound which has substantially the same structural and/or functional characteristics of the polypeptides of the invention. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic' s structure and/or activity. As with polypeptides of the invention which are conservative variants or members of a genus of polypeptides of the invention {e.g., having about 50% or more sequence identity to an exemplary sequence of the invention), routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered. Thus, in one aspect, a peptidomimetic composition is within the scope of the invention if it has binding activity sufficient to interfere with viral attachment and/or cell fusion.

[0101] Various pharmaceutical compositions and techniques for their preparation and use will be known to those of skill in the art in light of the present disclosure. For a detailed listing of suitable pharmacological compositions and associated administrative techniques one may refer to the detailed teachings herein, which may be further supplemented by texts such as REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY 20th Ed. (Lippincott, Williams & Wilkins 2003).

[0102] Pharmaceutically-acceptable materials, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0103] A peptide library of Henipavirus-G (i.e. HeV-G, NiV-G) can employed to identify potential candidates for vaccines to prevent or treat viral infection using conventional methods. See, e.g., EPITOPE MAPPING: A PRACTICAL APPROACH (Westwood et ah, eds. Oxford University Press 2001).

[0104] Kits comprising the compositions of the invention are also provided. Methods of using inhibitors to prevent or treat viral infection

[0105] In yet another aspect, provided herein is a method for inhibiting or preventing binding of a Henipavirus to a cell, wherein the method comprises contacting the cell with a composition as set forth above, thereby inhibiting or preventing binding of the Henipavirus to a cell. The cell can be a human or a pig cell. The cell can be contacted with the pharmaceutical composition in vivo, in vitro or ex vivo.

[0106] Further provided herein is a method for inhibiting or preventing binding of a Henipavirus to a cell, wherein the method comprises contacting the cell with a composition comprising a polypeptide comprising a soluble EphrinB2 or EphrinB3 or a Nipah Virus attachment (G) glycoprotein-binding fragment thereof, or soluble ephB4, thereby inhibiting or preventing binding of the Henipavirus to a cell.

[0107] Also provided herein is a method for inhibiting or preventing infection of a cell by a Henipavirus, wherein the method comprises contacting the cell with a composition comprising a polypeptide comprising a soluble EphrinB2 or EphrinB3 or a Nipah Virus attachment (G) glycoprotein-binding fragment thereof, or soluble ephB4,, thereby inhibiting or preventing binding of the Henipavirus to the cell and inhibiting or preventing infection of the cell by the Henipavirus.

[0108] In one aspect, provided herein is a method for inhibiting or preventing binding of a Henipavirus to a cell, wherein the method comprises contacting the cell with a composition comprising a Henipavirus attachment (G) glycoprotein-binding antibody or binding fragment thereof, thereby inhibiting or preventing binding of the Henipavirus to a cell.

[0109] Also provided herein is a method for reducing the infectivity by Henipavirus of a host cell susceptible to infection by Henipavirus, comprising administering to a subject in need thereof an antiviral agent that inhibits the ability of Henipavirus attachment (G) glycoprotein to attach to EphrinB2 or EphrinB3 and thereby reducing infectivity of the virus, hi some embodiments, the method is performed in vivo. The subject can be a human or a pig.

[0110] In one aspect, provided herein is a method for eliciting a protective immune response against Henipavirus, which comprises administering NiV-G, HeV-G or a fragment thereof to a subject in need thereof.

[0111] The subject treated by the present methods includes a subject having or being at risk of Henipavirus viral infection.

[0112] Exemplary compounds useful in the present invention include, but are not limited to compounds identified by the methods disclosed herein. Such compounds include soluble EphrinB2, EphrinB3, ephB4, NiV-G, HeV-G, or fragments thereof, antibodies specific for NiV-G, HeV-G, EphrinB3 or EphrinB2, antisense nucleic acids, siRNA, or small molecule inhibitors of attachment or cell signaling cascades. Other exemplary inhibitory compounds include 5- {[5-(4-nitrophenyl)-2-furyl]methylene}-3-(2-phenylethyl)-2-thioxo-l ,3-thiazolidin- 4-one (C22 H16 N2 04 S2), and 3-allyl-5-{[5-(3-bromophenyl)-2-furyl]methylene}-2-thioxo- l,3-thiazolidin-4-one (C17 H12 Br N O2 S2). The formulation and delivery methods will generally be adapted according to the site and the subject to be treated. Exemplary formulations include, but are not limited to, those suitable for parenteral administration, e.g., intravenous, intraarterial, intramuscular, or subcutaneous administration, including formulations encapsulated in micelles, liposomes or drug-release capsules (active agents incorporated within a biocompatible coating designed for slow-release); ingestible formulations; formulations for topical use, such as creams, ointments and gels; and other formulations such as inhalants, aerosols and sprays. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. The dosage of the compounds of the invention will vary according to the extent and severity of the need for treatment, the activity of the administered composition, the general health of the subject, and other considerations well known to the skilled artisan.

[0113] In some embodiments, the compositions of the invention are administered in a vaccine formulation adequate to stimulate a protective immune response. The compositions can be administered alone or in combination with one or more agents to enhance immunogenicity including but not limited to adjuvants and cytokines. See, e.g., VACCINES (Plotkin et al, eds. W.B. Saunders & Co. 2003).

[0114] The following examples are offered to illustrate but not to limit the invention.

Example 1 EphrinB2 is a receptor for NiV

[0115] Nipah virus (NiV) is an emergent paramyxovirus that causes fatal encephalitis in up to 70% of infected patients (Hsu, et al, (2004) Emerg Infect Dis 10, 2082-7), and there is evidence of human-to-human transmission (ICDDRB, (2004) HMh Sci Bull 2, 5-9). Endothelial syncytia is frequently found in NiV infections, and is mediated by the fusion (F) and attachment (G) envelope glycoproteins. Identification of the NiV receptor will shed light on the pathobiology of NiV infection, and spur the rational development of effective therapeutics. Here, the data demonstrate that EphrinB2, the membrane bound ligand for the ephB class of receptor tyrosine kinases (RTKs) (Poliakov, et al, (2004) Dev Cell 7, 465-80), specifically bound to the attachment (G) glycoprotein of NiV. Soluble Fc- fusion proteins of EphrinB2 but not EphrinBl effectively blocked NiV fusion and entry. Transfection of EphrinB2 into non-permissive cells rendered them permissive for NiV fusion and entry. EphrinB2 is expressed on endothelial cells and neurons (Poliakov, et al., (2004) Dev Cell 1, 465-80; Palmer & Klein, (2003) Genes Dev 17, 1429-50) consistent with the known cellular tropism for NiV (Wong, et al, (2002) Am J Pathol 161, 2153-67). Significantly, NiV envelope mediated infection of microvascular endothelial cells, and primary cortical rat neurons, was inhibited by soluble EphrinB2, but not the related EphrinBl protein. Cumulatively, the data showed that EphrinB2 was a functional receptor for NiV.

Cells and reagents

[0116] Primary rat cortical neurons were dissected and cultured from embryonic day 17 Sprague-Dawley rats as described (Estus, et al., (1997) JNeurosci 17, 7736-45), and plated 2 weeks prior to infection. Human microvascular endothelial cells (MVECs) immortalized with the human telomerase catalytic protein (hTERT) (Shao & Guo, (2004) Biochem Biophys Res Commun 321, 788-94) were also used. Soluble Fc-fusion proteins of ephrins and eph receptors were obtained from R&D Systems (Minneapolis, MN). Sequence-verified human EphrinB2 clones were obtained from Origene (CMV-driven) (Rockville, MD) and Open Biosystems (T7- driven) (Huntsville, AL). The open-reading frame of human EphrinB2 was also subcloned into pcDNA3 (Invitrogen, Carlsbad, CA) in frame with a C-terminal V5 epitope tag.

Immunoprecipitation and identification of EphrinB2

[0117] Production of Niv-G-Fc and sNiV-G-HA: NiV-G-Fc, sNiV-G-HA and 28 NiV-G-Fc DNA plasmids were transfected into 293T cells using 293fectin (Invitrogen, Carlsbad, CA) and serum-free supernatants were collected at two and four days post transfection. Production and normalization of NiV-G-Fc was measured by an Fc-specific ELISA. Briefly, biotinylated mouse monoclonal anti-human IgGl Fc (Caltag, Burlingame, CA) was captured onto pre-bound NeutraAvidin™ coated polystyrene plates (Pierce, Rockford, IL). 100 μl of unconcentrated supernatant from NiV-G-Fc or 28 NiV-G-Fc transfections were added to each well, and subsequently detected with HRP-conjugated polycolonal goat anti-human Fc antibodies (Pierce) using TMB substrate (Pierce). Concentrations of Fc-fusion proteins were calculated based on standards using purified human IgG. sMV-G-HA was detected by western blot using a HRP-conjugated anti-HA monoclonal antibody (Novus Biologicals, Littleton, CO) and the ECL plus chemiluminescent detection system (Amersham Biosciences, Piscataway, NJ).

[0118] 293T, Vero or CHO-pgsA745 cells were cell surface biotinylated using EZ-link Sulfo-NHS-LC-LC- Biotin reagent (Pierce, Rockford, IL). Each 100mm dish of cells were lysed (5OmM Tris-HCl, 15OmM NaCl, and 1% Triton X-100 pH 8.0 with protease inhibitors), clarified by centrifugation, and pre-cleared by one round of mock immunoprecipitation with Fc-only protein using protein G coupled magnetic beads (Dynal, Brown Deer, WI). Pre-cleared lysates were immunoprecipitated by NiV-G-Fc or Δ28 NiVG-Fc previously crosslinked to Dynal protein G beads (2OmM dimethyl-pimelimidate HCL in 0.2M triethanolamine) (Sigma St. Louis, MO), separated by non-denaturing SDS-PAGE, and analyzed by western blotting with HRP-conjugated streptavidin or anti-Human Fc (Pierce). 3x10⁷ 293T cells were used for preparative immunoprecipitation, and proteins visualized by Silver Stain Plus™ (Biorad, Hercules, CA). Parallel portions of the gel containing a specific band, immunoprecipitated by NiV-G-Fc, but not Δ28NiV-G-Fc, were excised, digested in-gel with sequencing grade trypsin, and subjected for peptide sequencing by tandem mass spectrometry. A Finnigan Ion Trap Mass Spectrometer LCQ coupled with a HPLC system running a 75 μM ID Cl 8 column was used. MS/MS spectra were used to search the most recent non-redundant protein database from GeneBank with the ProtQuest software suite (ProtTech, Inc. Norristown, PA).

Fusion assay

[0119] The fusion was performed essentially as described (Rucker, et ah, (1997) Methods Enzymol 288, 118-33; Bossart & Broder, (2004) Methods MoI Biol 269, 309-32). Briefly, effector cells (PKl 3) were transfected with 0.3 μg of codon-optimized NiV-F and G expression plasmids, 0.6 μg of T7-luciferase, and 0.8 μg of pcDNA3 per 6 well using Lipofectamine 2000 reagent (Invitrogen). The sequences of the codon-optimized genes have been deposited into GenBank (AY816748 and AY816746 for F and G, respectively based on the original sequences described for NiV-F and -G in Chua, et ah, (2000) Science 288, 1432- 5). The DNA amount was always kept constant with pDNA3. Target cells (293T, Vero, HMVECs) plated in a 24-well were infected with vTFl.l expressing T7 -polymerase (MOI of 5), and cultured overnight in DMEM/10% FCS. Rifampicin was added to reduce cytopathicity. 100,000 effector cells were mixed with target cells in a total volume of 250 μl, allowed to fuse for 6 hours, and then lysed with 180μl of lysis buffer (2OmM Tris pH 7.5, 10OmM NH₄SO₄, 0.1% BSA, 0.75% Triton X, and 0.001% sodium azide). Luciferase activity was detected by adding equal volumes (100 μl) of luciferase detection reagent (Promega E4030) and lysate, and the relative light units (RLU) were determined by luminometry (Turner Biosystems 998-9100). When Raji B cells were used as target cells, 2.5x10⁶ cells were transfected with 6μg of the indicated plasmid using Amaxa transfection in buffer V or T on program A23. These suspension target cells were added onto the adherent PKl 3 effector target cells prepared as described. Fusion was analyzed as above.

Binding of soluble NiV-G to EphrinB2

[0120] 3μg/ml of EphrinBl-Fc and EphrinB2-Fc diluted in ELISA buffer (2% BSA and 0.05% Tween-20 in TBS) were captured by biotinylated anti-human Fc (Caltag, Burlingame, CA) pre-bound to NeutrAvidin™ coated polystyrene plates (Pierce). Supernatant from sNiV- G-HA- or Δ28sNiV-G-HA-transfected 293T cells was added to each well, and detected with an HRP-conjugated anti-HA antibody (Novus biologicals, Littleton, CO) using TMB substrate (Pierce).

Infection assay

[0121] The VSV-ΔG-RFP virus is a recombinant VSV derived from a full-length cDNA clone of the VSV Indiana serotype in which the G-protein gene has been replaced with the red fluorescent protein (RFP) gene (Takada, et al., (1997) Proc Natl Acad Sci USA 94, 14764-9; a kind gift from Mike Whitt at GTx, Inc). Either VSV-G or NiV-F/G was provided in trans. NiV-F/G and VSV-G pseudotypes were purified via pelleting through a sucrose cushion and used to infect 293T, Vero, CHO-pgsA745, rat cortical neurons and HMVECs (MOI of 1 as titered on 293T cells). RFP production at 24 hours was analyzed by fluorescent microscopy or FACS.

Neutralization sera

[0122] NiV: NZW rabbits were genetically immunized with a mixture of codon-optimized NiV-M (matrix), NiV-F and NiV-G expression plasmids (Aldevron, Inc., Fargo, ND) using an electroporation protocol that results in increased antibody titers (Tollefsen, et al, (2003) Scand J Immunol 57, 229-38). A 1 :100 dilution of hyperimmune sera from the terminal bleed was used for neutralization studies. VSV: a VSV-G specific mouse monoclonal antibody (clone 8G5F II) was used. Pseudotyped viruses were pre-incubated with antibodies for 1 hour prior to use for infection.

[0123] To establish if NiV-G determines the known cell line tropism of NiV, an immunoadhesin, fusing the ectodomain of NiV-G with the Fc region of human IgGl was generated (Fig. 6). This NiV-G-Fc bound to fusion-permissive 293T, HeLa, and Vero cells (Bossart, et al, (2002) J Virol 16, 11186-98; Guillaume, et al., (2004) J Virol 78, 834-40), but not to non-permissive Chinese hamster ovary (CHO-pgsA745), pig kidney fibroblast (PKl 3) ((Bossart, et al., (2002) J Virol 76, 11186-98), and human Raji B cells (Fig.1 a). NiV-G-Fc immunoprecipitated a 48kDa band from the surfaces of permissive 293T and Vero but not non- permissive CHO-pgsA745 cells (Fig. Ib). Deletion analysis identified a deletion of 28 amino acids in the globular ectodomain of NiV-G that is produced as a Fc-fusion dimer at wild-type levels, but that no longer binds to the surfaces of permissive cells (Fig. Ib and Table I below). This deletion mutant was used as a negative control in preparative immunoprecipitation experiments to purify the putative NiV receptor. Parallel portions of the gel containing the 48kDa band, immunoprecipitated by NiV-G-Fc, but not Δ28NiV-G-Fc, were analyzed by trypsin digest and mass spectrometry. Only one transmembrane protein was uniquely identified in the NiV-G-Fc sample. Two independent tryptic fragments of 12 and 17 amino acids each identified the protein as EphrinB2 (Figure 5).

Table ϊ

US2006/023618

[0124] Table I above shows a deletion analysis of NiV-G. Based on the predicted folding patterns of the extracellular domain of Hendra G protein (Yu et al., Virology (1998) 251, 227-33), systematic deletions were made to the ectodomain of Nipah G to remove predicted units of secondary structures. The indicated amino acid residues were deleted using the QuickChange™ site-directed mutagenesis kit according to the manufacturer's directions (Stratagene, San Diego, CA). Protein production was measured by an Fc ELISA as described herein and values that were greater than 0.1 μg/ml of unconcentrated supernatant were considered positive. Each mutant was also analyzed for its ability to bind 293T in a flow cytometry assay. Deletion number 11 (residues 437-464) produced protein comparable to wild type NiV-G-Fc and did not bind to 293T cells. This construct, named 28 NiV-G-Fc, was chosen to serve as a negative control for immunoprecipitation experiments.

[0125] EphrinB2 is essential for vasculogenesis and axonal guidance, and is expressed on endothelial cells, neurons and smooth muscle cells surrounding small arteries/arterioles (Gale, et al., (2001) Dev Biol 230, 151-60; Shin, et al., (2001) Dev Biol 230, 139-50), an expression pattern highly concordant with the known cellular tropism of NiV (Wong, et ah, (2002) Am J Pathol 161, 2153-67). Using a soluble HA-tagged ectodomain of NiV-G (sNiV-G-HA), NiV- G bound was shown to bind directly to soluble EphrinB2-Fc, but not to EphrinBl-Fc in an ELISA based assay (Fig. 2a, Fig. 6). EphrinBl is the most closely related ephrin to EphrinB2. Additionally, EphrinB2-Fc, but not EphrinBl -Fc, competed readily for sNiV-G-HA binding on permissive 293T cells (Fig. 2b). Lastly, NiV-G-Fc bound to EphrinB2-transfected, but not to pcDNA3-transfected CHO-pgsA745 and human Raji B cells (Fig. 2c). Cumulatively, these data demonstrated a direct and specific association between NiV-G and EphrinB2.

[0126] Since endothelial syncytia are a hallmark of NiV disease (Wong, et al., (2002) Am J Pathol 161, 2153-67), the requirement for EphrinB2 for NiV envelope mediated syncytia formation was investigated. A T7 -polymerase driven, luciferase-reporter based fusion assay that has been used extensively to examine viral envelope mediated cell-cell fusion was used (Rucker, et al, (1997) Methods Enzymol 288, 118-33; Bossart & Broder, (2004) Methods MoI Biol 269, 309-32). Fig. 3a shows that NiV-F/G mediated fusion with permissive 293T or Vero, but not to non-permissive PKl 3 or human Raji B cells. No fusion was seen in the absence of NiV-G. Again, soluble EphrinB2, but not EphrinBl, significantly inhibited NiV-F/G mediated cell-cell fusion (Fig. 3b). Transfection of EphrinB2, but not EphrinBl or GFP, into human Raji B cells rendered them permissive for NiV envelope mediated fusion (Fig.3c). This fusion was inhibited by soluble EphrinB2 and ephB4, a cognate receptor for EphrinB2, but not EphrinBl (Fig. 3d). Significantly, NiV-F/G-expressing cells also fused with human microvascular endothelial cells (Fig.3e) in a manner inhibitable by soluble EphrinB2, and ephB4, but not EphrinBl . Thus, NiV fusion on cell lines, and on an in vivo target cell for NiV infection, was dependent on EphrinB2.

[0127] The ability of EphrinB2 to mediate NiV infection was then determined. Since NiV is a BSL-4 pathogen, a virion based infection assay that does not require the use of a BSL4 facility was developed. Heterologous viral envelopes can be pseudotyped onto a recombinant vesicular stomatitis virus (VSV) expressing red fluorescent protein (RFP), but lacking its own envelope (VSV-ΔG-RFP) (Takada, et al, (1997) Proc Natl Acad Sd USA 94, 14764-9). VSV-ΔG -RFP bearing NiV-F/G was used to infect permissive 293T or Vero cells, resulting in cells expressing RFP (Fig.4a-b). Viral entry was dependent on NiV-F/G as it was neutralized by NiV-F/G specific anti-serum (Fig.4a). VSV-F/G-RFP infection was blocked by EphrinB2- Fc but not EphrinBl -Fc, while infection by VSV-RFP bearing its own envelope (VSV-G) was not inhibited by either soluble ephrins (Fig.4b). Transfection of EphrinB2 into non-permissive CHO-pgsA745 cells rendered them permissive for viral entry (Fig. 4c). CHO-pgsA745 is a mutant CHO cell line that does not express cell surface heparan sulfate proteoglycans (Esko, et al., (1985) Proc Natl Acad Sd USA 82, 3197-201). Heparan sulfate has been described as an attachment or entry receptor for many viruses, and may confound the search for bonafide viral receptors that mediate membrane fusion (Liu & Thorp, (2002) Med Res Rev 22, 1-25). Thus, the observation that EphrinB2, in the absence of cell surface heparan sulfates, could mediate viral entry, strongly suggests that EphrinB2 was a functional receptor for NiV entry. Finally, NiV-F/G pseudotyped VSV was also able to infect primary cortical rat neurons and human microvascular endothelial cells (MVECs) (Fig.4d-e), two cell types that are infected in vivo (Wong, et al., (2002) Am J Pathol 161, 2153-67). NiV-F/G infection of rat neurons and MVECs was inhibited by soluble EphrinB2, but not EphrinBl . EphrinB2 inhibited NIV-F/G pseudotype infection of primary rat neurons by 76% compared to EphrinBl (average # of infected cells/field +/- S.D.: 5.7 +/-4.3 vs 23.5 +/- 12.7 for EphrinB2 vs EphrinBl inhibition, respectively; p<0.0001, Students' t test). Additionally, soluble ephB4 and ephB2, cognate receptors for EphrinB2, were shown to significantly inhibit NiV-F/G mediated infection of MVECs (Fig. 4e). The use of MVECs and primary rat neurons to show that NiV envelope mediated entry occurred in an EphrinB2 dependent manner strongly suggested EphrinB2 is a functional receptor for NiV in vivo.

[0128] In histopathological studies on patients who had succumbed to NiV infection, viral antigen can be detected in unequivocal amounts in relatively few cellular subtypes such as neurons, endothelial cells and smooth muscle cells surrounding small arteries (Wong, et al, (2002) Am J Pathol 161, 2153-67). This is in remarkable concordance with EphrinB2's expression pattern; in lacZ knock-in mice, EphrinB2 was specifically expressed in endothelial cells, neurons and in smooth muscle cells surrounding arterioles (Gale, et al, (2001) Dev Biol 230, 151-60; Shin, et al, (2001) Dev Biol 230, 139-50). The identification of EphrinB2 as the NiV receptor largely explains the in vivo tropism of the virus. EphrinB2 is a critical gene involved in embryogenic development, and has established roles in vasculogenesis and axonal guidance (Poliakov, et al., (2004) Dev Cell 7, 465-80; Palmer & Klein, (2003) Genes Dev 17, 1429-50). Ephrin genes are highly conserved and have been found in all animal species examined (Poliakov, et al, (2004) Dev Cell 1, 465-80). Thus, the conservation of EphrinB2 may also explain the unusually broad tropism of NiV.

[0129] Both EphrinB2 and its cognate receptor ephB4 have tyrosine signaling and PDZ binding motifs in their cytoplasmic domain (Kullander & Klein, (2002) Nat Rev MoI Cell Biol 3, 475-86). "Forward" signaling mediated by ephB4 mediates anti-adhesive and repulsive behavior upon contact with EphrinB2 expressing cells, while EphrinB2 "reverse" signaling mediates propulsive adhesion upon contact with ephB4 expressing cells. IfNiV-G acts like ephB4 and binds to EphrinB2 but lacks reverse signaling, perhaps only forward propulsion will ensue. This might act to recruit more endothelial cells to areas of NiV replication. Indeed, signaling deficient-ephB4 on tumor cells can promote invasion by EphrinB2-expressing endothelial cells (Noren, et al, (2004) Proc Natl Acad Sci USA 101, 5583-8). It will be interesting to re-examine pathological specimens for increased angiogenesis in areas of NiV replication. It is also possible that PDZ binding domains and other proteins known to interact with the cytoplasmic domain of EphrinB2 may play a role in the productive entry of NiV.

[0130] Hendra virus (HeV) appears to have a similar cellular tropism as NiV (Bossart, et al, (2002) J Virol 76, 11186-98), although NiV appears to be more pathogenic. The recent and repeated outbreaks of NiV in Bangladesh (Hsu, et al, (2004) Emerg Infect Dis 10, 2082- 7), underscores the search for vaccines and therapeutics against this emerging pathogen. Identifying the NiV receptor will contribute to these on-going efforts. Example 2 EphrinB3 is a receptor for NiV

[0131] EphrinB2 belongs to a large family of related molecules that are variably conserved in structure and function. Therefore, all known ephrins were screened. It was found that a closely related molecule, EphrinB3, also can function as an entry receptor for Nipah virus. Expression of human EplirinB3 in nonpermissive CHO-pgsA745 cells rendered them permissive to NiV entry and infection. While EphrinB2 was better used than EphrinB3 as an entry receptor, the same two critical amino acids in EphrinB2 and B3 were responsible for the viral receptor activity of these molecules.

Cells and culture conditions

[0132] CHO-pgsA745 is a mutant cell line derived from CHO cells that lack the endogenous expression of heparin sulfate proteogylcans (Huynh-Do, et ai, (2002) J Cell Sci 115, 3073-3081). CHO-pgsA745 cells and Vero (African green monkey kidney fibroblasts) cells were maintained in DMEM/F12 and α-MEM (Invitrogen, Carlsbad, California, United States), respectively, and both were supplemented with 10% fetal bovine serum (Omega Scientific, Tarzana, California, United States) and the antibiotics penicillin and streptomycin. CHO-pgsA745 cells expressing either wild-type or mutant ephrins were made by selecting for neomycin resistance with 0.5 mg/ml of G418 after transfection. Once selected, the ephrin- expressing populations were enriched using the magnetic bead selection (Miltenyi Biotech, Auburn, California, United States). Briefly, EphB3-Fc (R & D Systems, Minneapolis, Minnesota, United States) was coupled to protein G microbeads (Miltenyi Biotech), and then 23106 ephrin-expressing CHO-pgsA745 cells were added. Then, the cell-bead mixture was poured over a MACS MS column (Miltenyi Biotech), followed by positive cells elution.

Plasmids and reagents

[0133] Soluble Fc-fusion ephrin proteins (ephriiiAl-A5 and EphrinBl-B3) and Eph proteins (EphA2-Fc and EphB3-Fc) were purchased from R & D Systems. Human EphrinB2 and EphrinB3 plasmids were purchased from GeneCopoeia (Germantown, Maryland, United States), and human EphrinBl was obtained from Open Biosystems (Huntsville, Alabama, Unites States). Each ephrin open reading frame was subcloned into the pcDNA3.1 vector (Invitrogen) under CMV promoter-driven expression. In-house ephrin-Fc fusion constructs were made by subcloning the ectodomain of each ephrin into the pCR3-Fc vector, which contains the CH2 and CH3 domains of human IgGl. Mutations in both the full-length (pcDNA3.1 clones) and soluble (pCR3-Fc clones) (Negrete, et al, (2005) Nature 436, 401- 405; de Parseval, et al, (2004) J Virol 78, 2597-2600) were made using the QuikChange (Stratagene, La Jolla, California, United States) site-directed mutagenesis kit. All subclones and mutations were confirmed by sequencing.

Binding of soluble ephrins to NiV-G

[0134] Supernatant from NiV-GHA-transfected 293T cells was used to coat MaxiSorp high proteinbinding 96-well plates (NaIg Nunc International, Rochester, New York, United States) overnight at 4°C. The NiV-G-HA-coated plates were then blocked with 5% bovine serum albumin (BSA) in Trisbuffer saline (TBS) for 2 h at 37°C. The plates were rinsed with wash buffer (1% BSA, 0.05% Tween-20 in TBS), and ephrin-Fc proteins, diluted in wash buffer, were placed in each well to bind soluble NiV-G for 1 h at room temperature. The plates were washed three times with wash buffer and incubated with antihuman Fc monoclonal antibody conjugated with HRP for 30 min at room temperature. The plates were then washed three more times, and the amount of bound ephrin was assessed with 1-step Ultra TMB substrate (Pierce, Rockford, Illinois, United States). The colorimetric reading was performed on a spectrophotometer (Dynex Technologies, Chantilly, Virginia, United States). For each soluble ephrin-Fc, each experiment was performed three times, each time in triplicates.

Cell surface binding assays

[0135] The EphrinB2- and B3-exρressing CHO-pgsA745 cells (CHO-B2 and CHO-B3, respectively) were made as described above. Increasing amounts of the NiV-G-Fc were incubated with CHO-B2 or CHO-B3 cells for 1 h on ice. Then, the cells were washed with buffer and incubated with R-phycoerythrinconjugated anti-Fc antibodies for 30 min on ice. The cells were washed again and fixed with 2% paraformaldehyde, and the data were collected using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, New Jersey, United States). The data were analyzed using FCS Express V2 (DeNovo Software, Thornhill, Ontario, Canada). The cell surface K_d values were calculated using the GraphPad Prism software (San Diego, California, United States) by normalizing the highest mean fluorescent intensity value obtained to 100%. Results NiV-G Binds EphήnB3 at Lower Affinity than EphrinB2

[0136] Ephrins constitute a highly conserved class of proteins with many homologous members. Thus, we examined if any ephrins, other than EphrinB2, can bind similarly to NiV- G. Using an enzyme-linked immunosorbent assay (ELISA), the ability of soluble HA-tagged ectodomain of NiV-G (NiV-G-HA) to bind to all known ephrins (ephrinAl- A5 and EphrinBl-B3) was screened. It was found that EphrinB3-Fc, in addition to EphrinB2-Fc, bound to NiV-G-HA (Fig. 7a). To confirm these results with cell surface-expressed ephrins, CHO-ρgsA745 cells were stably transfected with human EphrinBl (CHO-Bl), EρhrinB2 (CHO-B2), or EρhrinB3 (CHO-B3). Chinese hamster ovary (CHO) cells do not express ephrins endogenously (Huynh-Do, et al, (2002) J Cell Sd 115, 3073-3081), while the CHO- pgsA745 cells derived from these CHO cells lack heparin sulfate proteoglycans (Esko, et al., (1985) Proc Natl Acad Sd USA 82, 3197-3201). Heparin sulfates are known to act as entry or attachment receptors for many viruses, which can confound viral receptor studies. Thus, NiV-G-Fc, a fusion construct between the ectodomain of NiV-G and the Fc region of human IgGl, was used to measure the binding of NiV-G to each of the ephrin B-class ligands stably expressed on CHOpgsA745 cells (Fig. 7b). Remarkably, the K_d for NiV-G-Fc binding to EphrinB2 and B3 was in the subnanomolar range (K_d=0.06 nM and 0.58 nM for EphrinB2 and B3, respectively). NiV-G-Fc did not bind to CHO-Bl cells when tested under the same conditions used for CHO-B2 and CHO-B3 cells (unpublished data).

[0137] To delineate the difference in binding affinities between EphrinB2 and B3, their binding kinetics to NiVG- Fc were examined. Surface plasmon resonance analysis was performed by coupling NiV-G-Fc to the sensor chip (as ligand) while using soluble EphrinB2 and B3 as analyte. BIAcore analysis of NiVG- Fc binding to EphrinB2-Fc and B3-Fc indicated that while NiV-G-Fc bound to EphrinB2 and B3 with similar on-rates (K_0n = 9.69 x 10 and 6.87 x 10⁵ for EphrinB2 and B3, respectively), their off-rates were significantly different (Figs. 7c and 7d). The K_0-T for EρhrinB2-Fc binding to NiV-G-Fc (l.OόxlO^"4 s^"1) was more than 10-fold slower than EphrinB3-Fc (1.94 x 10^"3 s^"1) binding (Fig. 7d). EphrinBl -Fc was also tested similarly to EphrinB2 and B3 and did not exhibit any binding to NiV-G-Fc even up to concentrations as high as 500 nM (unpublished data). As a control to determine the accuracy of our BIAcore measurements, the IQ of EphB4-Fc binding to EphrinB2-Fc was determined to be 0.37 nM, a value consistent with published values of approximately 0.5 nM (unpublished data; Blits-Huizinga, et al, (2004) IUBMB Life 56, 257-265). Moreover, NiV-G binds to EphrinB2 with higher affinity than the EphB4-Fc:EphrinB2 interaction (unpublished data). Cumulatively, the data show that the Nipah attachment protein bound to both EphrinB2 and EphrinB3 with different but significant affinities.

EphrinBS Supports NiV Entry and Infection

[0138] The high-affinity protein interaction seen between EphrinB3 and NiV-G was examined to determine whether it was sufficient to permit the entry of NiV. A panel of CHO- pgsA745 cells stably expressing EphrinBl, B2, and B3 was established. Since the EphB3 receptor binds to all ephrin B-class ligands with similar affinities (0.27—1.8 nM for EphrinBl, 0.28-0.78 nM for EρhrinB2, and 1.5 nM for EphrinB3) (Blits-Huizinga, et al, (2004) IUBMB Life 56, 257-265), saturating amounts of soluble EphB3 (EphB3-Fc) were used to determine the level of EphrinBl-B3 expression in the stable cell lines by flow cytometry (Fig. 8a). While CHO-Bl, CHO-B2, and CHO-B3 cells were found to be significantly positive for EphB3 binding (68%, 47%, and 47%, respectively), only CHO-B2 and CHO-B3 cells bound to NiV-G-Fc (46% and 31%, respectively). Soluble EphB3 did not bind to parental CHO- pgsA745 cells, nor did the ephrin A-class-specific EphA2-Fc bind any of the cell lines tested (CHOpgsA745, CHO-Bl, CHO-B2, or CHO-B3). These results confirmed the specific ephrin B-class expression on the panel of CHO-pgsA745 cells.

[0139] NiV entry was then quantitated using NiV envelope pseudotyped luciferase (Luc) reporter viruses. The inventors had previously shown that NiV envelopes can be successfully pseudotyped onto recombinant vesicular stomatitis virus (VSV) expressing a red fluorescent protein (RFP), but lacking its own envelope (NiV- VSV-ΔG -RFP) (Negrete, et al, (2005) Nature 436, 401—405). Here, the NiV- VSV-ΔG- Luc was made bearing the NiV fusion (F) and NiV-G and expressing the Renilla Luc reporter gene in place of the RFP gene. These NiV envelope pseudotyped VSV particles were used to infect CHO-pgsA745 parental cells (CHO), CHO-Bl, CHO-B2, and CHO-B3 cell lines. It was found that both CHOB2 and CHO-B3 allowed entry of NiV-VSV-ΔG-Luc virus (Fig. 8b), although viral entry into CHO-B2 cells reproducibly resulted in higher Luc levels (p = 0.05, paired t-test). In three independent experiments, the reduction in viral entry into CHO-B3 cells ranged from 21% to 46%. Since the EphB3-Fc binding data indicated similar levels of EphrinB2 and B3 expression on the CHO-B2 and CHO-B3 cells (Fig. 8a), NiV-VSV-ΔG-Luc virus entered CHO-B2 cells more efficiently than CHO-B3 cells. [0140] To examine whether EphrinB3 can support viral infection, live NiV infections were performed under Biosafety Level-4 conditions. The hallmark of NiV infection in humans is the presence of syncytial or multinucleated giant endothelial cells, and cell lines from many different species produced syncytia upon infection. Therefore, syncytia formation was examined in CHO, CHO-Bl, CHO-B2, CHO-B3, and Vero cells after infection with live NiV. Indeed, it was found that live NiV can infect EphrinB2- and B3 -expressing cells, although EphrinB2 appears to be used more efficiently (Fig. 8c). With any given multiplicity of infection (MOI), at 24 h post infection, there were always a greater number of syncytia in the CHO-B2 versus CHO-B3 cells. No syncytia were detected in CHO-Bl cells. At 48 h postinfection, syncytia were apparent at all MOIs tested in the CHO-B3 cells (unpublished data). Thus, EphrinB3 can serve as a bona fide alternative receptor for NiV entry.

NiV-G Binds EphrinB2 and B 3 via an Overlapping Site

[0141] To determine whether EphrinB2 and B3 interact with NiV-G in a distinct or overlapping manner, a competition assay was used where CHO-B2 and CHO-B3 cells were infected with NiV-VSV-ΔG-Luc viruses in the presence of soluble ephrin B-class ligands. As expected, EphrinB2-Fc inhibited pseudotyped NiV on CHO-B2 cells while EphrinBl-Fc did not inhibit entry (Fig. 9a). However, EphrinB3-Fc also inhibited pseudotyped NiV entry on CHO-B2 cells, suggesting that EphrinB3 blocked EphrinB2- dependent NiV entry by competing for a similar binding domain on NiV-G. Conversely, EphrinB2-Fc also inhibited pseudotyped NiV entry on CHO-B3 cells (Fig. 9b). In both CHO-B2 and CHO-B3 cells, EphrinB2-Fc was a more effective inhibitor of NiV-G entry than EphrinB3-Fc. In summary, these results suggest that EphrinB2 and B3 binding sites on NiV-G are overlapping.

Leu—Trp Residues in the G-H Loop ofEphrinB2 andB3 Are Critical for NiV-G Binding

[0142] Since EphrinB2 and B3 can support NiV entry, while EphrinBl cannot, it was hypothesized that conserved residues common in both EphrinB2 and B3 mediate specific interactions with NiV-G. Alignment of human, mouse, and rat EphrinBl, B2, and B3 sequences identified common residues in EphrinB2 and B3 not present in EphrinBl (Fig. 10a). Examination of the homologous G-H loop regions between EphrinBl, B2, and B3 revealed that the L-W (Leu-Trp) residues present in EphriiiB2 and B3 are replaced by Y-M (Tyr-Met) in EphrinBl (Fig. 10a). The cocrystal structure of EphrinB2 and the EphB2 receptor (an endogenous EphrinB2 receptor) indicates that the L-W (Leu₁₂₄- Trp₁₂₅) residues in the G-H loop of EphrinB2 insert deep into a hydrophobic pocket in EphB2 (Himanen, et ah, (2001) Nature 414, 933-938). This interaction is informative as soluble EphB2 inhibits NiV-G mediated infection (Negrete, et al., (2005) Nature 436, 401-405) and is likely to interact with a similar region on EphrinB2 as NiV-G. Thus, it was determined whether the G-H loop L-W residues in EphrinB2 and EphrinB3 also interact with NiV-G.

[0143] Full-length human EphrinBl, B2, and B3 clones were obtained, and the ectodomain of each was fused to the Fc region of human IgGl . Replacement of Y-M in EphrinBl with L- W from the homologous positions in EphrinB2 and B3 resulted in a soluble EphrinBl mutant, Bl _LW-Fc, which bound NiV-G almost as well as EphrinB2-Fc (Fig. 10b). Conversely, the L- W to Y-M mutations in soluble EphrinB2 (B2_YM-FC) and EphrinB3 (B3_YM-Fc) abrogated binding to NiV-G-HA, although at higher concentrations, B2_YM-FC appears to retain minimal NiV-G binding activity.

Leu—Trp Residues in the G-H Loop ofEphrinB3 Are Necessary for NiV Entry

[0144] Next, the effects of these L-WAT-M mutations were examined in the context of full-length ephrins and their ability to support NiV entry. To do so, stable CHOpgsA745 cells were made expressing the full-length ephrin mutants (CHO-B I_LW, CHO-B2_YM, and CHO- B3_YM). The level of mutant ephrin expression in the stable cell lines was compared to that of wild-type ephrin expression using EphB3-Fc in a flow cytometry analysis. It is unlikely that EphB3 binds to the ephrin LWAfM residues in question since EphB3 can bind EphrinBl, B2, and B3 with similar affinities (Blits-Huizinga, et al, (2004) IUBMB Life 56, 257-265). Therefore, using EphB3-Fc to measure cell surface expression, it was found that both wild- type ephrin and its relevant mutant were expressed at similar levels (Fig. l la; compare Bl to Bl Lw, B2 to B2γ_M, and B3 to B3_YM). Parental CHO-pgsA745 (CHO) served as a negative control and soluble EphB3 did not bind to these cells. In addition, NiV-G-Fc bound to the mutant ephrins in the expected patterns seen in the previous solid state ELISA experiment (Fig. 10b).

[0145] These cells were then infected with NiV-VSV-Luc pseudotyped viruses (Fig. 1 Ib). As expected, both EphrinB2 and B3 permitted NiV envelope-mediated entry as described previously (Fig. 8b). The B I _LW mutant now supported NiV entry, while entry into B2_YM cells was markedly reduced. Entry into B3_YM was completely abrogated. While entry into B2YM cells was reduced by about 85% compared to wild-type EphrinB2 cells, entry was still 44-fold over background (parental CHO-pgsA745 cells). These results suggest that while the L₁₂₄- W₁₂₅ residues in EphrinB3 appear to be critical for NiV entry, other residues in EphrinB2 likely have a supporting role in mediating NiV entry. Interestingly, the Bl _LW mutant could not fully restore entry equivalent to wild-type B2 or B3 levels, even though NiV-G bound to cell surface EphrinBlLW at wild-type EphrinB2 levels. Therefore, in addition to simple binding, additional residues in EphrinB2 likely mediate the subsequent conformational changes in NiV- G and/or F that leads to membrane fusion and entry.

[0146] Here, it has been shown that EphrinB3 is an alternate receptor for NiV and is independently able to support NiV entry and infection, albeit less efficiently than EphrinB2. NiV-G binds to both EphrinB2 and B3 with subnanomolar affinity, with the relatively weaker Kd of NiV-G for EphrinB3 explained by its faster off-rate. Finally, two residues (L-W) common in the G-H loop of EphrinB2 and B3 have been implicated as crucial for NiV receptor activity. Remarkably, replacement of the Y-M residues in the homologous positions in EphrinBl with L-W conferred wild-type NiV-G binding activity and substantial NiV receptor activity to a protein that is otherwise nonfunctional as a NiV receptor.

[0147] To our knowledge, there is no specific indication that EphrinB3 is expressed in the endothelium. At the minimum, EphrinB3 does not appear to be critical to vascular development since EphrinB3 knockout mice lack the overt defects in vascular morphogenesis seen in EphrinB2 knockout mice (Kullander, et at/., (2003) Science 299, 1889-1892; Wang, et al, (1998) Cell 93, 741-753). However, NiV entry into microvascular endothelial cells is almost completely abrogated by soluble ephB4-Fc (Negrete, et al., (2005) Nature 436, 401- 405), which binds to EphrinB2 but not B3, suggesting that EphrinB3 is likely not expressed on endothelial cells, at least not at levels that can support robust viral entry. In contrast, EphrinB3 is expressed in the CNS in overlapping and distinct patterns with EphrinB2 (Flenniken, et al., (1996) Dev Biol 179, 382-401; Bergemann, et al, (1998) Oncogene 16, 471-480). In the regions of the adult brain such as the cerebral cortex (Tang, et al., (1997) Genomics 41, 17—24; Liebl, et al., (2003) JNeurosci Res 71, 7-22) and the hippocampus (Grunwald, et al., (2001) Neuron 32, 1027—1040) where EphrinB2 and B3 exhibit overlapping expression, NiV could potentially use either receptor for entry with a possible preference for EphrinB2 based on the higher affinity of NiV-G for EphrinB2. However, in regions such as the corpus callosum (Liebl, et al, (2003) JNeurosci Res 71, 7-22) and the spinal cord (Yokoyama, et al, (2001) Neuron 29, 85-97; Kullander, et al, (2001) Genes Dev 15, 877-888), EρhrinB3 is distinctly expressed and could account for specific aspects of NiV pathology. [0148] EphrinB3 knockout mice studies indicate EphrinB3 is expressed in the spinal cord midline and functions to prevent corticospinal tract axons from recrossing the midline. Coincidently, in a histological study of NiV infection by Wong et al, ((2002) Am J Pathol 161, 2153-2167), three of eight patients examined showed pathological lesions in the spinal cord similar to other regions of the CNS (Wong, et al, (2002) Am J Pathol 161, 2153-2167). Indeed, clinical symptoms of segmental myoclonus and flaccid tetraplegia combined with nerve conduction studies have also suggested upper cervical and lower spinal cord involvement (Goh, et al, (2000) NEnglJMed 342, 1229-1235), and magnetic resonance imaging confirmation of a spinal cord lesion at the cognate C7 level in a patient who developed left-arm dysaesthesia and finger weakness has also been reported (Lim, et al, (2003) J Neurol Neurosurg Psychiatry 74, 131-133). In another study that reported magnetic resonance imaging findings in eight patients with NiV encephalitis, half the patients had numerous punctate lesions in the corpus callosum (Lim, et al, (2000) AJNR Am J Neuroradiol 21, 455- 461), where EphrinB3, but not EphrinB2, is expressed (Liebl, et al, (2003) JNeurosci Res 71, 7—22). Thus, although EphrinB2 seems to be the primary receptor for NiV, EphrinB3 can likely be used as an alternative receptor and may account for some of the CNS pathology seen in NiV infection.

[0149] In this study, it was established that NiV-G binding to EphrinB2 and B3 is dependent on the same L-W residues that are important for endogenous EphrinB2/EphB2 interactions (Blits-Huizinga, et al, (2004) IUBMB Life 56, 257-265; Himanen, et al, (2001) Nature 414, 933-938). Since NiV-G interacts with EphrinB2 in a similar fashion with at least some of the Eph B-class receptors, and NiV-G forms higher order oligomers (Levroney, et al, (2005) J Immunol 175, 413-420) analogous to Eph B-class receptors (Poliakov et al, (2004) Dev Cell 7, 465-480; Palmer & Klein, (2003) Genes Dev 17, 1429-1450), NiV-G could potentially induce "reverse-signaling" upon EphrinB2 or B3 binding.

[0150] In vivo, Eph-ephrin interactions cause bidirectional signaling that can direct the migration of endothelial cells and neuronal dendrites (Poliakov et al, (2004) Dev Cell 7, 465- 480; Palmer & Klein, (2003) Genes Dev 17, 1429-1450; Augustin & Reiss, (2003) Cell Tissue Res 314, 25-31 ; Kullander & Klein (2002) Nat Rev MoI Cell Biol 3, 475-486; Mellitzer, et al, (1999) Nature 400, 77-81). Therefore, NiV infection may not only target EphrinB2- or B3- expressing cells, but also disrupt normal Eph-ephrin signaling and possibly alter cellular migration patterns. Indeed, infusion of soluble EphB2-Fc has been reported to disrupt the migration of EphrinB2- and B3 -expressing cells of the sub ventricular zone region in an adult mouse (Conover, et al, (2000) NatNeurosci 3, 1091-1097). Although the levels of Ephs and ephrins in other regions of the adult brain are reduced compared to neonatal-stage expression (Liebl, et al, (2003) JNeurosci Res 71, 7-22), Eph and ephrins can alter their expression patterns after injury to the spinal cord (Miranda, et al, (1999) Exp Neurol 156, 218-222), hippocampus (Moreno-Flores & Wandosell, (1999) Neuroscience 91, 193-201), or after infection (Ivanov, et al, (2005) Physiol Genomics 21, 152-160; Masood, et al, (2005) Blood 105, 1310-1318). In this case, NiV infection may alter ephrin expression patterns in the CNS and disrupt the endogenous Eph-ephrin signaling resulting in the neuropsychiatric (Ng, et al, (2004) J Neuropsychiatry Clin Neurosci 16 500-504) or neuropathology sequelae seen in NiV infections.

Example 3 EphrinB2 and EphrinB3 are receptors for HeV

Cells and culture conditions [0151] CHOpgsA745 cells were derived and maintained as described in Example 2.

Plasmids and reagents

[0152] Soluble Fc-fusion EphrinB2 and B3 proteins were purchased from R & D Systems.

[0153] EphrinB2 and B3 Fc-fusion constructs were prepared as described in Example 2.

[0154] An HeV-G-Fc construct was generated by fusing residues 71 -604 of HeV-G, the designated entire ectodomain of HeV-G (Meng Yu et. al., Virology, 251:227-233), to the Fc constant region of IgGl, as described for NiV-G-Fc, in the pCR3-Fc vector.

Cell surface binding assays

[0155] The EphrinB2- and B3-expressing CHO-ρgsA745 cells (CHO-B2 and CHO-B3, respectively) were made and used as described in Example 2.

Results HeV and NiV bind EphrinB2 with a similar affinity

[0156] To determine whether HeV binds to EphrinB2, the binding kinetics of HeV and NiV to EphrinB2 were examined. Surface plasmon resonance analysis was performed by coupling HeV-G-Fc or NiV-G-Fc to the sensor chip (as ligand) while using soluble EphrinB2 as analyte (Figs. 12a and 12b). BIAcore analysis of NiV-G- Fc or HeV-G-Fc binding to EphrinB2-Fc indicated that NiV-G-Fc and HeV-G-Fc bound to ephrin B2 with similar on-rates (K_a = 1.18 x 10⁶ and 1.27 x 10⁶, respectively) (Fig. 12c). Additionally, their-off rates were also quite similar: the K_d for NiV-G-Fc was 5.76 x 10^"5 s^"1, and the Kd for HeV-G-Fc was 3.97 x 10^"5 s^'1 (Fig. 12c). Therefore, HeV-G and NiV-G appear to bind to EρhrinB2 with similar affinities.

He V binds EphrinB3

[0157] To confirm whether NeV-G binds to EphrinB2 and B3 with cell surface-expressed ephrins, CHO-pgsA745 cells were stably transfected with human EphrinB2 (CHO-B2), or EphrinB3 (CHO-B3). Chinese hamster ovary (CHO) cells do not express ephrins endogenously (Huynh-Do, et ah, (2002) J Cell Sd 115, 3073-3081), while the CHO-ρgsA745 cells derived from these CHO cells lack heparin sulfate proteoglycans (Esko, et ah, (1985) Proc Natl Acad Sd USA 82, 3197-3201). Heparin sulfates are known to act as entry or attachment receptors for many viruses, which can confound viral receptor studies. Thus, NiV- G-Fc, a fusion construct between the ectodomain of NiV-G and the Fc region of human IgGl, and HeV-G-Fc, a fusion construct between the ectodomain of HeV-G and the Fc region of human IgGl, were used to measure the binding of NiV-G and HeV-G to EphrinB2 and B3 stably expressed on CHOpgsA745 cells (Fig.13a and 13b). The K_d for NiV-G-Fc binding to EphrinB2 and B3 was in the subnanomolar range (0.27 and 0.75 K_d=0.27 nM and 0.75 nM for EphrinB2 and B3, respectively. However, while the Kd for HeV-G-Fc binding to EphrinB2 was also in the subnanomolar range and was similar to that for NiV-G-Fc (0.57 nM vs. 0.27 nM; Fig. 13a), the Kd for HeV-G-Fc binding to EphrinB3 was approximately 30 times higher than that for NiV-G-Fc (24 nM vs. 0.75 nM; Fig. 13b). Cumulatively, the data show that the Hendra attachment protein bound to both EphrinB2 and EphrinB3 with different but significant affinities.

[0158] Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably maybe practiced in the absence of any element(s) not specifically disclosed.

Claims

1. A pharmaceutical composition comprising a soluble polypeptide selected from the group consisting of ephB4 or a fragment thereof, a Henipaviras attachment (G) glycoprotein-binding protein, and a Henipaviras attachment (G) glycoprotein, and a pharmaceutically acceptable excipient, for preventing or inhibiting binding of the Henipaviras attachment (G) glycoprotein to a cell-bound EphrinB2 or EphrinB3 polypeptide.

2. The pharmaceutical composition of claim 1, wherein the Henipaviras attachment (G) glycoprotein-binding protein is selected from the group consisting of EphrinB2, EphrinB3, and a Henipaviras attachment (G) glycoprotein binding fragment of EρhrinB2 or EphrinB3.

3. The pharmaceutical composition of claim 1 , wherein the Henipaviras attachment (G) glycoprotein-binding protein comprises an antibody that specifically binds to the Henipaviras attachment (G) glycoprotein.

4. The pharmaceutical composition of claim 3, wherein the antibody comprises a recombinant, synthetic or humanized antibody.

5. The pharmaceutical composition of claim 1 , wherein the soluble polypeptide further comprises a heterologous domain.

6. The pharmaceutical composition of claim 5, wherein the heterologous domain comprises an Fc domain.

7. The pharmaceutical composition of claim 1 , wherein the soluble polypeptide is a human polypeptide.

8. The pharmaceutical composition of claim 1 , wherein the soluble polypeptide is a pig polypeptide.

9. The pharmaceutical composition of claim 1 , wherein the soluble polypeptide comprises a synthetic or recombinant polypeptide or a peptidomimetic.

10. The pharmaceutical composition of claim 1 , wherein the Henipavirus is a Nipah virus.

11. The pharmaceutical composition of claim 1 , wherein the Henipavirus is a Hendra virus.

12. A method for inhibiting or preventing binding of a Henipavirus to a cell, wherein the method comprises contacting the cell with the composition of claim 1, thereby inhibiting or preventing binding of the Henipavirus to the cell.

13. The method of claim 12, wherein the cell is a human or a pig cell.

14. The method of claim 12, wherein the contacting comprises administering the pharmaceutical composition in vivo.

15. The method of claim 12, wherein the contacting comprises contacting the composition with the cell in vitro or ex vivo.

16. The method of claim 12, wherein inhibiting or preventing binding of the Henipavirus also prevents or inhibits infection of the cell by the Henipavirus.

17. A method for identifying an antiviral agent, which comprises

providing a test agent; contacting a Henipavirus attachment (G) glycoprotein with Henipavirus attachment (G) glycoprotein-binding protein selected from the group consisting of an EphrinB2 polypeptide, a EphrinB3 polypeptide, a Henipavirus attachment (G) glycoprotein-binding fragment of EphrinB2 or EphrinB3, and an antibody that specifically binds to the Henipavirus attachment (G) glycoprotein in the presence or absence of the test agent; and detecting attachment between the Henipavirus attachment (G) glycoprotein with the Henipavirus attachment (G) glycoprotein-binding protein, whereby the test agent is identified as an antiviral agent when attachment in different in the presence of the test agent than in the absence of the test agent.

18. The method of claim 17, wherein the Henipavirus attachment (G) glycoprotein is present in a Henipavirus.

19. The method of claim 17, wherein the Henipavirus attachment (G) glycoprotein is present in a pseudo-type virus.

20. The method of claim 17, wherein the EphrinB2 polypeptide, EphrinB3 polypeptide, or Henipavirus attachment (G) glycoprotein-binding fragment of EphrinB2 or EphrinB3 is present in a host cell.

21. The method of claim 20, wherein the cells are human or pig cells.

22. The method of claim 20, wherein the Henipavirus attachment (G) glycoprotein is exogenously expressed.

23. The method of claim 20, wherein EphrinB2, EphrinB3 , or the Henipavirus attachment (G) glycoprotein-binding fragment of EphrinB2 or EphrinB3 is exogenously expressed.

24. The method of claim 20, wherein EphrinB2, EphrinB3, or the Henipavirus attachment (G) glycoprotein-binding fragment of EphrinB2 or EphrinB3 is endogenously expressed.

25. The method of claim 20, wherein contact of the host cell with the test agent and the Henipavirus prevents or inhibits Henipavirus-induced cell-cell fusion in comparison to a cell not contacted by the test agent.

26. The method of claim 25, wherein cell fusion is detected by the presence or absence of a signal produced by a functional reporter molecule.

27. The method of claim 26, wherein the functional reporter molecule is β- galactosidase.

28. The method of claim 17, wherein the test agent comprises a polypeptide, a carbohydrate or a small molecule.

29. The method of claim 17, wherein the test agent is a soluble EphrinB2 polypeptide or a fragment thereof; a soluble EphrinB3 polypeptide or a fragment thereof; an antibody specific for Henipavirus attachment (G) glycoprotein, EphrinB2, or EphrinB3; or siRNA.

30. The method of claim 17, wherein the test agent is a tyrosine kinase inhibitor or an ion channel inhibitor.

31. The method of claim 17, wherein a decrease in attachment of the Henipavirus attachment (G) glycoprotein to the Henipavirus attachment (G) glycoprotein-binding protein in the presence of the test agent corresponds to a decrease in entry of a Henipavirus into a host cell.

32. A Henipavirus anti-viral agent identified by the method of claim 17.

33. A method for reducing the infectivity by Henipavirus of a host cell susceptible to infection by Henipavirus, comprising administering to a subject in need thereof an antiviral agent that inhibits the ability of Henipavirus attachment (G) glycoprotein to attach to EphrinB2 or EphrinB3, thereby reducing infectivity of the Henipavirus.

34. The method of claim 33, wherein said method is performed in vivo.

35. The method of claim 33, wherein the subject is a human or a pig.

36. The method of claim 33, wherein the antiviral agent is a soluble EphrinB2 polypeptide or a fragment thereof; a soluble EphrinB3 polypeptide or a fragment thereof; an antibody specific for Henipavirus attachment (G) glycoprotein, EphrinB2, or EphrinB3; or siRNA.

37. A method for eliciting a protective immune response against Henipavirus, which comprises administering a Henipavirus attachment (G) glycoprotein or a fragment thereof to a subject in need thereof.