NZ617722B2 - Hendra and nipah virus g glycoprotein immunogenic compositions - Google Patents

Abstract

Discloses an immunogenic composition comprising a soluble fragment of the Hendra G glycoprotein, said fragment consisting of amino acids 73 to 604 of (SEQ ID NO: 2) or a sequence at least 90% identical thereto, an immunostimulatory complex (ISC), said ISC comprising a saponin, a phospholipid and a steroid, and one or more excipients, wherein the concentration of the soluble fragment of the Hendra virus G glycoprotein in the composition is 5-100 ?g per ml of ISC, wherein the sequences are as defined in the complete specification. teroid, and one or more excipients, wherein the concentration of the soluble fragment of the Hendra virus G glycoprotein in the composition is 5-100 ?g per ml of ISC, wherein the sequences are as defined in the complete specification.

Description

HENDRA AND NIPAH VIRUS G GLYCOPROTEIN IMMUNOGENIC COMPOSITIONS Field of the Invention The present invention relates to immunogenic and vaccine compositions comprising a G glycoprotein from Hendra virus (HeV) and/or Nipah virus (NW) and to s of use ‘ relating thereto.

Description of the ound Recurrent outbreaks of NW resulting in significant numbers of human fatalities have recently been problematic (see 6.9. Butler (2000) Nature 429, 7). HeV is also known to cause fatalities in human and animals and is genetically and immunologically closely related to NW. There are presently no vaccines or therapeutics for prevention of infection or disease caused by Nipah virus or Hendra virus. Both Nipah virus and Hendra virus are United States, National Institute of Allergy and Infectious Disease, category C priority agents of biodefense n. Further, as these viruses are zoonotic Biological Safety Level-4 agents (ESL—4), production of es and/or diagnostics, with safety is very costly and difficult. Thus, there is a need for Nipah virus or Hendra virus vaccines and diagnostics that allow for high throughput production of vaccines and/or diagnostics.

Paramyxoviruses such as HeV and NW s two major membrane-anchored glycoproteins in the envelope of the viral particle. One rotein is required for virion attachment to receptors on host cells and is designated as either hemagglutinin- neuraminidase protein (HN) or hemagglutinin protein (H), and the other is glycoprotein (G), which has neither lutination nor neuraminidase ties. The attachment glycoproteins are type II membrane proteins, where the molecule's amino (N) terminus is oriented toward the cytoplasm and the protein's carboxy (C) terminus is extracellular. The other major rotein is the fusion (F) rotein, which is a trimeric class I fusogenic envelope glycoprotein containing two heptad repeat (HR) s and a hydrophobic fusion peptide. HeV and NW infect cells though a pH-independent membrane fusion process into receptive host cells through the concerted action of their attachment G glycoprotein and F glycoprotein following receptor binding. The primary function of the HeV and NW attachment G glycoprotein is to engage appropriate ors on the surfaces of host cells, which for the ty of well-characterized paramyxoviruses are sialic acid moieties. The HeV and NW G glycoproteins utilize the host cell protein receptors ephrin B2 and/or ephrin B3 and antibodies have been developed which block viral attachment by the G glycoprotein (W)2006137931, Bishop (2008) J. Virol. 82: 11398-11409). Further, vaccines have been ped which also use the G glycoprotein as a means for generating an immunoprotective response against HeV and NiV infection (WO2009117035). ite reactivity is a major concern for both the veterinary and human use of Quil A in vaccine preparations. One way to avoid this toxicity of Quil A is the use of immunostimulating complexes (Rajput (2007) J. Zhejiang Univ. Sci. B, 8: 153-161). This is primarily because Quil A is less reactive when orated into immunostimulating complexes, because its association with cholesterol in the complex reduces its ability to extract terol from cell membranes and hence its cell lytic s. In on, a lesser amount of Quil A is required to generate a similar level of adjuvant effect. The immunomodulatory properties of the Quil A saponins and the addition benefits to be derived from these saponins when they are orated into an immunostimulating complex have been bed in WO2000041720.

The ation of HeV and/or NiV G glycoproteins with stimulating complexes in a single vaccine represents an advancement in developing effective HeV and NiV vaccines given the potential for enhanced immunoreactivity with decreased adjuvant side effects when these components are administered in combination.

Summary of the Invention The invention encompasses an immunogenic composition comprising Hendra and/or Nipah virus G protein, an immunostimulatory complex (ISC) and one or more excipients in an amount effective to elicit production of lizing antibodies against the Hendra and/or Nipah virus following administration to a subject. In some embodiments, the immunogenic composition comprises a saponin, a phospholipid, and a steroid. [0006a] In a first aspect there is provided an immunogenic composition comprising a soluble fragment of the Hendra G glycoprotein, said fragment consisting of amino acids 73 to 604 of (SEQ ID NO: 2) or a sequence at least 90% identical thereto, an immunostimulatory complex (ISC), said ISC comprising a saponin, a phospholipid and a steroid, and one or more ents, n the concentration of the soluble fragment of the Hendra virus G glycoprotein in the composition is 5-100 μg per ml of ISC. [0006b] In a second aspect there is provided use of an genic composition for the manufacture of a ment for producing a neutralizing antibody response against a Hendra and/or Nipah virus in a t, wherein the immunogenic composition comprises the immunogenic composition of any of claims 1 -11. [0006c] In a third aspect there is provided use of an immunogenic composition for the cture of a medicament for treatment of Hendra and/or Nipah virus infection, wherein the immunogenic composition comprises (1) a soluble fragment of the Hendra G glycoprotein in an amount of 5-100 μg in the immunogenic composition, said nt consisting of amino acids 73 to 604 of (SEQ ID NO: 2) or a sequence at least 90% identical thereto, and (2) an immunostimulatory complex (ISC), said ISC comprising a saponin,a phospholipid and a steroid, and one or more excipients, wherein the immunogenic composition is administered to the subject in an amount and duration effective to treat the subject for Hendra and/or Nipah virus infection. [0006d] It is to be noted that, throughout the description and claims of this ication, the word 'comprise' and variations of the word, such as 'comprising' and 'comprises', is not intended to exclude other variants or additional components, integers or steps. Modifications and ements to the ion will be readily apparent to those d in the art. Such modifications and ements are intended to be within the scope of this invention. [0006e] Any reference to or discussion of any document, act or item of knowledge in this specification is included solely for the purpose of providing a context for the present invention. It is not suggested or represented that any of these matters or any combination thereof formed at the priority date part of the common general knowledge, or was known to be relevant to an attempt to solve any problem with which this specification is concerned.

In some embodiments soluble Hendra virus G glycoprotein consists of amino acids 73 to 604 of the native Hendra G glycoprotein (SEQ ID NO: 2). In some embodiments, the soluble Hendra virus G glycoprotein is encoded by a tide sequence comprising nucleotides 64 to 1662 of SEQ ID NO: 16. In some embodiments, the soluble Hendra virus G protein is present in dimer form wherein each soluble Hendra virus G glycoprotein dimer subunit is connected by one or more disulfide bonds. In some embodiments, the soluble Hendra virus G protein is present in tetramer form. In some embodiments, the tetramer form exists as a dimer of dimers non-covalently linked and/or connected by one or more disulfide bonds. The concentration of soluble Hendra virus G protein can be about 5 to 100 ug/ml in the immunogenic composition.

In some embodiments, the n is isolated from Quillaja saponaria Molina and may be selected from QH-A, QH-B, QH-C or 0821. In some embodiments, the phospholipid is selected from the group consisting of phosphatidyl choline (PC), dipalmitoyl phosphatidyl choline , phosphatidic acid (phosphatidate) (PA), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (Pl) phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate (PIP2), phosphatidylinositol triphosphate (PlP3), phosphorylcholine (SPH), ceramide phosphorylethanolamine (Cer—PE) and ceramide phosphorylglycerol. In some embodiments the saponin is Quil A, the phospholipid is DPPC and the d is terol and the ratio of Quil A: DPPC: cholesterol in the composition is 521:1 by weight.

The invention also encompasses a method of producing a neutralizing antibody response against a Hendra and/or Nipah virus in a subject comprising administering to the subject the immunogenic composition described herein in an amount and duration ive to produce the neutralizing antibody response. In some embodiments, the neutralizing antibody response reduces Hendra and/or Nipah virus reproduction in the subject and may also reduce Hendra and/or Nipah virus shedding in the subject. In some embodiments, the subject has been d to Hendra and/or Nipah virus while in other embodiments, the subject is suffering from a Hendra and/or Nipah virus infection. In some embodiments, the ion encompasses a method of ing a neutralizing antibody se t a Hendra virus in a subject sing administering to the subject the immunogenic composition bed herein in an amount and on effective to produce the neutralizing antibody response. In some embodiments, the invention encompasses a method of producing a neutralizing dy response against a Nipah virus in a subject comprising administering to the subject the immunogenic composition described herein in an amount and duration effective to produce the neutralizing antibody response.

In some embodiments, the immunogenic composition is administered intramuscularly.

In some embodiments, the immunogenic composition is administered in multiple doses and the first dose is followed by a second dose at least about twenty-one days to about twenty- eight days after the first dose. In some embodiments, each dose contains about 50 or about 100 ug of soluble Hendra virus G protein.

The invention further encompasses a method of differentiating a subject vaccinated with the immunogenic composition described herein from a subject d to Hendra and/or Nipah virus comprising detecting the presence of an antibody in a biological sample isolated from the subject against at least one of any of the following HeV and/or NiV viral proteins selected from the group consisting of fusion protein (F), matrix protein (M), phosphoprotein (P), large n (L) and nucleocapsid n (N).

The immunogenic compositions and methods of the invention can be administered to a subject such as a human, horse, cow, sheep, pig, goat, chicken, dog or cat.

The invention also encompasses a method of ing a neutralizing antibody response against a Hendra and/or Nipah virus in a human subject comprising stering to the subject an immunogenic composition comprising a Hendra virus soluble G glycoprotein in an amount and duration effective to produce the neutralizing antibody response. In some ments, the immunogenic composition further ses an adjuvant.

Description of the Figures Figure 1 shows the rectal temperature over time for horses stered inant Hendra virus soluble rotein ($6) at 50 or 100 ug/dose adjuvanted with 250 pg of immune stimulating complex followed by exposure to live Hendra virus at day 0.

Figure 2 shows the heart rate over time for horses administered recombinant Hendra virus soluble glycoprotein (sG) at 50 or 100 ug/dose adjuvanted with 250 pg of immune stimulating complex followed by exposure to live Hendra virus at day 0.

Figure 3 depicts a schematic for the preparation of an lmmunostimulatory Complex.

Figure 4 depicts a schematic diagram of sGHeV vaccination and NW challenge le. Dates of sGHeV vaccination, NiV challenge and asia are indicated by arrows. Blood and swab specimens were collected on days —42, -7, 0, 3, 5, 7, 10, 14, 21 and 28 post-challenge as indicated (*). Gray text denotes challenge timeline (top row); black text denotes vaccination timeline (bottom row). African green monkey (AGM) number for subjects in each vaccine dose group and one control subject are shown.

Figure 5 depicts the survival curve of fected subjects. Data from control subjects (n=2) and sGHeV vaccinated subjects (n=9) were used to generate the Kaplan- Meier survival curve. Control included data from one additional historical control subject.

Vaccinated subjects received 10 pg, 50 pg or 100 pg sGHeV administered subcutaneously twice. Average time to end stage disease was 11 days in control subjects whereas all vaccinated subjects survived until euthanasia at the end of the study.

Figure 6 depicts NiV- and HeV- c Immunoglobulin (lg) in vaccinated subjects.

Serum and nasal swabs were collected from vaccinated subjects and lgG, lgA and lgM responses were ted using sGHeV, and sGNiV multiplexed microsphere assays. Sera or swabs from subjects in the same vaccine dose group (n=3) were d individually and the mean of microsphere median fluorescence intensities (M.F.|.) was calculated which is shown on the Y-axis. Error bars ent the standard error of the mean. Serum sG- specific lg is shown in black (sGHeV (open triangles), sGNiV (solid les» and mucosal sG-specific lgA is shown in gray s (sGHeV (open triangles), sGNiV (solid triangles)).

Description of the Invention Vaccine & genic Compositions The vaccine and immunogenic composition of the present invention induces at least one of a number of humoral and cellular immune responses in a subject who has been administered the composition or is effective in enhancing at least one immune response against at least one strain of HeV and/or NiV, such that the administration is suitable for vaccination purposes and/or prevention of HeV and/or NiV infection by one or more strains of HeV and/or NW. The composition of the present invention delivers to a subject in need thereof a G glycoprotein, including soluble G glycoproteins from HeV and/or NW and an lmmunostimulatory x (ISC) which acts as an adjuvant. In some embodiments, the amount of G glycoprotein includes, but is not limited to, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or 250 pg per ml which can also contain 100, 125, 150, 175, 200, 225, 250, 275 or 300 pg per ml of ISC. In some embodiments, the amount of G rotein is 5, 50 or 100 and the amount of ISC is 250 pg per ml.

A. HeV and NW G proteins In some embodiments, the vaccine and immunogenic compositions comprise one or more HeV and/or NiV G glycoproteins as described herein. The term protein is used broadly herein to include polypeptide or fragments thereof. By way of example, and not limitation, a HeV G glycoprotein may in e form and comprise amino acids 73-604 of the amino acid sequence for a HeV G glycoprotein in Wang (2000) J. Virol. 74, 9972-9979 (see also Yu (1998) Virology 251, 227-233). Also by way of example and not limitation, a NiV G glycoprotein may be in soluble form and comprise amino acids 71-602 of the amino acid sequence for a NiV G glycoprotein in Harcourt (2000) Virology 271: 9, 2000 (see also Chua (2000) e, 288, 1432-1).

Generally, the soluble forms of the HeV and NW G glycoproteins comprise all or part of the ectodomain (e.g. extracellular) of the G glycoprotein of a HeV or NW and are generally produced by deleting all or part of the transmembrane domain of the G glycoprotein and all or part of the cytoplasmic tail of the G glycoprotein. By way of e, a soluble G glycoprotein may comprise the complete main of a HeV or NiV G glycoprotein. Also by way of example, and not limitation a soluble G glycoprotein may se all or part of the ectodomain and part of the transmembrane domain of a HeV or NiV G glycoprotein.

The soluble HeV or NiV G glycoproteins of the invention, generally retain one or more characteristics of the corresponding native viral glycoprotein, such as, ability to interact or bind the viral host cell receptor, can be produced in oligomeric form or forms, orvthe ability to elicit antibodies (including, but not limited to, viral neutralizing antibodies) capable of recognizing native G glycoprotein. Examples of additional characteristics include, but are not limited to, the ability to block or prevent infection of a host cell. tional methodology may be utilized to evaluate soluble HeV or NiV G glycoproteins for one of more of the characteristics.

By way of e, and not limitation, a polynucleotide ng a soluble HeV G glycoprotein may comprise a cleotide sequence encoding about amino acids 73-604 of the amino acid sequence for an HeV G glycoprotein in Wang (2000) J. Virol. 74, 9972- 9979 (SEQ ID NO: 2). Also by way of example, and not limitation, a polynucleotide ng a soluble HeV G glycoprotein may comprise nucleotides 9129 to 10727 of the polynucleotide sequence for an HeV G glycoprotein in Wang (2000) J. Virol. 74, 9972-9979. In addition, codon optimized polynucleotide sequence encoding about amino acids 73-604 of the amino acid sequence for an HeV G glycoprotein (SEQ ID NO: 2) can also be utilized. In some embodiments, these codon optimized ces comprises or consist of nucleotides 64 to 1662 of SEQ ID NO: 16. In further embodiments, the codon optimized sequences comprises or consists of SEQ ID NO: 16 which includes nucleotides encoding an ng leader sequence.

By way of example, and not limitation, a NiV G glycoprotein may in soluble form and se amino acids 71-602 of the amino acid sequence for the NW G glycoprotein in Harcourt (2000) Virology 271, 334-349. Non—limiting examples of sequences that may be used to construct a soluble NiV G glycoprotein can be found in Harcourt (2000) Virology 271, 334-349. Generally, G glycoprotein sequences from any Nipah virus isolate or strain may be utilized to derive the polynucleotides and polypeptides of the invention.

By way of example, and not limitation, a polynucleotide encoding a e NiV G rotein may comprise a polynucleotide sequence encoding about amino acids 71—602 of the amino acid ce for an NiV G Glycoprotein in Harcourt (2000) Virology 271, 334- 349. Also by way of example, and not limitation, a cleotide encoding a soluble NiV G glycoprotein may comprise 234-2042 of the polynucleotide sequence for an NiV G glycoprotein in rt (2000) Virology 271, 334-349 (SEQ ID NO: 4). In addition, codon optimized polynucleotide sequence encoding about amino acids 71-602 of the amino acid sequence for an NiV G glycoprotein can also be utilized.

Functional equivalents of these G glycoproteins can be used in the immunogenic and vaccine compositions of the invention. By way of example and not tion functionally lent ptides possess one or more of the following characteristics: ability to interact or bind the viral host cell receptor, can be produced in dimeric or tetrameric form or forms, the ability to elicit antibodies (including, but not limited to, HeV and/or NiV viral neutralizing dies) capable of izing native G glycoprotein and/or the ability to block or prevent infection of a host cell.

In some embodiments, the G glycoprotein may be in dimeric and/or tetrameric form.

Such dimers depend upon the formation of disulfide bonds formed between cysteine residues in the G glycoprotein. Such disulfide bonds can correspond to those formed in the native G glycoprotein (e.g. location of es remains unchanged) when expressed in the surface of HeV or NiV or may be altered in the presence or location (e.g. by altering the location of cysteine(s) in the amino acid ce) of the G glycoprotein so as to form different dimeric and/or tetrameric forms of the G glycoprotein which enhance antigenicity.

Additionally, non-dimerized and tetramerized forms are also within the invention, again taking into t that G glycoprotein presents us conformation-dependent epitopes (i.e. that arise from a tertiary three dimensional structure) and that preservation numerous of such natural epitopes is highly preferred so as to impart a neutralizing dy response.

The HeV immunogenic and vaccine compositions of the invention may contain proteins of variable length but include the amino acid residues 73 to 604 of SEQ ID NO: 2.

In one embodiment of the present invention, envelope proteins of the ion are at least about 85, 90, 91, 92, 93, 94, 95 or 99% cal to the HeV glycoprotein of SEQ , 96, 97, 98, ID NO: 2 (including amino acids 73 to 604). Accordingly, the HeV G glycoproteins of the invention comprise immunogenic fragments of the native HeV G glycoprotein with sufficient number of amino acids to produce conformational epitopes. miting examples of immunogenic fragments include amino acid sequences which may be at least 530, 531, 532, 533, 534 or 535 or more amino acids in length. In some embodiments, the HeV G glycoprotein comprises or consists of SEQ ID NO: 2 or tic constructs further comprising an ng leader sequence (SEQ ID NO: 15).

The NiV immunogenic and vaccine compositions of the invention may contain proteins of variable length but include the amino acid residues 71 to 602 of SEQ ID NO: 4.

In one embodiment of the present invention, envelope proteins of the invention are at least about 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the NW glycoprotein of SEQ ID NO: 4 (including amino acids 71 to 602). Accordingly, the NW G glycoproteins of the invention comprise immunogenic fragments of the native NiV G glycoprotein with sufficient number of amino acids to e mational epitopes. Non-limiting examples of immunogenic fragments include amino acid ces which may be at least 528, 529, 530, 531, 532, or 533 or more amino acids in length. In some embodiments, the NW G glycoprotein comprises or consists of SEQ ID NO: 4 or synthetic constructs further sing a leader sequence. lmmunogenic fragments as described herein will contain at least one epitope of the antigen and display HeV and/or NiV antigenicity and are capable of raising an immune response when presented in a suitable construct, such as for example when fused to other HeV and/or NiV antigens or presented on a r, the immune response being directed against the native n. In one embodiment of the present invention, the genic fragments contain at least 20 contiguous amino acids from the HeV and/or NiV antigen, for example, at least 50, 75, or 100 contiguous amino acids from the HeV and/or NiV antigen.

HeV and NW G glycoprotein embodiments further include an isolated polypeptide comprising an amino acid sequence having at least a 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity to native HeV or NiV G glycoproteins, n said polypeptide sequence may be identical to the native HeV or NiV G glycoprotein amino acid sequence or may include up to a certain integer number of amino acid tions as compared to the native HeV or NiV G protein amino acid sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, tution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino— or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference ce or in one or more uous groups within the native HeV or NiV G rotein amino acid sequence.

Sequence identity or homology at the amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis using the thm employed by the ms blastp, blastn, blastx, tblastn and tblastx (Altschul (1997) Nucleic Acids Res. 25, 3389-3402 and Karlin (1990) Proc. Natl. Acad. Sci. USA 87, 2264-2268) which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments, with gaps (non-contiguous) and without gaps (contiguous), between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to ize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see ul (1994) Nature Genetics 6, 119-129.

The search ters for histogram, descriptions, alignments, expect (i.e., the tical significance old for reporting matches against database sequences), cutoff, matrix and filter (low complexity) are at the default settings. The default scoring matrix used by blastp, blastx, tblastn. and tblastx is the BLOSUM62 matrix (Henikoff (1992) Proc. Natl. Acad. Sci.

USA 89, 10915-10919), recommended for query sequences over 85 amino acids in length.

The vaccine and immunogenic compositions of the present invention may further se additional HeV and/or NiV G proteins from different strains that may further potentiate the immunization methods of the invention.

B. lmmunostimulatory Complexes Generally this invention provides immunogenic compositions, including vaccine compositions, comprising e forms of HeV and/or NiV G glycoprotein envelope protein in ation with an immune stimulatory complex (ISO) and to methods for using these compositions for ting and ng HeV and/or NiV_ infections in a subject. In the present invention, the vaccine and/or immunogenic composition comprise an immunostimulatory complex which acts as an adjuvant. As used herein, ant" refers to an agent which, while not having any specific antigenic effect in itself, may stimulate the immune system, increasing the response to an antigen.

ISO have a number of features that makes it an ideal adjuvant for certain applications: Antigen sparing: As noted for example in Wee (2008) Mucosal Immunol. 1, 489-496 in situations where antigen availability is limited or antigen costs are high, ISO has been shown to allow antigen sparing as much as 10 to 100 fold. Most likely this due to a ation of increased efficiency or more appropriate mechanism of action compared to other adjuvants.

Cross-presentation: As noted for e in Schnurr (2009) J. Immunol. 182, 1253- 1259, presentation of antigen by antigen presenting cells (APCs) usually follows one of two ys. n antigen is usually engulfed by APCs and then processed and reexpressed on the surface of APC in the context of Major Histocompatibility Complex (MHC) class II les. They are then able to be seen by lymphocytes and, if the right co- stimulatory s/signals are present, be responded to as appropriate. Self or cancer antigens and viral ns are normally processed and expressed in the context of Class I molecules as they are present in the cytoplasm of APCs. Effective immunity to cancer and viral antigens requires access to the Class | pathway. This occurs naturally during viral infection or cellular tasis (cellular turnover of internal antigens). Antigens (viral or self) introduced as vaccines need to find their way from outside the cell to antigen processing machinery of the cell and entry into the Class‘ II pathway to the Class I pathway. This can occur lly in Dendritic Cells (DCs - specialist APCs) or can be achieved by vaccinating with antigens mixed with ISO as adjuvant. This process of externally derived antigen finding its way into the Class | pathway of antigen presentation is called presentation. The precise mechanism by which lSC achieves cross-presentation of antigen has not been fully elucidated but may rely on membrane perturbation of ISO components. 2012/037839 Humoral and cell mediated responses: As noted, for example in Maraskovsky (2009) lmmunol. Cell Biol. 87, 371-376), by virtue of the mechanism of action of ISC both humoral and cellular arms of the adaptive immune system are engaged. In some species this is paralleled the profile of cytokines stimulated by ation with this adjuvant. Type 1 immune responses terized by Interleukin-2 and IFN-gamma expression and protection against intracellular pathogens (bacteria, protozoa and viruses) and type 2 responses characterized by expression of Interleukin-4 and generation of neutralizing antibody for anti- toxin and anti-pathogen related immunity. ISC provides a balanced cytokine profile between these two extremes allowing for greater breadth of immune response. Additionally, a number of studies have shown that ISC can be effective if vaccines are delivered asally. This allows for sensitization of mucosal surfaces and thus providing relevant immunity at the site of pathogen entry, of particular relevance in this case (mucosal immunity), see also Sjolander (2001) Vaccine 19, 4072-4080.

Sterile filterable and consistent manufacturing criteria: The size of the ISC particle is routinely 40 nm in diameter allowing it to pass through filters used to sterilize preparations late in ation. onally, the natural tendency for triterpenoid saponins as found in Quil A to associate with terol and phospholipids has been taken advantage of in developing manufacturing methods for ISC. Quil A species that do not form ISC particles are dialyzed away from the final product. By controlling ratios of the components a consistent t is generated from a heterogeneous spectrum of Quil A saponins. This ratio is important as ion leads to ures that are not characteristic 40 nm particles (helices, sheets etc.). The free-flowing nature of the ISC colloid and its y to be measured using transmission electron microscopy, HPLC and other techniques make this adjuvant amenable to pment of release assays and other measures of quality.

Thus, based on the above, in some embodiments, the formulation of an immunostimulating complex with an optimal amount of G glycoprotein includes a saponin, a phospholipid and a d molecule. In some embodiments, the molar ratio of saponin, olipid, steroid molecule in a ratio of 5:1 :1. An immunostimulating complex may contain, for example, 5 to 10% by weight saponin, 1 to 5% steroid molecule and phospholipid and the remainder comprising G glycoprotein. G glycoprotein can be incorporated into the immunostimulating complex either directly or by chemical coupling to a carrier protein (e.g. ic or fusion protein) after incorporation of protein into immunostimulating complexes.

Reference to an immunostimulating complex should be understood to e reference to derivatives, chemical equivalents and analogs thereof. In some embodiments, the ISC is admixed separately from the HeV and/or NiV G glycoprotein then the G glycoprotein is admixed with the ISC. In some embodiments, the G glycoprotein is admixed directly with the saponin, olipid and steroid molecule.

In some embodiments, the saponin for use in the present invention is Quil A and/or its derivatives. Quil A is a saponin preparation isolated from the South American tree ja saponaria Molina and was first described as having nt activity by Dalsgaard (1974) n adjuvants, Archiv. fiir die gesamte Virusforschung, Vol. 44, Springer Verlag, pp. 243-254. Purified nts of Quil A have been isolated by HPLC which retain adjuvant activity without the toxicity associated with Quil A (EP 8), for example 087 and 0821 (also known as QA7 and QA21). QSZ1 is a natural n derived from the bark of Quillaja saponaria Molina which induces CDB+ xic T cells (CTL), Th1 cells and a predominant lgG2a antibody response and is a saponin for use in the context of the present invention.

Other suitable saponins for use in the ISC e, but are not limited to, the QH-A, QH-B and QH-C subfractions of Quil A, those from species other than Quil/aia saponaria such as those from the genera Panax (ginseng), Astragalus, Achyranthes, Soy bean, Acacia and Codonopsis. In some embodiments, the saponin is isolated from a species other than Quillaia saponaria.

Non-limiting examples of phospholipids for use in the immunogenic and vaccine compositions of the invention include molecules with diacylglyceride structures and phosphosphingolipids. Non-limiting examples of phospholipids with diacyglyceride structures include phosphatidic acid (phosphatidate) (PA), atidylethanolamine (cephalin) (PE), phosphatidylcholine (lecithin) (PC), dipalmitoyl atidylcholine (DPPC) or phosphatidylserine (PS). Another non-limiting example of phospholipids with diacylgylceride structures includes phosphoinositides. Exemplary phosphoinositides include, but are not limited to, phosphatidylinositol (Pl), phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate (PlP2) or phosphatidylinositol triphosphate (PIP3). Non- limiting examples of ospingolipids include, ceramide phosphorylcholine (Sphingomyelin) (SPH), ceramide phosphorylethanolamine (Sphingomyelin) (Cer-PE) or ceramide phosphorylglycerol.

Steroid molecules for use in the genic and vaccine compositions of the invention include molecules which incorporate a steroid as part of their structure. Non- limiting examples of d molecules include cholesterol, pregnenolone, 17-alpha-hyrdroxy pregnenolone, dehydroepiandrosterone, androstenediol, progesterone, 17-alpha-hydroxy progesterone, androstenedione, testosterone, dihyrdroxytestorone, deoxycorticosterone, 11— deoxycorticosterone, cortisol, corticosterone, aldosterone, estrone, estradiol or estriol.

In some embodiments, immunostimulating complexes are lly, but not limited to, small cage like structures 30-40 nM in diameter. In some ments, the formulation of an immunostimulating complex has a molar ratio of Quil A, cholesterol, phosphatidylcholine and G glycoprotein in a ratio of 5:1 :1. An immunostimulating complex may contain, for example, 5 to 10% by weight Quil A, 1 to 5% cholesterol and phospholipids and the remainder comprising G glycoprotein. G glycoprotein can be incorporated into the immunostimulating x either directly or by coupling to a carrier protein (eg. a chimeric or fusion protein) after incorporation of protein into immunostimulating complexes. Reference to an immunostimulating complex should be understood to include reference to derivatives, chemical equivalents and analogs thereof. For example, reference to a tive of an immunostimulating complex es nce to an immunostimulating complex in which one or more of Quil A, terol, phosphatidylcholine or protein, for example, are deleted, substituted for, or where a component in addition to Quil A, cholesterol, phosphatidylcholine or protein is added to the complex. The functional equivalent of an immunostimulating complex may be an immunostimulating x in which one or more of its four components are replaced with a functional equivalent. In some embodiment of the t invention, the G glycoprotein component of the immunostimulating complex is deleted. This type of immunostimulating complex is herein referred to as a protein-free immunostimulating In some embodiments the present invention includes, but is not limited to, an genic composition sing an isolated HeV or NiV G protein capable of inducing the production of a cross-reactive neutralizing anti-serum against multiple strains of HeV and/or NiV in vitro and an nt comprising Quil A, DPPC and terol, for example wherein the composition contains: 5, 50 or 100 pg of soluble HeV or NiV G protein, and appropriate amounts of Quil A, DPPC, and terol. Further exemplary embodiments of immunostimulatory complexes, and the preparation thereof, are described in EP 024238081 and EP 481, and also W02000041720 (see, for example, pages 3 and 9 n, referring to: Cox & Coulter (1992) Advances in Adjuvant logy and Application in Animal te Control tJtilizing Biotechnology, Chapter 4, Yong (ed.), CRC Press; Dalsgard (1974) Gesamte Virusforsch, 44, 243-254; lian Patent Specification Nos. 558258, 589915, 590904 & . See also the entative protocols described in US. Patent 6,506,386, and reference therein to the well known fact that immunostimulatory xes can be used wherein the protein antigen is included in the immunostimulatory complex when formed (see EP 0109942B1), or alternatively, preformed immunostimulatory complexes are provided which are then mixed with a separately added aliquot of antigen to form the vaccine (see EP 043662081). As will be generally recognized, the protein antigen can also be covaltently attached to the immunostimulatory complex (see again EP 0180564B1). As is also well recognized in the art, stimulatory complexes may be administered via muscosal vaccination (see Mowat (1991) |mmun0logy 72, 317-322) and stimulatory complexes of the present invention may be further ed for al vaccination by inclusion of membrane targeting proteins (WO 9730728).

In some embodiments the t invention includes, but is not limited to, an immunogenic composition comprising an isolated HeV or NiV G protein capable of inducing the production of a cross-reactive neutralizing anti-serum against multiple strains of HeV and/or NiV in vitro and an adjuvant comprising Quil A, DPPC and cholesterol, for example wherein the composition contains: 5, 50 or 100 pg of soluble HeV or NiV G protein, and appropriate amounts of Quil A, DPPC, and cholesterol. Further exemplary embodiments of immunostimulatory xes are described in W02000041720.

In another embodiment of the invention, the vaccine and immunogenic compositions may be part of a pharmaceutical composition. The pharmaceutical compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising ents and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically for delivery to the site of action.

C. Excipients The immunogenic and vaccine compositions of the invention can further comprise pharmaceutically acceptable carriers, excipients and/or stabilizers (see eg. Remington: The Science and practice of Pharmacy (2005) cott VWliams), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are WO 58643 nontoxic to recipients at the dosages and concentrations, and may comprise buffers such as . phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as y((o-carboxyphenyl)thio)ethy| sodium salt (THlOMERSAL), octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl n; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such as serum albumin, gelatin, or globulins; hydrophilic polymers such as nylpyrrolidone; amino acids such as glycine, glutamine, gine, histidine, arginine, or ; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; ing agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG), TWEEN or PLURONICS.

The compositions of the invention can be in dosages suspended in any appropriate pharmaceutical e or carrier in sufficient volume to carry the dosage. Generally, the final volume, ing carriers, adjuvants, and the like, typically will be at least 1.0 ml. The upper limit is governed by the practicality of the amount to be administered, generally no more than about 0.5 ml to about 2.0 ml.

Methods of Use The invention encompasses methods of preventing and/or treating Hendra and/or Nipah virus infection comprising administering the immunogenic and vaccine compositions of the invention in any mammalian subject. Active immunity elicited by vaccination with a HeV and/or NiV G rotein with the adjuvants described herein can prime or boost a cellular or humoral immune response. An effective amount of the HeV and/or NiV G glycoprotein or nic fragments thereof can be prepared in an admixture with an adjuvant to prepare a vaccine.

The invention encompasses methods of preventing and/or treating Hendra and/or Nipah virus infection in a human subject comprising administering an immunogenic and/or , vaccine composition comprising a soluble HeV and/or NiV G rotein or combinations thereof either by itself or in combination with at least one adjuvant le for use in humans. Adjuvants suitable for use in humans may be used alone or in combination.

Examples of adjuvants suitable for use in humans e, but are not limited to, aluminum salts. Examples of aluminum salts include. but are not limited to, aluminum hydroxide, aluminium hydroxide gel (Alhydrogelm), aluminum phosphate, alum (potassium aluminum sulfate), or mixed aluminum salts. Additional examples of adjuvants suitable for use in humans include, but are not limited to, water-in-oil emulsions, oil-in-water emulsions, and A804 (combination of aluminum hydroxide and monophosphoryl lipid A) and CpG oligodeoxynucleotides. CpG eoxynucleotides are synthetic oligonucleotides that contain unmethylated CpG dinucleotides in particular sequence contexts (CpG motifs).

These CpG motifs are present at a d greater frequency in bacterial DNA compared to mammalian DNA. CpG oligodeoxynucleotides are ized by Toll-like or 9 (TLR9) leading to strong immunostimulatory effects.

The administration of a vaccine or immunogenic composition comprising HeV and/or NiV G glycoprotein with one or more adjuvants described herein, can be for either a prophylactic or therapeutic purpose. In one aspect of the present ion the composition is useful for prophylactic purposes. When provided prophylactically, the vaccine composition is provided in advance of any detection or symptom of HeV and/or NiV infection. The prophylactic administration of an effective amount of the compound(s) serves to prevent or attenuate any subsequent HeV and/or NiV infection.

When ed therapeutically, the vaccine is provided in an ive amount upon the detection of a symptom of actual infection. A composition is said to be “pharmacologically acceptable" if its administration can be tolerated by a recipient. Such a composition is said to be administered in a “therapeutically or prophylactically effective amount” if the amount stered is logically significant. A vaccine or immunogenic composition of the present invention is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient, for example, by ing a broadly reactive humoral or cellular immune response to one or more strains of HeV and/or NW. The protection provided need not be absolute (i.e., the HeV or NiV infection need not ' be totally ted or ated), provided that there is a statistically significant improvement relative to a control tion. Protection can be limited to mitigating the severity or rapidity of onset of symptoms of the disease.

A vaccine or immunogenic composition of the present invention can confer resistance to multiple s of HeV and/or NiV. As used herein, a e is said to prevent or 2012/037839 attenuate an infection if its administration to a subject results either in the total or partial ation (i.e., suppression) of a symptom or condition of the infection, or in the total or partial immunity of the individual to the infection.

At least one vaccine or immunogenic ition of the present invention can be administered by any means that achieve the intended purpose, using a pharmaceutical composition as described herein. For example, administration of such a composition can be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes. In one embodiment of the present invention, the composition is administered by subcutaneously. Parenteral stration can be by bolus injection or by gradual perfusion over time.

A typical n for preventing, suppressing, or treating a e or condition which can be alleviated by a cellular immune response by active specific cellular immunotherapy, ses administration of an effective amount of a vaccine composition as described above, administered as a single treatment, or ed as enhancing or booster s, over a period up to and including one week to about twenty-four months. Non-limiting examples include a first dose ed by a second dose about at least 10, 11, 12, 13, 14, , 16, 17, 18, 19, 20, 21, 22, 23 or 24 days after the first dose (day 0). The amount of the dose of the immunogenic or vaccine composition may be the less than, the same as, or greater than the first dose administered at day 0.

According to the present invention, an “effective amount" of a vaccine or immunogenic composition is one which is sufficient to achieve a desired biological effect, in this case at least one of cellular or humoral immune response to one or more strains of HeV and/or NiV. It is understood that the effective dosage will be dependent upon the age, sex, health, and weight of the subject, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The ranges of effective doses provided below are not intended to limit the invention and represent examples of dose ranges which may be suitable for administering compositions of the present invention. However, the dosage may be tailored to the dual subject, as is understood and determinable by one of skill in the art, without undue experimentation.

The ents of the vaccine and immunogenic itions of the present invention can be any t which can acquire specific immunity via a cellular or humoral immune response to HeV and/or NiV, where the cellular response is mediated by an MHC class i or WO 58643 class ii protein. Among s. the recipients may be mammals of the orders primata (including , chimpanzees, apes and monkeys). In one embodiment of the present invention there is provided a method of treating humans with the vaccine or immunogenic compositions of the invention. The subjects may be infected with HeV and/or NiV or provide a model of HeV or NiV infection as in experimental studies. In some embodiments, the subject is a domesticated mammal including, but not limited to, a horse, cow, oxen, water o, sheep, pig (Mingyi (2010) Vet. Res. 41, 33), goat, dog curity Alert — Hendra Virus Update, 27 July 2011, Press Release, Biosecurity Queensland) or cat. In some embodiments, the subject is a fowl, including a chicken.

Vaccines of the present invention also provide for cross-protection against Nipah virus ion at doses used to protect against Hendra virus infection and thus also provide ive vaccination against Nipah virus.

Reference to an effective immune response should be understood as a reference to an immune response which either directly or indirectly leads to a beneficial prophylactic or therapeutic effect. In the case where the immunogen comprises a HeV or NiV G glycoprotein as described herein, such a response includes the reduction or blocking of viral reproduction and/or viral shedding and/or reduction in disease symptoms in an animal. It should be understood that efficacy is a functional measure and is not defined by reference to eV and/or iV antibody titre alone since the presence of circulating antibody alone is not arily indicative of the capacity of said circulating antibody to block viral reproduction and shedding.

Also by way of example, and not limitation, if a e G protein ptide of the invention is being administered to augment the immune response in a subject ed with or suspected of being infected with Hendra or Nipah and/or if antibodies of the present invention are being administered as a form of passive immunotherapy the composition can further comprise, for example, other therapeutic agents (e.g., anti-viral ).

Example 4 below provides information on certain preferred compositions for use in vaccinating horses. In regard of other s that may be infected with Hendra virus, and which therefore warrant vaccination to protect both animals and thus humans from both Hendra and Nipah virus infection, the following information is generally applicable and can readily be adapted by those skilled in the art. Generally speaking, companion animals (dogs and cats) warrant approximately 25 micrograms of Hendra antigen, and can benefit from an ISO adjuvant in the range of 25-150 micrograms, with a 5:1 :1 ratio of saponin, olipid and sterol being among the preferred ISC itions while using any of the component species as disclosed herein. For companion s it is preferred that the final dose be about 1 ml. nTM (MVP Technologies), a mer based adjuvant, may also be used at preferably about 5-15% (v/v).

Generally speaking, for larger farm animals (sheep, cows, pigs, etc.) the antigen and adjuvant dosing (and final dosing volume) s othenivise provided herein for horses are applicable, that is, from 50-100 micrograms of antigen, and typically about 250 micrograms of ISO may be used, final volume 1-3 ml for example). In regard of pigs, an alternative and effective adjuvant formulation involves (for approximately the same amount of antigen) a blend of lSC and ionic polysaccharide, cally 100 mg DEAE dextran and 800 micrograms ISO in 1-3 ml final dose volume (again 5:1 :1 of Quil Azphoshatidyl e:cholesterol (see )).

Differentiation of Vaccinated Animals The invention also encompasses methods of differentiating healthy vaccinated animals from animals exposed to, or infected with HeV and/or NiV. During viral infection, HeV and NW express additional ns other than G glycoprotein (G) including fusion protein (F), matrix protein (M), phosphoprotein (P), large protein (L) and nucleocapsid protein (N). These additional proteins have the potential to induce immune responses in s in the form of antibodies which bind to these proteins or T cell immunity. The level of antibody response to these other proteins can normally be measured by assays such as enzyme- linked immune assay (EIA). The immunogenic and vaccine formulations of the present invention, in some embodiments, contain only G glycoprotein as an HeV and/or NiV antigen and will therefore induce immune responses with antibodies only to the G glycoprotein of HeV and/or NiV. Animals vaccinated with the immunogenic compositions described herein which are subsequently ed by HeV or NiV will mount a booster immune se to the G glycoprotein, but will also show changes of antibody presentation to some other HeV and NW proteins other than G glycoprotein. Thus, the presence of antibodies to any of the fusion protein (F), matrix protein (M), phosphoprotein (P), large protein (L) and nucleocapsid protein (N) can be measured in an EIA to ine the presence or absence of antibodies specific to these proteins in serum samples. If antibody to any of these other proteins (i.e. other than G glycoprotein) is detected, then the animal hasbeen exposed to HeV and/or NiV.

Alternatively, if no antibody to these other proteins is found and only antibodies binding to G protein are detected, then the animal has only been ated.

The ElA of the present ion are both highly specific and highly selective in detecting and differentiating between animals infected with HeV and/or NW and healthy animals which have been vaccinated with the immunogenic compositions described herein.

The present invention may utilize a variety of assay procedures including ELISA in both homogenous and heterogenous environments. The assay ures may be conducted on samples such as blood, serum, milk, or any other body fluid containing antibodies.

In some embodiments, the antibodies used in the EIA may uniquely compete with antibodies induced by vaccination with the G rotein, but not antibodies induced in animals by infection with HeV and/or NiV. This allows not only serologic diagnosis of HeV and NW infection, but differentiation of vaccination from infection in a single assay. The EIA procedure may be performed on standard blood serum samples or any body fluids or secretions containing antibodies. The EIA procedure may employ either monoclonal and/or onal antibodies to G glycoprotein and any other HeV and/or NiV viral protein (e.g. fusion protein (F), matrix protein (M), oprotein (P), large protein (L) and capsid protein (N) as such proteins are not t in ated healthy animals which have not been exposed to HeV and/or NW). The EIA may be carried out in any number of commercially available fixed or portable-manual, semi-automated or robotics-automated ELISA equipment with or without computer assisted data analysis reduction re and hardware. In some embodiments, the methods of differentiating healthy vaccinated animals from animals exposed to, or infected with HeV and/or NiV may be conducted on a biological sample isolated from a domesticated mammal including, but not limited to, a horse, cow, sheep, pig, goat, dog or cat. In some embodiments, the subject is a fowl, including a chicken.

In some embodiments, the subject is a human.

Examples The ing examples illustrate only certain and not all embodiments of the ion, and thus, should not be viewed as limiting the scope of the invention.

Example 1: Vector Constructs Vectors were constructed to express transmembrane/cytoplasmic tail-deleted HeV G or NiV G. The cloned cDNA of ength HeV or NiV G protein were amplified by PCR to generate nts about 2600 nucleotides encoding the embrane domain/cytoplasmic tail-deleted HeV or NiV G protein.

The following oligonucleotide primers were synthesized for amplification of HeV G. sHGS: 5'-GTCGACCACCATGCAAAATTACACCAGAACGACTGATAAT—3' (SEQ ID NO: 5). sHGAS: 5'-GTTTAAACGTCGACCAATCAACTCTCTGAACATTG GGCAGGTATC-3'. (SEQ ID NO: 6).

The following oligonucleotide primers were synthesized for amplification of NW G. sNGS: 5'-CTCGAGCACCATGCAAAATTACACAAGATCAACAGACAA-3' (SEQ ID NO: 7). sNGAS: 5'-CTCGAGTAGCAGCCGGATCAAGCTTATGTACATT GTATC-S'. (SEQ ID NO: 8).

All PCR ons were done using Accupol DNA polymerase (PGS Scientifics Corp) with the following settings: 94°C for 5 minutes initially and then 94°C for 1 minute, 56°C for 2 minutes, 72°C for 4 minutes; 25 cycles. These primers generated a PCR product for the sHeV G ORF flanked by Sal 1 sites and the sNiV G ORF flanked by Xho 1 sites. PCR products were gel purified (Qiagen). After gel purification, sHeV G and sNiV G were subcloned into a TOPO vector (lnvitrogen).

PSectagZB (lnvitrogen) was purchased and d to contain a S-peptide tag or a myc-epitope tag. Overlapping oligonucleotides were sized that encoded the sequence for the S-peptide and ed Kpn 1 and EcoR1 overhangs.

SPEPS: 5'-CAAGGAGACCGCTGCTGCTAAGTTCGAACGCCAGCACATGGATT CT-3' (SEQ ID NO: 9). SPEPAS: 5'AATTAGAATCCATGTGCTGGCGTTCGAACTT AGCAGCAGCGGTCTCCTTGGTAC-3’. (SEQ ID NO: 10).

Overlapping oligonucleotides were synthesized that encoded the sequence for the myc-epitope tag and digested Kpn 1 and EcoR1 overhangs.

MTS: 5'-CGAACAAAAGCTCATCTCAGAAGAGGATCTG-s' (SEQ ID NO: 11). MTAS '-AATTCAGATCCTCTTCTGAGATGAGCTTTTGTTCGGTAC-B' (SEQ ID NO: 12). 64 pmol SPEPS and 64 pmol SPEPAS were mixed and heated to 65°C for 5 minutes and cooled slowly to 50°C. 64 pmol MTS and 64 pmol MTAS were mixed and heated to 22 . 65°C for 5 minutes and cooled slowly to 50°C. The two mixtures were diluted and cloned into Kpn1-EcoR1 digested pSecTagZB to te S-peptide modified pSecTagZB or myc- epitope modified pSecTagZB. All constructs were initially screened by restriction digest and further verified by sequencing.

The TOPO sG construct was digested with Sal 1 gel d (Qiagen) and ned in frame into the Xho 1 site of the S-peptide modified pSecTagZB or myc-epitope modified pSecTang. All constructs were initially screened by restriction digest and further verified by cing.

The IgK Ieader—S-peptide—s HeVG (sGs.,ag) and the IgK leader-myc tag-sHeVG (sGmyc, tag) constructs were then subcloned into the vaccinia shuttle vector pMC02. Oligonucleotide SEQS: ACCCACCATGGAGACAGACACACTCCTGCTA-B' (SEQ ID NO: 13) was synthesized and used in combination with ucleotide sHGAS to amplify by FOR the sGS- tag and sGmymg. All PCR reactions were done using l DNA polymerase (PGS Scientifics Corp.) with the following settings: 94°C for 5 s initially and then 94°C for 1 minute, 56°C for 2 minutes, 72°C for 4 minutes; 25 cycles. These primers generated PCR products flanked by Sal 1 sites. PCR products were gel d (Qiagen). After gel purification, sGstag and sGmyG.£19 were subcloned into a TOPO vector (lnvitrogen). sG S-tag and sG myc-tag were digested with Sal 1 and ned into the Sal 1 site of pMC02. All constructs were initially screened by restriction digest and further verified by sequencing. A codon optimized nucleotide sequence was subsequently generated to facilitate production in euckaryotic cell lines which is depicted in SEQ lD NO: 16.

Example 2: Protein Production of Soluble G Protein using Vaccinia For protein production the genetic constructs ning the codon optimized sequences were used to generate recombinant us vectors (vaccinia virus, strain WR).

Recombinant poxvirus was then obtained using standard techniques employing tk-selection and GUS staining. Briefly, CV—1 cells were transfected with either pMC02 sHeV G fusion or pMC02 sNiV on using a calcium phosphate transfection kit ga). These monolayers were then infected with Western Reserve (WR) wild-type strain of vaccinia virus at a multiplicity of infection (MOI) of 0.05 PFU/cell. After 2 days the cell pellets were collected as crude recombinant virus stocks. TK‘ cells were infected with the recombinant crude stocks in the presence of 25 ug/ml o—2'-deoxyuridine (BrdU) (Calbiochem).

After 2 hours the virus was replaced with an EMEM-10 overlay containing 1% low melting point (LMP) agarose (Life Technologies) and 25 ug/ml BrdU. After 2 days of incubation an additional EMEM-10 overlay containing 1% LMP agarose, 25 pig/ml BrdU, and 0.2 mg/ml 5- Bromo—4-chloroindolyI-B-D—glucuronic acid (X-GLUC) (Clontech) was added. Within 24-48 hours blue s were evident, picked and subject to two more rounds of double selection plaque purification. The recombinant vaccinia viruses vKB16 (sHeV G fusion) and vKBZ2 (sNiV G fusion) were then amplified and purified by standard methods. Briefly, recombinant vaccinia viruses are purified by plaque purification, cell-culture amplification, sucrose cushion pelleting in an ultracentrifuge and ion by plaque assay. Expression of sHeV G was verified in cell lysates and e supernatants.

Example 3: Protein Production of e G Protein using 293F Cells c constructs containing the codon zed ces were used to transform 293F cells (lnvitrogen) to produce a stable cell line which expresses HeV soluble G glycoprotein. CHO~S cells (lnvitrogen) may also be used for transformation and expression of HeV soluble G glycoprotein. Transformed cells are plated on 162 cm2 tissue culture flask with ml DMEM-10. Cells were allowed to adhere and grow at 37°C with 5-8% 002 for l days. When cells were confluent, they were split into multiple flasks with DMEM-10 with 150 ug/ml ycin B (30 ml per flask). When the cells are 70-80% confluent, they were washed twice with 30 ml PBS, then 20 ml of 293 SFM || (lnvitrogen) was added and the cells were incubated at 37°C with 5-8% C02 overnight. On the next day, cells were transferred into Erlenmeyer flasks with 200 ml SFM || media. Cells were allowed to grow at 37°C with 5- 8% CO; at 125 rpm for 5-6 days until cells started to die. At that time, the supernatant is collected.

Media from each Erlenmeyer flask is centrifuged at 3,500 rpm for 30 minutes. The supernatant was then transferred into 250 ml centrifuge s and spun at 10,000 rpm for one hour. The resulting supernatant is collected and protease inhibitor is added according to manufacturer’s recommendation along with Triton X-100 to final concentration of 0.1%. The supernatant is then filtered through a 0.2 pm low protein binding filter membrane.

HeVsG is purified through use of an S-protein agarose affinity column. A 20 ml bed volume of S-protein agarose (Novagen) is loaded into a XK 26 column (GE Healthcare). The column is washed with 10x bed volumes of Bind/Wash buffer (0.15 M NaCl, 20 mM Tris-HCI, pH 7.5 and 0.1% Triton X-100). The prepared supernatant of HeV $6 is applied to the column to maintain a flow rate of 3 ml/min. The column is washed with 10x bed volumes (200 ml) of ash buffer I followed by 6x bed volumes (120 ml) of wash buffer 1x Wash Buffer (0.15 M NaCl, and 20 mM Tris-HCI, pH 7.5).

The pump is then stopped and the Wash Buffer is allowed to drain until it reaches the surface of the beads when 30 ml of Elution Buffer (0.2 M Citric Acid, pH 2) is added. The first 10 ml of flow through (this should still be the wash ) is collected and then the elution buffer is incubated with the beads for 10 s. Next, 15 ml of the eluate is collected into a 50 mL sterile conical fuge tube containing 25 ml of neutralization buffer (1 M Tris, pH 8). The pH is adjusted to neutral and the elution and tion is repeated three times. All of the neutralized eluate is combined and concentrated to about 4 ml. The collected HeV 3G (4 ml) is purified through a 0.2 pm low protein binding filter membrane (Acrodisc 13 mm Syringe Filter with 0.2 pm HT Tuffryn Membrane.

Gel tion can be utilized to further purify the HeV sG. After quality l analysis and confirmation of purity and oligomeric state, aliquot HeV sG pooled fractions of tetramer+dimer, dimer and monomer are stored at -80°C.

Example 4: Preparation of Vaccine Formulation A schematic summarizing the preparation of ISO is set forth in Figure 3 and is further described below.

Step 1: A solution of 90 g/L decanoyl-n-methylglucamide (Mega-1O detergent) is prepared in Water For Injection-(WFI). The solution is heated to ensure total dissolution of Mega 10 then it is either used immediately in Step 2 or filter sterilized.

Step 2: A solution containing 25 g/L cholesterol and 25 g/L dipalmitoyl phosphatidyl choline (DPPC) is prepared by ving these components in the stock solution of Mega 10 ent. The solution is heated to dissolve all components then either used immediately in Step 3 or filter sterilized.

Step 3: Buffered ic Saline, 10 mM phosphate buffer, pH 6.2 i 1 (BIS) is prepared with WFI and sterile filtered if not used ately.

Step 4: Quil A is prepared in BIS to final concentration of 100 g/L and sterile filtered if not used immediately.

Step 5: ISO is formulated in an agitated temperature controlled vessel (22-37°C) by tial addition of pre-heated BIS, cholesterol/DPPC in 0 on (160 ml/L), and Quil A solution (200 ml/L). The reaction is brought to target volume by addition of BIS.

Step 6: The entire formulation is equilibrated to the required temperature (Target 27°C with acceptable operating range 22-37°C) then incubated for 15 minutes with agitation to tate ISC formation. The ISC solution is either processed further in Step 7 or sterile filtered for intermediate storage.

Step 7: The ISC on mixture is washed by dialysis (Membrane: Hydrosart 30 kDa (Sartorius AG ngen)) for a minimum of 20 volume exchanges against BIS under temperature control (Target 27°C with acceptable operating range 21-37°C) to remove uncomplexed components.

Step 8: Dialyzed ISO is concentrated approximately 2-fold by ultra-filtration using the same membrane as that used for dialysis. The filtration system is rinsed with BIS to restore ISO to original volume.

Step 9: ISO is transferred to a sterile storage container via sterile filtration through a 0.22 pm cellulose acetate filter.

Step 10: ISO adjuvant is stored at 2-8°C until released for use in vaccine formulation.

The immunostimulatory composition (250 ug/ml) is then combined with riate amounts of soluble HeV G glycoprotein (eg. 5, 50, 100 pg/ml) and ed to volume in BIS.

Example 5: First Clinical Experiment in Horses Test vaccine 1: Recombinant Hendra virus soluble glycoprotein (sG) at 100 ug/dose adjuvanted with 250 pg of immune stimulating complex; volume is adjusted to 1 ml/dose with saline on.

Test vaccine 2: Recombinant Hendra virus soluble glycoprotein (sG) at 50 pg/dose nted with 250 pg of immune stimulating complex; volume is ed to 1 ml/dose with saline solution.

Test vaccine 3: Recombinant Hendra virus soluble glycoprotein (sG) at 5 pg/dose adjuvanted with 250 ug of immune stimulating complex; volume is adjusted to 1 mI/dose with saline solution.

Serological and challenge protection data from horses has been collected from two lots of horses given the vaccines containing the higher levels of antigen (50 ug/dose and 100 pg/dose). gy: Two horses were each immunized with two vaccine doses (100 pg 3G with ISO) 21 days apart. Post-priming and pre-challenge serology confirmed vaccine-induced seroconversion to HeV (Table 1). Pre-challenge virus neutralizing antibody levels were comparable to those which had been found to be protective in cats exposed to an otherwise lethal dose of the closely related Nipah virus. The horse receiving adjuvant only (negative control) did not develop antibody to HeV prior to viral challenge.

Table 1 Horse No. Baseline titre rime Pre-challenge titre titre V1 32 1024 V2 32 512 V3 (Control) <2 Accordingly, each horse was exposed to live HeV in a BSL4 containment facility 27 days after ing the booster immunization. Virus was administered intranasally (1 x 10 6TCle) and orally (1 x 10 6TCleo). At the time of challenge and for the period of observation thereafter, the identity of the control horse was not known by staff involved in this part of the work.

CliniCal observations for V1: This horse remained clinically well during the period of observation ing exposure to HeV apart from a zed ion of the entry site of the indwelling jugular catheter noted on day 8 hallenge. This was not associated with any constitutional signs of disease. The horse was vely euthanized on day 9 after viral challenge. Abnormalities at gross post mortem examination were confined to a 10 cm mesenteric lipoma (incidental finding) and mild dilation of lymphatic vessels at the l tip of the left cardiac lung lobe that was attributed to barbiturate. lnitial screening of tissues has found no evidence of either lesions or HeV antigen in this horse.

Clinical observations for V2: This horse developed a mild transient nasal discharge on day 3, but then remained otherwise well until a temperature rise on day 6 associated with a localized atory reaction at the site of her indwelling jugular catheter. The catheter was removed, but the lesion continued to enlarge and the horse was becoming quite irritable so on the following day (d 7) the mare was treated with long-acting penicillin. Both her temperature and her temperament had returned to normal on day 8 and she was vely euthanized. Abnormalities at gross post mortem examination were confined to mild dilation of lymphatic vessels at the ventral tip of the right cardiac lung lobe that was attributed to barbiturate. l screening of tissues has found no evidence of either lesions or HeV n in this horse; detailed examination is currently being ted.

Clinical observations for V3: This horse developed a mild transient nasal discharge on day 4, but then remained otherwise well until a temperature rise on day 6 without localizing signs. Her heart rate had also risen, and she had slight tenting of the skin tent with mild dehydration and a tucked-up appearance. This constellation of signs is typical of acute HeV infection under our laboratory conditions. Her temperature and heart rate continued to increase over the g 12 hours (Figure 1 and 2), she was slightly depressed, and so she was euthanized on humane grounds on day 7. At post mortem examination there was moderately severe dilation of lymphatic vessels on the cardiac lobes of the lung with involvement of the ventral 8-10 cm accompanied by pleural ning and edema.

On histological examination there was ary vasculitis with id necrosis of vascular walls, edema of interlobular septa and fOCal necrotizing alveolitis. There was extensive deposition of HeV antigen in the endothelium and media of blood vessels in the lung; meninges; brain parenchyma; trigeminal ganglion; submandibular, bronchial, inguinal and renal lymph nodes; spleen; liver; heart; soft ; adrenal gland; renal glomeruli; small and large intestines; ovary; pharynx and turbinates as well as germinal centers in the spleen and occasional cardiac myocytes. Spinal cord, guttural pouch, bladder, and ory pole of the brain were negative. The histology and immunohistology was consistent with peracute HeV infection. lar analysis of clinical samples. There was no ce for shedding of HeV in any biological sample collected from immunized horses V1 and V2 throughout the period of clinical observation. Specifically, no genome was red from either deep nasal swabs or from blood on any day post-exposure.

In contrast, in the non-immunized horse V3, viral genome was detected in nasal swabs from day 3 after challenge. Decreasing Ct values on successive ng days is suggestive of viral replication in upper respiratory tract and is consistent with earlier observations from our laboratory following exposure of naive horses to HeV Redlands 2008.

The finding of viral genome in blood ately prior to the onset of fever, and in all secretions thereafter, coinciding with the earliest recognition of other al signs such as depression is also consistent with r observations.

A V _ ESamp_le dayi V _i U U U U U U U u u U U u U U u U U u U U U U U U u U U U Faeces U U U U U U U ‘ l EDTAblood Ur ,U “WU U ,U U U ,.

= I i ' HorseWZ E l 2 _ l, Oral swab U U U U U 4-241, U U .

Rectal swab U U U U U U U U i Nasal swab U U U U U U U U Urine u u U U U u U u Faeces U U U U U U U U y A EDTA blood UN cam , U U U a 7U _ _ U, U , . h _ . _._.% ; , , -. _c_,, .3. V , ,wfil i, -.

-Jiqrzettvé,,- ' A , , , . , ; A._9.'3L5_“LaL” U U U U U _ .; -_ {Jectgljm_ U U U U U U U _____: L,_Na§§|.§vxyb__ U U l Urine U U U u U u «1.34; i Faeces . U U U U U U U m i EDrAbIood ~ AL, U U ,___ i U a U m- Post mortem samples. TaqMan PCR (HeV N-gene) confirmed replication of the challenge virus in V3 (Control) with dissemination of infection to multiple s (Table 3). Highest levels of replication ed to be present in lung, spleen, kidney, myocardium, and lymphoid tissues associated with the upper and lower respiratory tracts as previously reported. There was no evidence of virus replication in tissues of immunized horses (V1 and V2).

Table 3 Tissue Type Horse #V1 Horse # V2 Horse #V3 Adrenal Bladder Brain Gutteral Pouch Heart Horse Kidney Horse Large lntestine Liver Lung Lymph-Bronchial Lymph-Head {:5a Inguinal Lymph- Man cfibular Lymph—Renal Meninges NasaI turbinate Nerve Olfactory Lobe Ovaries , 411] Pharynx Small Intestine Spinal Cord Spleen Trigeminal ganglion Uterus ECCCCCCCCCCCCCCCCCCC CCCCCCCCCCCCCCéCCCCCCCCCCC Vin—o i U= Negative LficafiQn) V [001 O8] Post-challenge serology. lmmunized horses V1 and V2 did not have a boost in titre following HeV challenge (Table 4). This is consistent with no significant replication of the challenge virus in these animals. No dy was detected in the control horse V3 at the time of euthanasia on hallenge day 7. It was considered that there had been insufficient time between virus exposure and death of this animal for generation of detectable antibody, and is consistent with previous observations in our laboratory with HeV Redlands in the horse.

Table 4 Horse No. Baseline Post-prime Pre-challenge Terminal titre titre titre titre V1 32 1024 128, 128 da 9 V2 32 512 128, 256 (day 8) V3 (Control) <2 <2, <2 (day 7) Two horses (V1 and V2) that were vaccinated with 100 ug sG + ISC adjuvant in a prime-boost regime seroconverted to HeV prior to HeV exposure. One horse (V3) that received ISC only ed seronegative to the challenge virus.

Following challenge with an othen/vise lethal dose of HeV, immunized horses ed ally well throughout the period of observation, which surpassed the time of onset of all experimentally induced cases of HeV in horses. The horse with no gical evidence of immunity (V3) was ized after developing clinical signs consistent with acute HeV. No boosting of antibody titre was ed following challenge in zed horses, consistent with no ation of the challenge virus in these animals.

There was no evidence of viral shedding by immunized horses, as reflected by PCR negative test results on all daily clinical samples. In the non-immunized control, viral genome was detected in nasal swabs from day 3 after exposure to virus, in blood immediately prior to the onset of fever, and in all clinical samples from the time fever was established. This pattern of shedding is tent with that found in naive horses exposed to HeV in an earlier study at this facility.

There was no evidence of HeV viral replication in any tissue of immunized horses collected at post mortem examination, following euthanasia during what would be expected to be the period of acute infection. In contrast, HeV genome and antigen were buted throughout the tissues of the control horse in a pattern consistent with acute HeV infection, and vasculopathy typical of HeV infection was also identified.

Example 6: Second Clinical Trial in Horses Three horses were each immunized with two vaccine doses (50 pg 56 with ISO) 21 days apart. Post-priming and pre-challenge serology confirmed vaccine-induced seroconversion to HeV (Table 5). Pre—challenge virus neutralizing antibody levels were comparable to those which had been found to be protective in cats d to an otherwise lethal dose of the closely related Nipah virus and to horses exposed to HeV in the first clinical trial described herein. A horse receiving adjuvant only did not develop antibody to HeV prior to viral challenge of immunized horses (data not displayed).

Table 5 Baseline titre Post-prime Pre—challenge titre titre 256/128 2048/>8192 512/1024 Accordingly, each immunized horse was exposed to live HeV in a BSL4 nment facility 27 days after receiving the booster immunization. Virus was administered intranasally (1 x 106 TCIDso) and orally (1 x 106 . Four guinea pigs were employed in this study as pathogenicity controls with the expectation that at least one of these would b to HeV disease. Guinea pigs were exposed to 50,000 TCIDso HeV by the intraperitoneal route.

Clinical ations for V4: This horse remained clinically well during the period of observation following re to HeV and temperature and heart rate remained within normal limits. The horse was electively euthanized on day 8 after viral challenge. No abnormalities were noted at gross postmortem examination. Initial ing of tissues has found no evidence of either lesions or HeV antigen in this horse; detailed examination is currently being completed. [001 16] Clinical observations for V5: This horse remained clinically well during the period of ation following exposure to HeV and temperature and heart rate remained within normal limits (Figure 2). The horse was electively euthanized on day 7 after viral challenge. No abnormalities were noted at gross post mortem examination. Initial screening of tissues has found no ce of either lesions or HeV antigen in this horse; detailed ation is currently being completed.

Clinical observations for V6: This horse ed clinically well during the period of observation following re to HeV and temperature and heart rate remained within normal limits (Figure 2). The horse was electively euthanized on day 9 after viral challenge. No alities were noted at gross post mortem examination. initial screening of tissues has found no evidence of either s or HeV antigen in this horse; detailed examination is currently being completed.

Guinea pigs: One of 4 guinea pigs (No. 3) started to lose weight on day 3 after HeV challenge. Weight loss progressed until day 5 when the animal exhibited neurological signs (head tilt, tremor) and was euthanized. Abnormalities at post mortem examination were confined to edema of the retroperitoneal tive tissues.

On histological examination there was pulmonary vasculitis, vasculitis of peri— renal blood vessels, oophoritis, and non-suppurative encephalitis associated with deposition of HeV antigen. The histology and immunohistology were consistent with acute HeV infection and confirmed the pathogenicity of the challenge virus.

There was no evidence for ng of HeV in any biological sample collected from V4, V5 or V6 hout the period of clinical observation apart from a rectal swab from V6 on day 3 in which a Ct value (HeV N gene) of 36.2 was observed by TaqMan PCR in one of two replicate wells with the second well exhibiting no amplification (Table 6). Specifically, no genome was recovered from either deep nasal swabs or from blood on any day post- exposure.

WO 58643 Table 6 , 'e'n'ei‘lia'o Mangare‘émtgsgfi 1 .Deily samples day , Sample l 0 N u 4 01 0') Horse #V4 Oral swab Rectal swab —_ uasal swab Urine Faeces ,_ ., ; "E-DTA blood cccccc “cccccc ‘jcccccc, ,cccccc Horse W5 Oral swab u u Rectal swab u u l Nasal swab u u Urine u u fleeces u U EDTA blood CCCCCC U _U, -Jgraewe-‘ . , ,Qra'éwk M Bscglswab _ ‘. ,Nssabwab ., Urine Faeces EDTA blood ‘CCCCCC CCCCCC, , _ Colour code: Post mortem s. There was no evidence of virus replication in tissues of immunized horses V4, V5 or V6. In one guinea pig (No. 3), viral genome was detected in blood (Ct 34.2), brain, lung, and spleen on day 5 after challenge corroborating the clinical, histological and immunohistological findings of acute HeV infection in this animal (Table 7).

Table 7 {TjgyeIme ,.

H. rse #V4i“ Arjl-jor—se # V5 Home _#V6 . 7—..- "TGuinea-pig #3 _- , _ oer... __r_.e.-._ fiv-’ - i__ _._ ,4-» __ Kaenal —__‘ H Blgddfl U _ _ ,Bgm1_ U , - ‘csr U ESSLtteeLEwL ,_._ _ U %.H§§r1591§e___ U :Kidggy. Horse ____~ U {Lstgflniesfinem ._§ U éLiver ____- U , ,__-~ lymph-Bronchial U lymph-Head U inal .. CCCCCCCCCCCCCC U iymph- Mandibular U :Lyrnph-Ben.a1__ :3 U LMeAi299§____ U ymdzioete.--‘ w U =N_er_V.e U :Olfactory_l_.o_b_e _ U :9y1ri55m_ ,,____. U flew! U 3Small Intestine I U f§pinal Cord 5 U 3Spleen g ’IvU- ”V {ETg‘geminalAggnglion 1 § Uterus. fccccccccccc‘ "CCCCCCCCCCCCCCCCCCCCCCCCCCI Colour code: Post-challenge serology. lmmunized horses V4, V5 and V6 did not have a boost in titre following HeV challenge (Table 8). This is tent with no significant replication of the challenge virus in these animals.

Table 8 Horse No. Baseline titre Post-prime Pre-challenge Terminal titre ' titre V4 256/128 256/32 da 8 V5 2048l>8192 1024/512 da 7 V6 512/1024 28/256 da 9 Three horses (V4, V5 and V6) that were vaccinated with 50 pg sG + ISC adjuvant in a prime-boost regime nverted to HeV prior to HeV exposure. One horse that received ISC only remained seronegative to the challenge virus.

Following challenge with an otherwise lethal dose of HeV, immunized horses remained clinically well throughout the period of ation, which surpassed the time of onset of all experimentally induced cases of HeV in horses. One guinea pig used as a pathogenicity control was euthanized after developing clinical signs consistent with acute HeV. No boosting of dy titre was detected following challenge in immunized horses, consistent with no replication of the challenge virus in these animals.

] There was no evidence of viral ng by immunized horses, as reflected by PCR negative test results on all daily clinical s apart from one replicate from a rectal swab from V6 on day 3. This test is being repeated; should a similar result be observed one explanation is that this represents a low level of residual inoculum. In one non-immunized guinea pig, viral genome was ed in major organs and blood on day 5 after exposure to virus.

There was no evidence of HeV viral replication in any tissue of immunized horses collected at post mortem examination, following euthanasia during what would be ed to be the period of acute infection. In contrast, HeV genome and antigen were distributed throughout the tissues of a susceptible guinea pig in a pattern consistent with acute HeV infection, and vasculopathy typical of HeV infection was also identified in this animal.

Example 7: Clinical Trial in Primates for Nipah Virus Statistics. Conducting animal studies, in particular non-human e studies, in biosafety level 4 (ESL-4) ly restricts the number of animal subjects, the volume of biological s that can be obtained and the ability to repeat assays independently and thus limit statistical analysis. Consequently, data are presented as the mean or median calculated from replicate s, not replicate assays, and error bars represent the standard deviation across replicates.

Viruses. NiV—Malaysia (GenBank Accession No. AF212302) was ed from the Special Pathogens Branch of the Centers for Disease Control and Prevention, Atlanta, Georgia. NiV was propagated and titered on Vero cells as described for HeV in Rockx et al. (2010) J. Virol. 84, 9831.

Vaccine formulation. Three e formulations of sGHeV were employed (10 pg, 50 pg or 100 pg). Production and purification of sGHeV was done as previously bed in Pallister (2011) Vaccine 29, 5623. Each vaccine formulation also contained AllhydrogelTM (Accurate Chemical & Scientific Corporation) and CpG oligodeoxynucleotide (ODN) 2006 (lnvivogen) containing a fully phosphorothioate backbone. Vaccine doses containing fixed amount of ODN 2006, g amounts of sGHeV and aluminum ion (at a weight ratio of 1:25) were formulated as follows: 100 pg dose: 100 pg sGHeV, 2.5 mg aluminum ion and 150 pg of ODN 2006; 50 pg dose: 50 pg sGHeV, 1.25 mg aluminum ion and 150 pg of ODN 2006; and 10 pg dose: 5 pg sGHeV, 250 pg aluminum ion and 150 pg of ODN 2006. For all doses, ogelTM and sGHeV were mixed first before ODN 2006 was added. Each vaccine dose was adjusted to 1 ml with PBS and es were incubated on a rotating wheel at room ature for at least two to three hours prior to injection. Each t received the same 1 ml dose for prime and boost and all vaccine doses were given via intramuscular injection.

Animals. Ten young adult African Green Monkeys (AGM) ocebus aethiops), weighing 4-6 kg (Three Springs Scientific Inc.) were caged individually. Subjects were anesthetized by uscular injection of ketamine (10-15 mg/kg) and vaccinated with sGHeV on day -42 (prime) and day —21 (boost). Three subjects received two 10 pg doses (AGM 16, AGM 17, AGM 18), three subjects received two 50 pg doses (AGM 13, AGM 14, AGM 15), three animals received two 100 pg doses (AGM 10, AGM 11, AGM 12) and one subject (AGM 9) received adjuvant-alone. On day 0, subjects were anesthetized and inoculated intratracheally with 1 x 105 TCIDso n tissue culture infectious dose) of NW in 4 ml of Dulbecco’s minimal essential medium (DMEM) (Sigma-Aldrich). Subjects were anesthetized for clinical examinations including temperature, respiration rate, chest radiographs, blood draw and swabs of nasal, oral and rectal mucosa on days 0, 3, 5, 7, 10, 14, 21 and 28 post-infection (p.i.). The control subject (AGM 9) had to be euthanized according to approved humane end points on day 10 post-infection. All other subjects survived until the end of the study and were euthanized on day 28 post-infection. Upon necropsy, various s were collected for virology and histopathology. Tissues sampled include: conjunctiva, tonsil, oro/naso pharynx, nasal mucosa, trachea, right bronchus, left bronchus, right lung upper lobe, right lung middle lobe, right lung lower lobe, light lung upper lobe, light lung middle lobe, light lung lower lobe, bronchial lymph node (LN), heart, liver, spleen, kidney, adrenal gland, as, jejunum, colon transversum, brain (frontal), brain (cerebellum), brain stem, cervical spinal cord, pituitary gland, mandibular LN, salivary LN, inguinal LN, axillary LN, mesenteric LN, urinary bladder, testes or ovaries, l bone marrow. Vaccination was done under BSL-2 containment. A timeline of the ation schedule, challenge and biological specimen collection days is shown in Figure 4.

Vaccination and NW challenge. Previously, we have trated that intratracheal inoculation of AGMs with 105 TCleo (median tissue culture infectious dose) of NW caused a uniformly lethal outcome (Rockx et al. (2010) J. Virol. 84, 9831). Rapidly ssive clinical illness was noted in these studies; clinical signs included severe depression, respiratory disease leading to acute respiratory ss, severe ogical disease and severely reduced mobility; and time to reach approved humane endpoint ia for euthanasia ranged from 7 to 12 days. Here we sought to determine if vaccination with sGHeV could prevent NiV infection and disease in AGMs. Doses of 10, 50 or 100 pg sGHeV were mixed with alum and CpG moieties as described in the Methods. Each vaccine formulation was administered subcutaneously to three subjects on day 0 (prime) and again on day 21 (boost) and one control t (AGM 9) received an adjuvant alone prime and boost on the same days. On day 42. all subjects were inoculated intratracheally with 105 TCleo NW. The control subject (AGM 9) showed loss of appetite, severe sustained behavior s (depression, decreased activity, hunched e), decreases in platelet number and a gradual increase in respiratory rate at end-stage disease. Subsequently, AGM 9 ped acute respiratory distress and had to be euthanized according to approved humane end points on day 10 post-infection. In contrast, none of the vaccinated ts had al disease and all survived until the end of the study. A Kaplan-Meier survival graph is shown in Figure 5.

W0 2012/158643 NiV-mediated disease in the control t. Gross pathological changes in the control subject were tent with those found previously in NiV-infected AGMs (Geisbert et al. (2010) PLoS One 5, ). Splenomegaly and congestion of blood vessels on surface of brain were present and all lung lobeswere wet and heavy. NiV RNA and infectious virus were not recovered from AGM 9 blood samples and there was no evidence of a. AGM 9 had significant levels of NiV-specific lgM and detectable NiV- specific lgG and lgA. Further analysis of tissue samples ed an extensive NiV tissue tropism similar to the wide-spread NiV infection seen previously in AGMs (Geisbert et al. (2010) PLoS One 5, e10690). AGM 9 had NiV RNA in the majority of tissues as ted and infectious virus was recovered from numerous tissues. Significant lesions included interstitial pneumonia, subacute encephalitis and necrosis and hemorrhage of the splenic white pulp. Alveolar spaces were filled by edema fluid, fibrin, karyorrhectic and cellular debris, and alveolar macrophages. Multifocal encephalitis was characterized by ion of Virchow-Robins space by moderate s of lymphocytes and fewer neutrophils.

Smaller numbers of these inflammatory cells extended into the adjacent parenchyma.

Numerous neurons were swollen and ated (degeneration) or were fragmented with karyolysis (necrosis). Multifocal germinal centers of follicles/in splenic white pulp were effaced by hemorrhage and fibrin, as well as small numbers of neutrophils and cellular and karyorrhectic . These findings were consistent with is and loss of the germinal centers in the spleen. Extensive s of viral antigen were present in the brainstem ght the extensive damage NiV causes in the central nervous system.

Protection of sGHeV-vaccinated subjects. All biological specimens, including all blood samples collected following challenge and all tissues collected upon necropsy, were negative for NiV RNA and infectious virus was not isolated from any specimen. Upon closer examination of tissue sections from vaccinated subjects, tissue architecture appeared normal and NW antigen was not detected in any tissue using immunohistochemical techniques. To further dissect the vaccine-elicited mechanisms of tion, serum and mucosal sGNiV— and specific lgM, lgG and lgA as well as NW and HeV serum neutralization titers were ed in vaccinated animals. As demonstrated in Figure 6, seven days prior to challenge, subjects receiving the lowest sGHeV dose had detectable antigen-specific serum lgM and the highest level of sGHeV-specific serum lgG. Subjects given 50 ug sGHeV also had detectable levels of serum lgM and their highest levels of serum lgG seven days prior to challenge. High dose subjects had no able serum lgM and serum lgG levels were significantly less on day -7 as compared to the other two groups. By the day of NW nge, serum lgG levels in the high dose subjects had increased and all vaccinated subjects had similar lgG levels. Serum lgM levels did not change in any subject following NiV challenge. Serum lgG levels decreased in the medium dose subjects the day of NW challenge and lgG levels decreased in low dose subjects just after NiV challenge. stingly, lgG levels increased in both of these groups by day 3 and day 5 pi. but never surpassed the lgG levels present seven days prior to challenge and in both groups titer decreased significantly by day 28 pi.

Conversely, serum lgG levels in the high dose group remained high and were at their highest of day 28 pi. Antigen-specific serum lgA was detectable in all subjects following vaccination; however, levels were very low and pre- and hallenge levels did not appear to be significantly different (Figure 6). A minimal increase in mucosal antigen- specific lgA was detected in nasal swabs from low dose subjects on day 14 p.i., however, the levels were so low these mucosal antibodies likely played no role in preventing the spread of NW following challenge. Results from serum neutralization tests (SNTs) are shown in Table 9. For all vaccinated subjects, ecific neutralization titer remained the same or sed by day 28 pi. and NiV-specific neutralization titer did not change significantly by day 7 p.i., even in ts that had the lowest titer prior to challenge. One low dose and one high dose subject had a log se in NW SNT titer by day 14 pi. and one medium dose subject had a log increase in NW SNT titer by day 21 pi. For all other vaccinated animals, changes in SNT titer were either inconsistent (titer would increase and then decrease) or insignificant (titer increased by 3—4 fold but not more than a log). y, seroconversion to the NW fusion (F) pe glycoprotein was measured in vaccinated subjects following NiV challenge. Minimal levels of serum anti-NiV F lgM were detected in the low and medium dose subjects on day 10 and day 21 p.i., respectively, and these low M.F.l. values t a weak primary antibody response following NiV challenge. Serum anti-NiV-F lgM was not detected in the high dose subjects suggesting these animals had little to no circulating virus following challenge. <20 <20 < <20 379 226 2147 <20 134 134 453 537 <20 189 134 189 453 ' 13 <20 >2560 >2560 >2560 757 <20 379 189 189 50 H9 14 <20 1514 >2560 >2560 537 <20 28 47 226 4 134 <20 2147 757 >2560 905 <20 67 95 757 1074- <20 >2560 2147 1810 453 <20 67 113 268 453 100 HQ 11 <20 >2560 >2560 >2560 1514 <20 134 189 905 1514 12 <20 >2560 >2560 >2560 757 <20 189 226 >2560 1514 Example 8: Clinical Trial in Primates for Hendra Virus ] A second clinical trial was conducted in AGM to assess vaccination and challenge with Hendra virus. The same formulation as set forth in Example 7 was utilized as a vaccine but was also compared to another group that received sGHeV with AlhydrogelTM alone as an adjuvant (no ODN 2006 was present). Animals were vaccinated day -21, boosted on day 0, and challenged on day 21. Unless othenNise indicated, all conditions were the same as those on Example 7. An experimental y is below: Ema—I__ 100 ug/dose Hendra sG vaccine + ane +. 1 b°ngigparated by 3 nts (150 ug CpG ODN 2006 + 119 I Alh dro-el 100 ug/dose Hendra sG vaccine + Prime + 1 boost separated by 3 nt 250 I Alh dro-el weeks ane + 1 bofizgiiparated by 3 Adjuvant only (150 pg CpG ODN 2006) u Ad'uvants onl 250 IAIh dro-el Same schedule as Grou-s A-B _—IEI_ Result: All animals (n=4) in both groups (A and B) survived Hendra virus challenge after being inoculated intratracheally with 105 TCleo Hendra virus. Control subjects died on day 8. No clinical illness was observed in any of the ated subjects and they remained healthy and well until study endpoint.

Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the cation and practice of the invention disclosed herein. All references cited herein, including all publications, US. and foreign patents and patent applications, are cally and entirely incorporated by reference. It is intended that the cation and examples be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.

Claims (27)

We Claim

1. An immunogenic composition comprising a e fragment of the Hendra G

glycoprotein, said fragment consisting of amino acids 73 to 604 of (SEQ ID NO: 2) or a

sequence at least 90% identical thereto, an stimulatory complex (ISC), said ISC

sing a saponin,a phospholipid and a steroid, and one or more excipients, wherein

the concentration of the soluble fragment of the Hendra virus G glycoprotein in the

composition is 5-100 μg per ml of ISC.

2. The immunogenic composition of claim 1, wherein the concentration of soluble Hendra

virus G n is 10-50 μg per ml of ISC.

3. The immunogenic composition of claim 1, wherein the concentration of e Hendra

virus G protein is 25-50 μg per ml of ISC.

4. The immunogenic composition of claim 1, wherein the tration of soluble Hendra

virus G protein is 25-100 μg per ml of ISC.

5. The immunogenic composition of any one of claims 1 to ,4 wherein the soluble Hendra

virus G glycoprotein is encoded by a nucleotide ce comprising nucleotides 64 to

1662 of SEQ ID NO: 16.

6. The immunogenic composition of any one of claims 1 to 5 wherein the soluble Hendra

virus G glycoprotein is present in dimer form.

7. The immunogenic composition any one of claims 1 to 5, wherein the soluble Hendra

virus G glycoprotein is present in tetramer form.

9. The immunogenic composition of any one of claims 1 to 7, wherein the phospholipid is

selected from the group consisting of phosphatidyl choline (PC), dipalmitoyl phosphatidyl

choline , phosphatidic acid (phosphatidate) (PA), phosphatidylethanolamine

(PE), phosphatidylserine (PS), phosphatidylinositol (PI) Phosphatidylinositol phosphate

(PIP), phosphatidylinositol bisphosphate (PIP2), phosphatidylinositol sphate

(PIP3), phosphorylcholine (SPH), ceramide phosphorylethanolamine (Cer-PE) and

ceramide phosphorylglycerol.

10. The immunogenic composition of any one of claims 1 to 9, n the saponin is Quil

A, the phospholipid is DPPC and the steroid is cholesterol.

11. The immunogenic composition of claim 10, wherein the ratio of Quil A: DPPC:

cholesterol in the composition is 5:1:1 by .

12. Use of an immunogenic ition for the manufacture of a medicament for producing

a neutralizing antibody response against a Hendra and/or Nipah virus in a subject,

wherein the immunogenic composition comprises the genic ition of any

of claims 1 -11.

13. The use of claim 12, n the neutralizing antibody response reduces Hendra and/or

Nipah virus reproduction in the subject.

14. The use of claim 12 or 13, wherein the neutralizing dy response reduces Hendra

and/or Nipah virus shedding in the subject.

15. The use of any one of claims 12 to 14, wherein the subject has been exposed to Hendra

and/or Nipah virus.

16. The use of any one of claims 12 to 15, wherein the subject is suffering from a Hendra

and/or Nipah virus infection.

17. The use of any one of claims 12 to 16, wherein the immunogenic composition is

administered intramuscularly.

18. The use of any one of claims 12 to 17, wherein the immunogenic composition is

administered in multiple doses.

19. The use of claim 18, wherein the first dose is followed by a second dose at least about

twenty-one days to about twenty-eight days after the first dose.

20. The use of claim 18 or 19, wherein each dose contains about 50 or about 100 μg of

soluble Hendra virus G glycoprotein in oneml of ISC.

21. The use of any one of claims 12-20, wherein the subject is a horse.

22. Use of an immunogenic composition for the manufacture of a medicament for treatment

of Hendra and/or Nipah virus infection, wherein the immunogenic composition comprises

(1) a e fragment of the Hendra G glycoprotein in an amount of 5-100 μg in the

immunogenic composition, said fragment consisting of amino acids 73 to 604 of (SEQ ID

NO: 2) or a sequence at least 90% identical thereto, and (2) an immunostimulatory

x (ISC), said ISC comprising a saponin, a phospholipid and a steroid, and one or

more excipients, wherein the immunogenic composition is administered to the subject in

an amount and duration ive to treat the t for Hendra and/or Nipah virus

infection.

23 The use of claim 22, n the immunogenic composition is administered

intramuscularly.

24. The use of claim 22 or 23, wherein the immunogenic composition is administered in

multiple doses.

25. The use of any one of claims 22 to 24, wherein the first dose is followed by a second

dose at least about twenty-one days to about twenty-eight days after the first dose.

26. The use of claim 24 or 25, wherein each dose contains about 50 or about 100 μg of

soluble Hendra virus G glycoprotein in one ml of ISC.

27. The use of any one of claims 22-26, wherein the subject is a horse.