HK1120730B - Nipah virus vaccines - Google Patents

Description

Nipah virus vaccine

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application serial No. 60/674,583, filed on 25/4/2005.

Incorporated references

All documents cited or referenced herein ("herein cited documents"), as well as all documents cited or referenced in herein cited documents, are hereby incorporated by reference, along with any manufacturer's specifications, descriptions, product specifications, and product statements for any product in any document mentioned or incorporated by reference herein, and which may be employed in the practice of the present invention.

Technical Field

The present invention relates to recombinant vaccines against Nipah virus and the administration of such vaccines.

Background

Nipah virus is a member of the paramyxovirus (Paramyxoviridae) family and is associated with the Hendra virus (formerly known as equine measles virus). Nipah virus was originally isolated in 1999 from examination samples of encephalitis and respiratory disease that developed in adult males in Malaysia and Singapore (see, e.g., Chua et al, Lancet.1999 Oct 9; 354 (9186): 1257-9 and Paton et al, Lancet.1999 Oct 9; 354 (9186): 1253-6). The host for Nipah virus is not known, but flying foxes (bats of the genus flying fox (Pteropus)) are suspected to be natural hosts.

Flying foxes are increasingly in contact with humans and domestic animals because of changes in ecological conditions. Thus, it is therefore conceivable that viruses in flying foxes may infect domestic animals and humans, which may lead to more toxic, potentially fatal diseases. Nipah virus causes relatively mild disease in pigs in malaysia and singapore, and the virus is transmitted to humans, cats and dogs via close contact with infected pigs.

Nipah virus infection in humans has been accompanied by encephalitis characterized by fever and lethargy and more serious central nervous system disorders such as coma, seizures, and inability to sustain respiration (see, e.g., Lee et al, Ann neurol.1999 Sep; 46 (3): 428-32). Nipah virus disease begins with fever and headache on days 3-14, followed by lethargy and disorientation characterized by confusion. These features and symptoms can develop into coma within 24-48 hours. Some patients have respiratory disease in the early stages of their infection. Severe neuropathy with Nipah viral encephalitis has been marked by certain sequelae, such as persistent convulsions and personality changes. During the outbreak of the Nipah virus disease in 1998-.

Thus, the goal of animal health is to improve the health of humans by preventing the spread of disease between animals and/or humans.

Nipah virus infection can be prevented by avoiding animals known to be infected and using appropriate personal protective equipment devices when they must come into contact with potentially infected animals. The drug ribavirin has been shown to be effective against the Nipah virus in vitro, however, controlled drug investigations have not been performed and clinical effectiveness remains uncertain.

If an effective program is to be developed to prevent or treat Nipah virus infection, viral antigens important in inducing protective responses must be defined and effective immunoprophylaxis treatments designed. The attachment (G) and fusion (F) glycoproteins of the Nipah virus have been implicated as viral antigens (see, e.g., Bossart et al, J Virol.2002 Nov; 76 (22): 11186-98 and Guillame et al, J Virol.2004 Jan; 78 (2): 834-40). Nipah virus glycoproteins (G and F), when expressed as recombinant vaccinia viruses, have induced immune responses in hamsters that protect against lethal challenge by Nipah virus (see, e.g., Guillame et al, J Virol.2004 Jan; 78 (2): 834-40). However, it was observed that the antibody response against Nipah virus was strongly stimulated in both active and passive immunization, suggesting that the efficacy of immunization is related to the vector replication capacity.

Thus, there is a need in the art for an effective and reliable Nipah virus vaccine in which the heterologous protein has limited or no expression for productive replication.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

Invention of the inventionSUMMARY

The present invention is based in part on the development of an effective recombinant vaccine that immunizes pigs against Nipah virus using an attenuated canarypox or attenuated fowlpox (fowlpox) vector encoding the Nipah virus glycoprotein, and thus can have limited or no productive replication (productive replication) heterologous protein expression.

The invention may comprise an avipox expression vector comprising a polynucleotide encoding a Nipah virus glycoprotein. In one embodiment, the Nipah virus glycoprotein may be an attachment (G) protein. Advantageously, the polynucleotide may comprise SEQ ID NO: 1, nucleotide base sequence from nucleotide 8943 to nucleotide 10751, or a polynucleotide encoding the polypeptide of SEQ id no: 8. In another embodiment, the Nipah virus glycoprotein may be a fusion (F) protein. Advantageously, the polynucleotide may comprise SEQ ID NO: 1, nucleotide 6654-nucleotide 8294 of SEQ ID NO: 7. In yet another embodiment, the Nipah virus glycoprotein may be an attachment (G) protein and a fusion (F) protein. Advantageously, the polynucleotide may comprise SEQ ID NO: 1 nucleotide 6654-nucleotide base sequence of nucleotide 8294 and nucleotide 8943-nucleotide base sequence of nucleotide 10751, or the polynucleotide encodes the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8.

The fowlpox expression vector may be an attenuated fowlpox expression vector. In one embodiment, the fowlpox expression vector may be a canarypox vector. Advantageously, the canarypox viral vector may be ALVAC. In another embodiment, the fowlpox expression vector can be a fowlpox (fowlpox) vector. Advantageously, the fowlpox virus (fowlpox) vector may be a TROVAC.

The present invention includes formulations for delivery and expression of Nipah virus glycoproteins, wherein the formulation may comprise any of the vectors described above and a pharmaceutically or veterinarily acceptable carrier (carrier), vehicle or excipient. In one embodiment, the carrier, vehicle or excipient may facilitate transfection of the vector and/or improve its preservation. The invention also includes a method of delivering a Nipah virus glycoprotein to an animal comprising administering the formulation of the paragraph above to the animal. Advantageously, the animal is a pig.

The invention also includes a method of eliciting an immune response in an animal which may comprise administering a composition which may comprise any of the vectors described above in an amount effective to elicit an immune response. The invention also relates to a method of eliciting an immune response in an animal, which may comprise administering a composition that may comprise cells, wherein the cells may comprise any one of the vectors described above in an amount effective to elicit an immune response. Advantageously, the animal is a pig.

The invention further includes a method of inducing an immune or protective response in an animal, which can comprise administering a composition that can comprise any of the vectors described above in an amount effective to induce an immune response. The invention further relates to a method of inducing an immune or protective response in an animal, which may comprise administering a composition that may comprise cells, wherein the cells may comprise any one of the vectors described above, in an amount effective to induce an immune response. Advantageously, the animal is a pig.

The invention also provides a kit for performing any of the methods described above, comprising any of the compositions described above, and optionally instructions for performing the methods.

It should be noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprising" and "comprises" may have the meaning attributed to it in U.S. patent law; for example, they may mean "including"; and terms such as "consisting essentially of … …" have the meaning ascribed to them in U.S. patent law, e.g., they encompass elements not expressly recited, but exclude elements found in the prior art or affecting the basic or novel features of the present invention.

These and other embodiments are disclosed in, or are obvious from and encompassed by, the following detailed description.

Brief Description of Drawings

The following detailed description, given by way of example and not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, in which:

FIG. 1 illustrates the nucleotide (FIG. 1A) and amino acid (FIG. 1B) sequences of the Nipah virus. See, e.g., GenBank accession No. NC _002728, Chua et al, science.2000may 26; 288(5470): 1432-5; harcourt et al, virology.2001 Aug 15; 287(1): 192-; chan et al, J Gen Virol.2001 Sep; 82(Pt 9): 2151-5 and Chua et al, Microbes Infect.2002 Feb; 4(2): 145-51, the disclosure of which is incorporated by reference in its entirety.

FIG. 2 illustrates the construction of plasmid pSL-6802-1-4. FIG. 2A is a diagram of the coding region of the Nipah virus. FIG. 2B illustrates PCR oligonucleotides used to amplify the Nipah G gene. FIG. 2C illustrates the construction of pSL-6802-1-4. FIG. 2D is the nucleotide sequence of the left and right arms and the expression cassette for translation of the Nipah virus G gene.

FIG. 3 illustrates the construction of plasmid pSL-6802-2-5. FIG. 3A illustrates PCR oligonucleotides for amplification of the Nipah G gene. FIG. 3B illustrates the construction of pSL-6802-2-5. FIG. 3C is the nucleotide sequence of the left and right arms and the expression cassette for translation of the Nipah virus G gene.

FIG. 4 illustrates the construction of plasmid pSL-6839-1. Figure 4A is a diagram of Nipah virus and vaccine antigens. FIG. 4B illustrates PCR oligonucleotides for amplification of the nepaF gene (11456. SL and 11457.SL are disclosed as SEQ ID NOS: 30 and 31, respectively, for clarity). FIG. 4C illustrates the construction of pSL-6839-1. FIG. 4D is the nucleotide sequence of the left and right arms and the expression cassette for translation of the Nipah virus F gene.

FIG. 5 illustrates the construction of plasmid pSL-6851-29. FIG. 5A illustrates PCR oligonucleotides for amplification of the Ni pah F gene (11456. SL and 11457.SL are disclosed as SEQ ID NOS: 30 and 31, respectively, for clarity). FIG. 5B is a schematic diagram of the plasmid pSL-6851-29. FIG. 5C is the nucleotide sequence of the left and right arms and the expression cassette for translation of the Nipah virus F gene.

FIG. 6 illustrates a Nipah G Western blot. Lane 1 is the ALVAC supernatant, lane 2 is the vCP2199 supernatant (ALVAC Nipah G), lane 3 is the vCP2199 supernatant (ALVAC Nipah G), lane 4 is the fowlpox virus (fowlpox) supernatant, lane 5 is the vFP2200 supernatant (fowlpox virus (fowlpox) Nipah G), lane 6 is the vFP2200 supernatant (fowlpox virus (fowlpox) Nipah G), lane 7 is the marker (177.6, 113.9, 81.2, 60.7, 47.4, 36.1, 25.3, 19.0, 14.7, 6.1), lane 8 is the ALVAC precipitate, lane 9 is the vCP2199 precipitate, lane 10 is the vCP2199 precipitate, lane 11 is the fowlpox virus (fowlpox) precipitate, lane 12 is the vFP2200 precipitate, and lane 13 is the vFP2200 precipitate.

Figure 7 illustrates a Nipah F immunoblot. FIG. 7A blots with guinea pig antiserum. Figure 7B was blotted with pig antiserum. Gel #1 was Nipah F recombinant (only pellet). Lane 1 is blank, lane 2 is fowlpox virus (fowlpox), lane 3 is vFP2207, lane 4 is vFP2207, lane 5 is blank, lane 6 is ALVAC, lane 7 is cvCP2208, lane 8 is markers 170, 130, 100, 72, 55, 40, 33, 24kDa, and lanes 9 and 10 are blank. Gel #2 was Nipah F recombinant (supernatant only). Lane 1 is blank, lane 2 is fowlpox virus (fowlpox), lane 3 is vFP2207, lane 4 is vFP2207, lane 5 is marker 170, 130, 100, 72, 55, 40, 33, 24kDa, lane 6 is blank, lane 7 is ALVAC, lane 8 is blank, lane 9 is vCP2208, and lane 10 is blank.

Detailed Description

The present invention is based in part on the development of an effective recombinant vaccine against the Nipah virus. Thus, the invention includes, in part, recombinant vaccines against Nipah virus.

In one embodiment of the invention, the Nipah viral gene is encoded within an expression vector. In an advantageous embodiment, the Nipah virus gene encodes a glycoprotein. In a particularly advantageous embodiment, the Nipah virus gene encodes the attachment (G) glycoprotein. In another particularly advantageous embodiment, the Nipah virus gene encodes the fusion (F) glycoprotein.

In an advantageous embodiment, the expression vector is a viral vector. In a particularly advantageous embodiment, the viral vector is a fowlpox viral vector. In a more advantageous embodiment, the avipox viral vector is a canarypox viral vector or a fowlpox (fowlpox) vector. More advantageously, the fowlpox viral vector is an attenuated fowlpox viral vector. In a particularly advantageous embodiment, the attenuated fowlpox virus vector is an attenuated canarypox virus or an attenuated fowlpox virus (fowlpox) vector. Advantageously, the attenuated canarypox virus vector is ALVAC and the attenuated fowlpox virus (fowlpox) vector is TROVAC.

In another embodiment, the Nipah viral protein is any Nipah viral protein or fragment thereof having a known protein sequence. In an advantageous embodiment, the Nipah virus protein is a glycoprotein. In a particularly advantageous embodiment, the Nipah virus protein is an attachment (G) glycoprotein, advantageously having the amino acid sequence of SEQ ID NO: 8 in sequence (b). In another particularly advantageous embodiment, the Nipah viral protein is a fusion (F) glycoprotein, advantageously having the amino acid sequence of SEQ ID NO: 7.

In a particularly advantageous embodiment of the invention, the recombinant constructs are an ALVAC construct expressing Nipah G designated vCP2199, an ALVAC construct expressing Nipah F designated vCP2208, a TROVAC construct expressing Nipah G designated vFP2200, and a TROVAC construct expressing Nipah F designated vFP 2207.

In another embodiment of the invention, the Nipah viral proteins include, but are not limited to, nucleocapsid protein (advantageously SEQ ID NO: 2), phosphoprotein (advantageously SEQ ID NO: 3), V protein (advantageously SEQ ID NO: 4), C protein (advantageously SEQ ID NO: 5), matrix protein (advantageously SEQ ID NO: 6), or polymerase (advantageously SEQ ID NO: 9).

The terms "protein," "peptide," "polypeptide," and "polypeptide fragment" are used interchangeably herein to refer to a polymer of amino acid residues of any length. The polymer may be linear or branched, it may comprise modified amino acids or amino acid analogues, and it may be interrupted by chemical moieties other than amino acids. The term also includes amino acid polymers that have been modified, either naturally or by intervention; such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a label or bioactive component.

In another embodiment, the Nipah viral gene is any Nipah viral gene having a known nucleotide sequence. In an advantageous embodiment, the Nipah virus gene encodes a glycoprotein. In a particularly advantageous embodiment, the Nipah viral gene encodes an attachment (G) glycoprotein, advantageously SEQ ID NO: nucleotide 8943-10751 of 1. In another particularly advantageous embodiment, the Nipah viral gene encodes a fusion (F) glycoprotein, advantageously the amino acid sequence of SEQ id no: nucleotide 6654 and 8294 of 1.

In another embodiment of the invention, the Nipah viral gene may encode a nucleocapsid protein (advantageously nucleotide 113 of SEQ ID NO: 1-1711), a phosphoprotein (advantageously nucleotide 2406 of SEQ ID NO: 1-4535), a V protein (advantageously nucleotide 2406 of SEQ ID NO: 1-3775), a C protein (advantageously nucleotide 2428 of SEQ ID NO: 1-2928), a matrix protein (advantageously nucleotide 5108 of SEQ ID NO: 1-6166), or a polymerase (advantageously nucleotide 11259 of SEQ ID NO: 1-18213).

A "polynucleotide" is a polymeric form of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides, and the like, in any combination. The polynucleotide may have a three-dimensional structure and may perform any known or unknown function. The term "polynucleotide" includes double-stranded, single-stranded and triple-helical molecules. Unless otherwise stated or required, any embodiment of the polynucleotides of the invention described herein includes the double stranded form as well as each of the 2 complementary forms, which are known or expected to form the double stranded form of DNA, RNA or hybrid molecules.

The following are non-limiting examples of polynucleotides: a gene or gene fragment, exon, intron, mRNA, tRNA, rRNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, and primer. Polynucleotides may comprise modified polynucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars or linking groups such as fluororibose and thiolate, and nucleotide branches. The nucleotide sequence may be further modified after polymerization, for example by conjugation with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more naturally occurring nucleotides with analogs, and introduction of means for attaching polynucleotides to proteins, metal ions, labeling components, other polynucleotides, or solid supports.

An "isolated" polynucleotide or polypeptide is a polynucleotide or polypeptide that is substantially free of materials with which it is associated in its natural environment. Substantially free means at least 50%, advantageously at least 70%, more advantageously at least 80%, and still more advantageously at least 90% free of these materials.

The invention further comprises the complementary strand of a Nipah virus polynucleotide, advantageously a Nipah virus glycoprotein polynucleotide.

The complementary strand can be polymeric and of any length, and can contain deoxyribonucleotides, ribonucleotides, and the like in any combination.

Hybridization reactions can be performed under different "stringency" conditions. Conditions that increase the stringency of hybridization reactions are well known. See, e.g., "Molecular Cloning: ALaborory Manual ", second edition (Sambrook et al, 1989). Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25 ℃,37 ℃,50 ℃ and 68 ℃; buffer concentrations of 10XSSC, 6XSSC, 1XSSC, 0.1XSSC (where SSC is 0.15M NaCl and 15mM citrate buffer), and equivalents thereof using other buffer systems; formamide concentrations of 0%, 25%, 50% and 75%; an incubation time of 5 minutes to 24 hours; 1.2 or more washing steps; 1. wash incubation times of 2 or 15 minutes; and a wash solution of 6xSSC, 1xSSC, 0.1xSSC, or deionized water.

The invention further includes polynucleotides encoding functionally equivalent variants and derivatives of Nipah virus polypeptides, as well as functionally equivalent fragments thereof that may enhance, reduce, or not significantly affect the properties of the polypeptides encoded thereby. These functionally equivalent variants, derivatives and fragments show the ability to retain the activity of a Nipah virus polypeptide, advantageously a Nipah virus glycoprotein. For example, changes in the DNA sequence that do not alter the encoded amino acid sequence, as well as those that result in conservative substitutions of amino acid residues, one or a few amino acid deletions or additions, and substitutions of amino acid residues by amino acid analogs, are those that do not significantly affect the properties of the encoded polypeptide. Conservative amino acid substitutions are glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and phenylalanine/tyrosine/tryptophan.

For the purposes of the present invention, sequence identity or homology is determined by comparing sequences when aligned, which enables the maximization of overlap and identity while minimizing sequence gaps. In particular, sequence identity can be determined using any of a number of mathematical algorithms. One non-limiting example of a mathematical algorithm for comparing 2 sequences is Karlin and Altschul, proc.natl.acad.sci.usa 1990; 87: 2264. sup. 2268, by Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1993; 90: 5873-5877.

Another example of a mathematical algorithm for sequence comparison is Myers and Miller, cabaos 1988; 4: 11-17. Such algorithms are incorporated within the ALIGN program (version 2.0) as part of the GCG sequence alignment software package. When using the ALIGN program for comparing amino acid sequences, a PAM120 weighted residue table, a gap length penalty of 12 and a gap penalty of 4 can be used. Another useful algorithm for identifying local sequence similarity and alignment regions is, for example, Pearson and Lipman, proc.natl.acad.sci.usa 1988; 85: 2444 along with the FASTA algorithm described in 2448.

Advantageously used in accordance with the present invention is WU-BLAST (Washington university BLAST) version 2.0 software. WU-BLAST version 2.0 executables for several UNIX platforms can be downloaded from ftp:// BLAST. This program is based on WU-BLAST version 1.4, which is based on the public domain NCBI-BLAST version 1.4(Altschul and Gish, 1996, Localalignment standards, Doolitled, Methods in Enzymology 266: 460-480; Altschul et al, Journal of Molecular Biology 1990; 215: 403-410; Gish and States, 1993; Nature Genetics 3: 266-272; Karlin and Altschul, 1993; Proc. Natl. Acad. Sci. USA 90: 5873-5877; all of which are incorporated herein by reference).

In general, comparison of amino acid sequences is accomplished by aligning the amino acid sequence of a polypeptide of known structure with the amino acid sequence of a polypeptide of unknown structure. The amino acids in the sequences are then compared and the homologous amino acid groups are assigned. This approach detects conserved regions of polypeptides and takes into account amino acid insertions and deletions. Homology between amino acid sequences can be determined by using commercially available algorithms (see also the description of homology above). In addition to those otherwise mentioned herein, the programs BLAST, gapped BLAST, BLASTN, BLASTP and PSI-BLAST provided by the national center for biotechnology information may also be mentioned. These programs are widely used in the art for this purpose and can align regions of homology of 2 amino acid sequences.

In all search programs of the suite, the gap alignment routine is part of the database search itself. The notch can be closed if necessary. The default penalty (Q) for a gap of length 1 is Q-9 for proteins and BLASTP and Q-10 for BLASTN, but can vary to any integer. The default per residue penalty (R) for extending a gap is R-2 for proteins and BLASTP and R-10 for BLASTN, but can vary to any integer. Any combination of values for Q and R can be used for aligning sequences that maximizes overlap and identity while minimizing sequence gaps. The default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices such as PAM may be used.

Alternatively or additionally, the term "homology" or "identity", e.g. in terms of nucleotide or amino acid sequences, may refer to a quantitative measure of homology between 2 sequences. The percentage of sequence homology can be calculated as (N)_ref-N_dif)*100/N_refIn which N is_difIs the total number of residues in the 2 sequences that are not identical when aligned, and wherein N is_refIs the number of residues in one of the sequences. Thus, the DNA sequence AGTCAGTC will have 75% sequence identity (N) to the sequence AATCAATC_ref＝8；N_dif＝2)。

Alternatively or additionally, "homology" or "identity" with respect to sequence can refer to the number of positions having the same nucleotide or amino acid divided by the number of nucleotides or amino acids of the shorter of 2 sequences, wherein the alignment of the 2 sequences can be determined according to the Wilbur and Lipman algorithm (Wilbur and Lipman, Proc Natl Acad Sci USA 1983; 80: 726, incorporated herein by reference), e.g., using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation (including alignment) of the sequence data can use commercially available programs (e.g., Intelligenetics)^TMSuite, intelligentics inc. ca). When an RNA sequence is said to be similar to, or have a degree of sequence identity or homology with, a DNA sequence, thymidine (T) in the DNA sequence is considered to be equivalent to uracil (U) in the RNA sequence. Thus, RNA sequences are within the scope of the invention and may be identified by considering thymidine (T) in DNA sequences as equivalentUracil (U) in RNA sequences is obtained from DNA sequences.

And without undue experimentation the skilled person can refer to a number of other programs or references for determining sequence homology.

The invention further comprises Nipah viral proteins, advantageously Nipah viral glycoproteins, contained in a vector molecule or expression vector, and operably linked to enhancer and/or promoter elements, if necessary. In an advantageous embodiment, the promoter is the Cytomegalovirus (CMV) promoter. In another embodiment, the enhancer and/or promoter includes various cell or tissue specific promoters, various viral promoters and enhancers, and various Nipah virus DAN sequences isogenic specific for each animal species.

"vector" refers to a recombinant DNA or RNA plasmid or virus comprising a heterologous polynucleotide to be delivered to a target cell in vitro or in vivo. The heterologous polynucleotide may comprise a sequence of interest for therapeutic use and may optionally be in the form of an expression cassette. As used herein, a vector need not be capable of replication in the final target cell or subject. The term includes cloning vectors for the translation of polynucleotide coding sequences. Also included are viral vectors.

The term "recombinant" refers to a polynucleotide of genomic cDNA, semisynthetic, or synthetic origin that does not exist in nature or that is linked to another polynucleotide in a manner not found in nature.

"heterologous" refers to an entity that is derived from a genetically different entity than the rest of the entity to which it is being compared. For example, a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a different source and be a heterologous polynucleotide. A promoter that is removed from its native coding sequence and operably linked to a coding sequence other than the native sequence is a heterologous promoter.

Polynucleotides of the invention may comprise additional sequences, such as additional coding sequences within the same transcription unit, control elements such as promoters, enhancers, ribosome binding sites, polyadenylation sites, transcription terminators, additional transcription units under the control of the same or different promoters, sequences that allow for cloning, expression, homologous recombination, and transformation of host cells, and any such construct may be desirable to provide embodiments of the invention.

Elements for the expression of Nipah viral proteins, advantageously Nipah viral glycoproteins, are advantageously present in the vectors of the invention. At a minimum, this comprises, consists essentially of, or consists of the following elements: an initiation codon (ATG), a stop codon and a promoter, and optionally also polyadenylation sequences for certain vectors, such as plasmids, and certain viral vectors, such as viral vectors other than poxviruses, and transcription terminators for poxviruses. When the polynucleotide advantageously encodes a polyprotein fragment, such as a Nipah virus protein, in a vector, the ATG is placed 5 'of the reading frame and the terminator is placed 3'. Other elements for controlling expression may be present, such as enhancer sequences, stabilizing sequences and signal sequences which allow secretion of the protein.

The method for preparing and/or administering the vector or recombinant or plasmid for expressing the gene product of the gene of the invention in vivo or in vitro may be any desired method, for example, the method disclosed by or similar to the following documents: U.S. Pat. nos. 4,603,112; 4,769,330; 4,394,448, respectively; 4,722,848; 4,745,051; 4,769,331, respectively; 4,945,050; 5,494,807; 5,514,375, respectively; 5,744,140, respectively; 5,744,141, respectively; 5,756,103; 5,762,938; 5,766,599; 5,990,091, respectively; 5,174,993; 5,505,941; 5,338,683, respectively; 5,494,807; 5,591,639, respectively; 5,589,466; 5,677,178; 5,591,439, respectively; 5,552,143, respectively; 5,580,859; 6,130,066, respectively; 6,004,777, respectively; 6,130,066, respectively; 6,497,883, respectively; 6,464,984, respectively; 6,451,770, respectively; 6,391,314, respectively; 6,387,376, respectively; 6,376,473, respectively; 6,368,603, respectively; 6,348,196, respectively; 6,306,400, respectively; 6,228,846, respectively; 6,221,362, respectively; 6,217,883, respectively; 6,207,166, respectively; 6,207,165, respectively; 6,159,477, respectively; 6,153,199, respectively; 6,090,393; 6,074,649, respectively; 6,045,803, respectively; 6,033,670, respectively; 6,485,729, respectively; 6,103,526, respectively; 6,224,882, respectively; 6,312,682, respectively; 6,348,450 and 6; 312,683, respectively; U.S. patent application serial No. 920,197 filed on 16/10/1986; WO 90/01543; WO 91/11525; WO 94/16716; WO 96/39491; WO 98/33510; EP 265785; EP 0370573; andreansky et al, proc.natl.acad.sci.usa 1996; 93: 11313-11318; ballay et al, EMBO J.1993; 4: 3861-65; felgner et al, j.biol.chem.1994; 269: 2550, 2561; frolov et al, proc.natl.acad.sci.usa 1996; 93: 11371-11377; graham, Tibtech 1990; 8: 85-87; grunhaus et al, sen.virol.1992; 3: 237-52; ju et al, Diabetologia 1998; 41: 736-739; kitson et al, j.virol.1991; 65: 3068-3075; McClements et al, proc.natl.acad.sci.usa 1996; 93: 11414-11420; moss, proc.natl.acad.sci.usa 1996; 93: 11341-11348; paoletti, proc.natl.acad.sci.usa 1996; 93: 11349-11353; pennock et al, mol.cell.biol.1984; 4: 399-; richardson (Ed), Methods in Molecular Biology 1995; 39, "Baculoviral Expression Protocols," Humana Press Inc.; smith et al, (1983) mol.cell.biol.1983; 3: 2156 and 2165; robertson et al, proc.natl.acad.sci.usa 1996; 93: 11334, 11340; robinson et al, sem. immunol.1997; 9: 271; and Roizman, proc.natl.acad.sci.usa 1996; 93: 11307-11312. Thus, the vector of the invention can be any suitable recombinant virus or viral vector, such as poxviruses (e.g., vaccinia virus, fowlpox virus, canarypox virus, fowlpox virus (fowlpox), raccoon poxvirus, swinepox virus, etc.), adenoviruses (e.g., canine adenovirus), herpes viruses, baculoviruses, retroviruses, and the like (as in the documents incorporated by reference herein); alternatively, the vector may be a plasmid. Documents cited and incorporated herein by reference, in addition to providing examples of vectors useful in the practice of the present invention, may also provide a source of non-Nipah viral proteins or fragments thereof, e.g., non-Nipah viral proteins or fragments thereof, cytokines, etc., expressed by one or more vectors in or included in the compositions of the present invention.

The one or more cytokines may be in the form of proteins in an immunogenic or vaccine composition, or may be co-expressed in a host with one or more immunogens or epitopes thereof. Preference is given to coexpressing one or more cytokines by the same vector as the one or more immunogens or epitopes thereof, or by a separate vector.

The cytokine may be selected from: interleukin 18(IL-18), interleukin 12(IL-12), interleukin 15(IL-15), MIP-1 α (macrophage inflammatory protein 1 α; Marshall E. et al, Br. J. cancer, 1997, 75(12), 1715-. Preferably, cytokines of the species to be vaccinated are used; that is, advantageously, the cytokine matches the target or host species, and mention is made, for example, of porcine GM-CSF (S. Inumaru et al, immunol. cell biol.1995, 73(5), 474-.

WO00/77210 provides nucleotide and amino acid sequences corresponding to equine GM-CSF, in vitro production of GM-CSF, and construction of vectors (e.g., plasmids and viral vectors) that allow for in vivo expression of equine GM-CSF.

The invention also relates to formulations, e.g., therapeutic compositions, comprising the vectors, e.g., expression vectors. The formulation may comprise, consist essentially of, or consist of one or more vectors, such as expression vectors, e.g., in vivo expression vectors, comprising, consisting essentially of, or consisting of (and advantageously expressed) one or more Nipah virus polynucleotides, and advantageously, the vectors contain and express polynucleotides comprising, consisting essentially of, or consisting of coding regions encoding Nipah virus proteins (advantageously Nipah virus glycoproteins), in a pharmaceutically or veterinarily acceptable carrier, excipient, or vehicle. Thus, according to one embodiment of the invention, the one or more additional vectors in the formulation comprise, consist essentially of, or consist of a polynucleotide encoding, and under suitable circumstances the vector expresses, one or more additional proteins of a Nipah virus glycoprotein, or fragments thereof.

According to another embodiment, the one or more vectors in the formulation comprise, consist essentially of, or consist of a polynucleotide encoding one or more proteins of a Nipah virus protein, advantageously a Nipah virus glycoprotein, or a fragment thereof. The preparation of the invention advantageously comprises, consists essentially of, or consists of at least 2 vectors comprising, consisting essentially of, or consisting of polynucleotides from different Nipah virus isolates, and advantageously also expressing said polynucleotides, advantageously in vivo under suitable conditions or in a suitable host cell, said polynucleotides encoding the same protein and/or different proteins, but advantageously encoding the same protein. With regard to formulations comprising one or more vectors comprising, consisting essentially of, or consisting of a polynucleotide encoding, and advantageously expressing, advantageously in vivo, a Nipah viral protein, advantageously a Nipah viral glycoprotein, or an epitope thereof, advantageously the expression product is from 2, 3 or more Nipah viral isolates, advantageously a strain. The invention also relates to a vector mixture comprising, consisting essentially of, or consisting of sequences encoding different Nipah virus glycoproteins, and expressing the same.

In an advantageous embodiment, the vector is a viral vector, advantageously an avipox viral vector, containing a Nipah viral gene, advantageously a Nipah viral glycoprotein gene. In a particularly advantageous embodiment, the avipox viral vector is a canarypox viral vector, advantageously an attenuated canarypox viral vector such as ALVAC. Attenuated canarypox viruses are described in U.S. Pat. No. 5,756,103(ALVAC) and WO 01/05934. In another particularly advantageous embodiment, the fowlpox viral vector is a fowlpox (fowlpox) vector, advantageously an attenuated fowlpox (fowlpox) vector such as TROVAC. Reference is also made to U.S. Pat. No. 5,766,599, which relates to the attenuated fowlpox virus (fowlpox) strain TROVAC.

In one embodiment, the viral vector is a poxvirus, such as a vaccinia virus or an attenuated vaccinia virus (e.g., MVA, a modified Ankara strain obtained after more than 570 passages of the Ankara vaccine strain on chicken embryo fibroblasts; see Stickl and Hochstein-Mintzel, Munch.Med.Wschr., 1971, 113, 1149-1153; Sutter et al, Proc.Natl.Acad.Sci.U.S.A., 1992, 89, 10847-10851; available as ATCC VR-10851; or NYVAC, see U.S. Pat. No. 5,494,807, e.g., U.S. Pat. Nos. 1-6 of U.S. Pat. No. 5,494,807, et al, which discuss the construction of NYVAC, and recombinant strains having additional ORFs deleted from the genome of the present Hayan strain vaccinia virus, and recombinant nucleic acids encoding heterologous variants of such attenuated vaccinia virus, and further use of compatible promoters of fowlpox viruses (see, e.g., WO 96), canary pox, fowlpox (fowlpox), dovepox (dovepox), pigeon pox, quail pox, ALVAC or TROVAC; see also U.S. Pat. nos. 5,505,941, 5,494,807), swinepox, raccoon pox, camel pox, or myxomatosis virus.

According to another embodiment of the invention, the poxvirus vector is a canarypox or fowlpox (fowlpox) vector, advantageously an attenuated canarypox or fowlpox (fowlpox) virus. In this regard, canarypox is available from the ATCC under accession number VR-111. Attenuated canarypox viruses are described in U.S. Pat. No. 5,756,103(ALVAC) and WO 01/05934. Numerous strains of chicken pox virus (fowlpox) vaccination are also available, for example the DIFTOSEC CT strain marketed by MERIAL, and the NOBILIS VARIOLE vaccine marketed by INTERVET; and also to us patent No. 5,766,599, which relates to attenuated fowlpox virus (fowlpox) strain TROVAC.

For information on the methods of producing recombinants thereof and how to administer recombinants thereof, the skilled person can refer to the documents cited herein and WO90/12882, for example as to vaccinia viruses mention may be made of U.S. Pat. nos. 4,769,330, 4,722,848, 4,603,112, 5,110,587, 5,494,807 and 5,762,938, among others; as regards fowlpox (fowlpox), mention may be made of U.S. Pat. Nos. 5,174,993, 5,505,941 and 5,766,599, among others; as regards canary jaundice, mention may be made of U.S. Pat. No. 5,756,103 and others; as regards swinepox, mention may be made of us patent No. 5,382,425 and others; and as regards raccoon poxviruses, mention may be made of WO00/03030 and others.

When the expression vector is a vaccinia virus, the insertion site or sites for the polynucleotide or polynucleotides to be expressed are advantageously in the Thymidine Kinase (TK) gene or insertion site, the Hemagglutinin (HA) gene or insertion site, the region encoding the a-type inclusion bodies (ATI); see also documents cited herein, in particular those relating to vaccinia virus. In the case of canarypox, advantageously the insertion site or sites are orf(s) C3, C5 and/or C6; see also documents cited herein, in particular those relating to canarypox virus. In the case of fowlpox (fowlpox), the insertion site or sites are advantageously ORFs F7 and/or F8; see also the documents cited herein, in particular those relating to fowlpox virus (fowlpox). One or more insertion sites for MVA viruses are advantageously as in various publications, including Carroll m.w. et al, Vaccine, 1997, 15(4), 387-; stittelaar K.J. et al, J.Virol, 2000, 74(9), 4236-; sutter G. et al, 1994, Vaccine, 12(11), 1032-; and it should also be noted in this regard that the complete MVA genome is described in Antoine g., Virology, 1998, 244, 365-.

Advantageously, the polynucleotide to be expressed is inserted under the control of a particular poxvirus promoter, for example vaccinia promoter 7.5kDa (Cochran et al, J.virology, 1985, 54, 30-35), vaccinia promoter I3L (Riviere et al, J.virology, 1992, 66, 3424-3434), vaccinia promoter HA (Shida, Virology, 1986, 150, 451-457), vaccinia promoter Genati (Funahashi et al, J.Virol, 1988, 69, 35-47), vaccinia promoter H6(Taylor J. et al, Vaccine, 1988, 6, Geneva 508; Guo P. et al, J.Virol, 1989, 63, 4189-4198; Perkus M. et al, J.Virol, 1989, 3863, 3829, among others.

Advantageously, for vaccination of mammals, the expression vector is canary jaundice or fowlpox virus (fowlpox). In this way, heterologous proteins with limited or no productive replication can be expressed.

According to one embodiment of the invention, the expression vector is a viral vector, in particular an in vivo expression vector. In an advantageous embodiment, the expression vector is an adenoviral vector, such as a Human Adenovirus (HAB) or a Canine Adenovirus (CAV). Advantageously, the adenovirus is a human Ad5 vector, an E1-deleted and/or disrupted adenovirus, an E3-deleted and/or disrupted adenovirus, or an E1-and E3-deleted and/or disrupted adenovirus. Optionally, E4 may be deleted and/or fragmented from any of the adenoviruses described above. For example, the human Ad5 vector described in Yarosh et al and Lutze-Wallace et al can be used to express the Nipah virus glycoprotein gene according to the methods of the invention (see, e.g., Yarosh et al, vaccine.1996 Sep; 14 (13): 1257-64 and Lutze-Wallace et al, biologicals.1995 Dec; 23 (4): 271-7).

In one embodiment, the viral vector is a human adenovirus, particularly a serotype 5 adenovirus rendered replication-incompetent by a deletion in the E1 region of the viral genome. Deleted adenoviruses were propagated in 293 cells or PER cells expressing E1, in particular PER. C6 (F. Falloux et al Human Gene Therapy 1998, 9, 1909-. Human adenovirus can be deleted in The E3 region and also in The E1 region (see, e.g., J.Shriver et al, Nature, 2002, 415, 331-335, F.Graham et al, Methods in molecular Biology Vol.7: Gene Transfer and expression protocols Edied by E.Murray, The Human Press Inc, 1991, page 109-128, Y.Ilan et al, Proc.Natl.Acad.Sci.1997, 94, 2587-2592, S.Tripathy et al, Proc.Natl.Acad.Sci.1994, 91, 11557-11561, B.Tapne Adll.Drug Deliv.Rev.1993, 12, 185, X.Danthine et al, Gene Thrply, 2000, 1717, 11557-11561, B.Tapne.Dr.70-1717, Bei.1986, Bei.11 K.19811, beer K. 19811-16, beer K. 19811, beer K. 19811, beer K. 11, 19811, beer K. 19811, and 19811). After partial or complete deletion of the E1 and/or E3 regions, the insertion site may be finally the E1 and/or E3 loci. Advantageously, when the expression vector is an adenovirus, the polynucleotide to be expressed is inserted under the control of a promoter functional in eukaryotic cells, such as a strong promoter, preferably the cytomegalovirus immediate early gene promoter (CMV-IE promoter). The CMV-IE promoter is advantageously of murine or human origin. The promoter for elongation factor 1 α may also be used. In a particular embodiment, a promoter regulated by hypoxia may be used, such as the promoter HRE described in K.Boast et al Human Gene Therapy 1999, 13, 2197-. Muscle-specific promoters can also be used (X.Li et al nat. Biotechnol.1999, 17, 241-245). Strong promoters are also discussed herein with respect to plasmid vectors. The poly (A) sequence and termination sequence may be inserted downstream of the polynucleotide to be expressed, for example the bovine growth hormone gene or the rabbit beta globin gene polyadenylation signal.

In another embodiment, the viral vector is a canine adenovirus, particularly CAV-2 (see, e.g., L.Fischer et al, Vaccine, 2002, 20, 3485-. For CAV, the insertion site may be in region E3 and/or in the region between region E4 and the right ITR region (see U.S. Pat. No. 6,090,393; U.S. Pat. No. 6,156,567). In one embodiment, the insert is under the control of a promoter, such as the cytomegalovirus immediate early gene promoter (CMV-IE promoter) or a promoter already described for human adenovirus vectors. The poly (A) sequence and termination sequence may be inserted downstream of the polynucleotide to be expressed, for example the bovine growth hormone gene or the rabbit beta globin gene polyadenylation signal.

In another embodiment, the viral vector is a herpesvirus such as Canine Herpesvirus (CHV) or Feline Herpesvirus (FHV). For CHV, the insertion site may be specifically in the thymidine kinase gene, ORF3, or UL43 ORF (see U.S. patent No. 6,159,477). In one embodiment, the polynucleotide to be expressed is inserted under the control of a promoter functional in eukaryotic cells, advantageously a CMV-IE promoter (murine or human). In a particular embodiment, a promoter regulated by hypoxia may be used, for example the promoter HRE described in K.Boast et al HumanGene Therapy 1999, 13, 2197-. The poly (A) sequence and termination sequence may be inserted downstream of the polynucleotide to be expressed, for example the bovine growth hormone gene or the rabbit beta globin gene polyadenylation signal.

According to a still further embodiment of the invention, the expression vector is a plasmid vector or a DNA plasmid vector, in particular an in vivo expression vector. In a specific non-limiting example, pVR1020 or 1012 plasmids (VICAL Inc.; Luke C. et al, Journal of infectious Diseases, 1997, 175, 91-97; Hartikka J. et al, Human Gene Therapy, 1996, 7, 1205-1217) can be used as vectors for inserting polynucleotide sequences. The pVR1020 plasmid is derived from pVR1012 and contains the human tPA signal sequence.

The term plasmid encompasses any DNA transcription unit comprising a polynucleotide according to the invention and the elements required for its expression in vivo in one or more cells of a desired host or target; also, in this regard, it should be noted that supercoiled or non-supercoiled circular plasmids, as well as linear forms of plasmids, are contemplated to be within the scope of the present invention.

Each plasmid comprises or consists essentially of a polynucleotide encoding a Nipah virus protein, advantageously a Nipah virus glycoprotein, variant, analogue or fragment, operably linked to or under the control of or dependent on a promoter. In general, it is advantageous to employ strong promoters which function in eukaryotic cells. Preferred strong promoters are immediate early cytomegalovirus promoters (CMV-IE) of human or murine origin, or optionally of another origin such as rat or guinea pig. The CMV-IE promoter may comprise the actual promoter portion, which may or may not be combined with the enhancer portion. Reference may be made to EP-A-260148, EP-A-323597, U.S. Pat. Nos. 5,168,062, 5,385,839 and 4,968,615, and PCT application No. WO 87/03905. The CMV-IE promoter is advantageously human CMV-IE (Boshart M. et al, Cell, 1985, 41, 521-530) or murine CMV-IE.

More generally, the promoter is of viral or cellular origin. Strong viral promoters other than CMV-IE that may be usefully employed in the practice of the present invention are the early/late promoters of SV40 virus, or the LTR promoters of Rous sarcoma virus. Strong cellular promoters which can be usefully employed in the practice of the present invention are promoters of cytoskeletal genes, for example, the desmin promoter (Kwissa M. et al, Vaccine, 2000, 18, 2337-.

Functional sub-fragments of these promoters, i.e., portions of these promoters that maintain sufficient promoter activity, are included in the present invention, e.g., truncated CMV-IE promoters according to PCT application number WO98/00166 or U.S. Pat. No. 6,156,567 may be used in the practice of the present invention. Promoters in the practice of the present invention thus include derivatives and sub-fragments of the full-length promoter which maintain sufficient promoter activity and thus function as a promoter, preferably promoter activity substantially similar to that of the actual or full-length promoter from which the derivative or sub-fragment is derived, for example similar to that of the truncated CMV-IE promoter of us patent No. 6,156,567, or the full-length CMV-IE promoter. Thus, the CMV-IE promoter in the practice of the present invention may comprise, consist essentially of, or consist of the promoter portion of the full-length promoter and/or the enhancer portion of the full-length promoter, as well as derivatives and subfragments.

Advantageously, the plasmid comprises, or essentially consists of, other expression control elements. It is particularly advantageous to incorporate one or more stabilizing sequences, for example one or more intron sequences, preferably intron 1 of hCMV-IE (PCT application No. WO89/01036), intron II of the rabbit β globin gene (van Ooyen et al, Science, 1979, 206, 337-344).

As for the polyadenylation signal (poly a) of plasmids and viral vectors other than poxviruses, the poly (a) signal of bovine growth hormone (bGH) gene (see U.S. patent No. 5,122,458), or the poly (a) signal of rabbit β globin gene, or the poly (a) signal of SV40 virus can be used.

According to another embodiment of the invention, the expression vector is an expression vector for expressing a protein in vivo in a suitable cell system. Protein production may occur by: mammalian cells are transfected with plasmids, by expression of viral vectors, with or without productive replication, on mammalian or avian cells, or by baculovirus replication on insect cells (e.g., Sf9 Spodoptera frugiperda cells, ATCC CRL 1711; see also U.S. Pat. Nos. 6,228,846, 6,103,526) (see, e.g., U.S. Pat. No. 4,745,051; Vialard J. et al, J.Virol., 199064 (1), 37-50; Verne A., Virology, 1988, 167, 56-71), such as Autographa californica nuclear polyhedrosis virus NPV. Mammalian cells which may be used are advantageously hamster cells (e.g.CHO or BHK-21) or monkey cells (e.g.COS or VERO). The expressed protein may be harvested in or from the culture supernatant after or without secretion (if not, this typically occurs or is subject to cell lysis), optionally concentrated by a concentration method such as ultrafiltration and/or purified by a purification method such as affinity, ion exchange or gel filtration type chromatography methods.

It will be appreciated by those skilled in the art that the conditions for culturing the host cells will vary depending on the particular gene, and routine experimentation will sometimes be required to determine the optimal conditions for culturing the Nipah viral proteins, advantageously the Nipah viral glycoproteins, depending on the host cell. "host cell" refers to a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by the administration of an exogenous polynucleotide, such as a recombinant plasmid or vector. When referring to a genetically altered cell, the term refers to the originally altered cell and its progeny.

Polynucleotides comprising the desired sequences may be inserted into suitable cloning or expression vectors, and the vectors may subsequently be introduced into suitable host cells for replication and amplification. The polynucleotide may be introduced into the host cell by any method known in the art. Vectors containing the polynucleotide of interest can be introduced into the host cell by any of a number of suitable methods, including direct uptake, endocytosis, transfection, f mating, electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; bombardment of particles; lipofection; and infection (where the vector is infectious, e.g., a retroviral vector). The choice of vector or polynucleotide to introduce will generally depend on the characteristics of the host cell.

In an advantageous embodiment, the invention provides for the administration of a therapeutically effective amount of a formulation for the delivery and expression of a Nipah viral protein, advantageously a Nipah viral glycoprotein, in a target cell. Determination of a therapeutically effective amount is routine experimentation for one of ordinary skill in the art. In one embodiment, the formulation comprises an expression vector comprising a polynucleotide expressing a Nipah virus protein, advantageously a Nipah virus glycoprotein, and a pharmaceutically or veterinarily acceptable carrier, vehicle or excipient. In an advantageous embodiment, the pharmaceutically or veterinarily acceptable carrier, vehicle or excipient facilitates transfection and/or improves preservation of the vector or protein.

Pharmaceutically or veterinarily acceptable carriers, or vehicles or excipients are well known to those skilled in the art. For example, the pharmaceutically or veterinarily acceptable carrier, or vehicle or excipient may be a 0.9% NaCl (e.g., saline) solution or a phosphate buffer. Other pharmaceutically or veterinarily acceptable carriers, or vehicles or excipients that may be used in the methods of the invention include, but are not limited to, poly- (L-glutamic acid) or polyvinylpyrrolidone. The pharmaceutically or veterinarily acceptable carrier, or vehicle or excipient may be any compound or combination of compounds that facilitates administration of the vector (or protein expressed in vitro by the vector of the invention); advantageously, the carrier, or vehicle or excipient may facilitate transfection of the vector (or protein) and/or improve its preservation. Dosages and dose volumes are discussed in the general description herein and can also be determined by the skilled artisan from the present disclosure, without any undue experimentation, in conjunction with the knowledge of the art.

Cationic lipids containing quaternary ammonium salts which are advantageously, but not exclusively, suitable for plasmids are advantageously those having the formula:

wherein R is₁Is a saturated or unsaturated, straight-chain aliphatic radical having from 12 to 18 carbon atoms, R₂Is another aliphatic group containing 2 or 3 carbon atoms and X is amino or hydroxyl, e.g. DMRIE. In another embodiment, the cationic lipid may be conjugated to a neutral lipid, such as DOPE.

Of these cationic lipids, preference is given to DMRIE (N- (2-hydroxyethyl) -N, N-dimethyl-2, 3-ditetradecyloxy) -1-propanaminium; WO96/34109), advantageously in combination with a neutral lipid, advantageously DOPE (dioleoyl-phosphatidyl-ethanolamine; Behr J.P., 1994, Bioconjugate Chemistry, 5, 382-Asonic 389), to form DMRIE-DOPE.

Advantageously, the mixture of plasmid and adjuvant is formed temporarily and advantageously simultaneously with or shortly before administration of the formulation; for example, it may be advantageous to form the plasmid-adjuvant mixture prior to or shortly before administration, so as to give sufficient time for the mixture to form a complex prior to administration, e.g., about 10 to about 60 minutes prior to administration, e.g., about 30 minutes prior to administration.

When DOPE is present, the DMRIEDOPE molar ratio is advantageously from about 95: about 5 to about 5: about 95, more advantageously about 1: about 1, e.g., 1: 1.

DMRIE or DMRIE-DOPE adjuvants: the plasmid weight ratio may be from about 50: about 1 to about 1: about 10, for example from about 10: about 1 to about 1: about 5, and advantageously from about 1: about 1 to about 1: about 2, for example 1: 1 to 1: 2.

In a specific embodiment, the pharmaceutical composition is administered directly in vivo, and the encoded product is expressed in a host by a vector. Methods for in vivo delivery of vectors encoding Nipah viral proteins, advantageously Nipah viral glycoproteins (see, e.g., U.S. Pat. No. 6,423,693; patent publication EP 1052286, EP 1205551, U.S. patent publication 20040057941, WO 9905300 and Draghia-Akli et al, Mol ther. 2002Dec; 6 (6): 830-6, the disclosure of which is incorporated by reference in its entirety) can be modified to deliver the Nipah viral proteins, advantageously Nipah viral glycoproteins, of the present invention. In vivo delivery of the vectors of the present invention encoding Nipah viral proteins, advantageously Nipah viral glycoproteins, can be accomplished by one of ordinary skill in the art following the teachings of the references mentioned above.

Advantageously, the pharmaceutical and/or therapeutic compositions and/or formulations according to the invention comprise, consist essentially of, or consist of, an effective amount of one or more expression vectors and/or polypeptides as discussed herein to elicit a therapeutic response; also, an effective amount can be determined without undue experimentation in light of the present disclosure, including the documents incorporated herein, and the knowledge of one skilled in the art.

The immunogenic compositions and vaccines according to the invention may comprise, or consist essentially of, one or more adjuvants. Particularly suitable adjuvants for use in the practice of the present invention are (1) acrylic or methacrylic acid polymers, maleic anhydride and alkenyl derivative polymers, (2) immunostimulatory sequences (ISS), for example oligodeoxyribonucleotide sequences comprising one or more unmethylated CpG units (Klinman D.M. et al, Proc. Natl. Acad. Sci., USA, 1996, 93, 2879-, for example, SPT emulsions described on page 147 of "vaccine design, The Subunit and Adjuvant apparatus", published by M.Powell, M.Newman, Plenum Press 1995, and emulsion MF59 described on page 183 of the same article, (4) cationic lipids containing quaternary ammonium salts, (5) cytokines, (6) aluminum hydroxide or phosphate or (7) other adjuvants discussed in any of the documents cited in the present application and incorporated by reference, or (8) any combination or mixture thereof.

Oil-in-water emulsions (3) particularly suitable for viral vectors may be based on:

light liquid paraffin oil (typical of europathies),

isoprenoid oils such as squalane, squalene,

-oils resulting from the oligomerization of olefins such as isobutene or decene,

esters of acids or alcohols containing straight-chain alkyl groups, such as vegetable oils, ethyl oleate, propylene glycol, di (caprylate/caprate), glycerol tri (caprylate/caprate) and propylene glycol dioleate, or

Esters of branched fatty alcohols or acids, in particular isostearates.

The oil is used in combination with an emulsifier to form an emulsion. The emulsifier may be a nonionic surfactant, for example:

esters of sorbitan, mannitol (e.g. mannitol oleate), glycerol, polyglycerol or propylene glycol on the one hand and oleic, isostearic, ricinoleic or hydroxystearic acid on the other hand, said esters optionally being ethoxylated,

polyoxypropylene-polyoxyethylene block copolymers, such as Pluronic, e.g. L121.

Among the adjuvant polymers of type (1), preference is given to crosslinked acrylic or methacrylic acids, in particular polymers crosslinked by polyalkenyl ethers of sugars or polyols. These compounds are known as carbomers (Pharmeuropa, vol.8, No.2, June 1996). One skilled in the art can also refer to U.S. patent No.2,909,462, which provides such acrylic polymers crosslinked by a polyhydroxy compound containing at least 3 hydroxyl groups, preferably no more than 8 such groups, the hydrogen atoms of at least 3 hydroxyl groups being substituted with unsaturated aliphatic groups containing at least 2 carbon atoms. Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. Unsaturated groups may also contain other substituents, such as methyl. Products sold under the name Carbopol (BF Goodrich, Ohio, USA) are particularly suitable. They are crosslinked by allyl sucrose or by allyl pentaerythritol. Among them, mention may be made of carbomers 974P, 934P and 971P.

As regards the maleic anhydride-alkenyl derivative copolymers, ema (monsanto), which are linear or crosslinked vinyl-maleic anhydride copolymers, and which are crosslinked, for example, by divinyl ether, are preferred. Reference may also be made to j.fields et al, Nature 186: 778-780, June4, 1960.

With respect to the structure, the acrylic or methacrylic polymer and the EMA are preferably formed by a basic unit having the following formula:

wherein:

-R₁and R₂Which may be the same or different, represents H or CH₃

-x-0 or 1, preferably x-1

-y-1 or 2, while x + y-2.

For EMA, x ═ 0 and y ═ 2 and for carbomer x ═ y ═ 1.

These polymers are soluble in water or physiological salt solution (20g/l NaCl) and the pH can be adjusted to 7.3-7.4 by, for example, soda (NaOH) to provide an adjuvant solution into which the expression vector can be incorporated. The concentration of the polymer in the final vaccine composition may be 0.01-1.5% w/v, advantageously 0.05-1% w/v, and preferably 0.1-0.4% w/v.

One skilled in the art, based on the present disclosure and knowledge in the art, can determine the effective plasmid dose for each immunization or vaccination regimen and species.

In an advantageous embodiment, the pharmaceutical and/or therapeutic composition and/or formulation according to the invention is administered by injection, such as, but not limited to, Intramuscular (IM), Intradermal (ID) or Subcutaneous (SC) injection.

With respect to such therapeutic compositions, the skilled artisan can determine, without any undue experimentation, the number of administrations, the route of administration, and the dosage for each injection regimen in light of the disclosure herein and the knowledge in the art.

In an advantageous embodiment, the recombinant vaccine may be administered in each dose, for example, about at least 10 per 2ml dose³The amount of pfu is administered to the pig or infected or transfected into the cell; more preferably about 10⁴pfu-about 10¹⁰pfu, e.g. about 10⁵pfu-about 10⁹pfu, e.g. about 10⁶pfu-about 10⁸pfu. In a particularly advantageous embodiment, the dosage is about 10 per dose⁸ pfu。

The method comprises administering to the animal at least once an effective amount of a therapeutic composition according to the invention. The animal may be male, female, pregnant female and newborn. Such administration may be accomplished in particular by Intramuscular (IM), Intradermal (ID) or Subcutaneous (SC) injection or via intranasal or oral administration. In an advantageous embodiment, the therapeutic composition according to the invention may be administered by means of a syringe or a needleless device, such as, for example, Pigjet, Biojector or Vitajet (Bioject, Oregon, USA). Another method of administering plasmids is the use of electroporation, see, e.g., s.tollefsen et al, Vaccine, 2002, 20, 3370-3378; s.tollefsen et al, scan.j.immunol., 2003, 57, 229-; babiuk et al, Vaccine, 2002, 20, 3399-; PCT application No. WO 99/01158.

The present invention relates to the use of a pharmaceutical composition for vaccination against Nipah virus infection in animals. The present invention relates to the use of a pharmaceutical composition for vaccination against Hendra virus infection in an animal. In a specific embodiment, the pharmaceutical composition according to the invention comprising Nipah F and Nipah G is used for vaccination against infections caused by Nipah or Hendra viruses in animals. In an advantageous embodiment, the animal is a pig. In other advantageous embodiments, the animal is a cat, dog, horse or human.

The invention also provides a method for preventing transmission of a Nipah virus between a first animal and a second animal comprising immunizing or eliciting an immune response in the first animal using any of the methods described herein to prevent transmission of disease to the second animal. The invention also provides a method for preventing transmission of a Hendra virus from an infected animal to another animal comprising immunizing in a first animal or eliciting an immune response to prevent transmission of the disease to a second animal using any of the methods described herein. In a specific embodiment, the pharmaceutical composition according to the invention comprising Nipah F and Nipah G is used for vaccinating the first animal against an infection caused by Nipah or Hendra virus. In an advantageous embodiment, wherein the first animal is a pig. The second animal is a cat, dog, or horse, advantageously a human.

The invention also provides a kit for performing any of the methods described above, comprising any of the compositions described above, and optionally instructions for performing the methods.

The invention will now be further described by way of the following non-limiting examples.

Examples

Example 1: construct

Construction of plasmid pSL-6802-1-4. pSL-6802-1-4 comprises the flanking sequences of the C5 locus, the H6 vaccinia promoter and the G Nipah virus gene to produce VCP 2199. Nipah virus was isolated from human CSF. The Nipah G gene was PCR amplified and inserted into plasmid pTM1, yielding pTM1Nipah G. The objective was to construct the pC5H6p Nipah G donor plasmid for the generation of recombinant ALVAC canarypox virus expressing Nipah G. The plasmid name is pC5H6p NipahG, pSL-6802-1-4. The plasmid backbone was pCXL-148-2, pC5H6p containing the H6 vaccinia promoter, corresponding to the left and right arms of the C5 locus of the insert. Plasmid pCXL-148-2 was obtained from plasmid pNVQH6C5LSP-18 by a single base mutation from T to C in the right arm of C5. Plasmid pNVQH6C5LSP-18 in S.Loosmore et al US 2005/0031641.

The Nipah G gene was PCR amplified using pTM1Nipah G as template and primers 11470.SL and 11471.SL (fig. 2B). The 1.8kb PCR fragment was cloned into pCR2.1, resulting in clone pSL-6771-1-1 (pCR2.1H 6p Nipah G), which was confirmed by sequence analysis (FIG. 2C). The 1.8kb Nru I-Xho IH6p Nipah G fragment from pSL-6771-1-1 was cloned into pCXL-148-2(pC 5H 6p) to yield pSL-6802-1-4(pC 5H6p Nipah G), which was confirmed by sequence analysis (FIGS. 2C and 2D).

Construction of plasmid pSL-6802-2-5. pSL-6802-2-5 comprises the flanking sequences of the F8 locus, the H6 vaccinia promoter and the G Nipah virus gene to produce VFP 2200. Nipah virus was isolated from human CSF. The Nipah G gene was PCR amplified and inserted into plasmid pTM1, yielding pTM1Nipah G. The aim was to construct the pF8H6p Nipah G donor plasmid for the generation of Nipah G expressing recombinant fowlpox virus (fowlpox). The plasmid name is pF8H6p NipahG, pSL-6802-2-5. The plasmid backbone was pSL-6427-2-1, pF8H6p containing the H6 vaccinia promoter, the left and right arms of the F8 locus of the insert. Plasmid pSL-6427-2-1 was obtained from plasmid pSL-5440-5-1 by a single base mutation from C to T in the left arm of F8. Plasmid pSL-5440-5-1 is described in S.Loosmore et al US 2005/0031641.

The Nipah G gene was PCR amplified using pTM1Nipah G as template and primers 11470.SL and 11471.SL (fig. 3A). The 1.8kb PCR fragment was cloned into pCR2.1, resulting in clone pSL-6771-1-1 (pCR2.1H 6p Nipah G), which was confirmed by sequence analysis (FIG. 3B). The 1.8kb Nru I-Xho IH6p Nipah G fragment from pSL-6771-1-1 was cloned into pSL-6427-2-1(pF 8H 6p) to yield pSL-6802-2-5(pF 8H6p Nipah G), which was confirmed by sequence analysis (FIGS. 3B and 3C).

Construction of plasmid pSL-6839-1. pSL-6839-1 contains flanking sequences of the F8 locus, the H6 vaccinia promoter, and the F Nipah viral gene to produce VFP 2207. Nipah virus was isolated from human CSF. The Nipah F gene was PCR amplified and inserted into plasmid pTM1, yielding pTM1Nipah F. The aim was to construct the pF8H6p Nipah F donor plasmid for the generation of recombinant fowlpox virus (fowlpox) expressing NipahF. Plasmid name: pF8H6p Nipah F, pSL-6839-1. The plasmid backbone was pSL-6427-2-1, pF8H6p containing the H6 vaccinia promoter, the left and right arms of the F8 locus of the insert.

The internal T5NT sequence in Nipah F was removed by site-directed mutagenesis. Fragments encoding the 3 'end of the H6 promoter and the 5' end of the Nipah F gene were PCR amplified using primers 11457.SL and 11458. SL. In the amplified fragment, the T5NT sequence was removed and an ApaI site was introduced for cloning purposes (fig. 4B). The fragment was cloned into pCR2.1, resulting in pSL-6797-3-1 (pCR2.1H 6p 5' -Nipah F, T5NT), which was confirmed by sequence analysis (FIG. 4C). The 3' -Nipah F fragment was PCR amplified using primers 11456.SL and 11459. SL. In the amplified fragment, the T5NT sequence was removed and an ApaI site was introduced for cloning purposes (fig. 4B). The fragment was cloned into pCR2.1, resulting in pSL-6797-4-1 (pCR2.13' -Nipah F, without T5NT), which was confirmed by sequence analysis (FIG. 4C). The-0.7 kb Nru I-BamH I H6p5 '-Nipah F fragment from pSL-6797-3-1 was inserted into pSL-6427-2-1(pF 8H 6p) to yield pSL-6830-1(pF 8H6p 5' -Nipah F). The 1.0kb Apa I-BamHI 3' -Nipah F fragment from pSL-6797-4-1 was inserted between Apa I and Bam H I of pSL-6830-1 to yield pSL-6839-1(pF 8H6p Nipah F), which was confirmed by sequence analysis (FIGS. 4C and 4D).

Construction of plasmid pSL-6851-29. pSL-6851-29 contains flanking sequences of the C5 locus, the H6 vaccinia promoter and the FNipah virus gene to produce VCP 2208. Nipah virus was isolated from human CSF. The Nipah F gene was PCR amplified and inserted into plasmid pTM1, yielding pTM1Nipah F. The objective was to construct the pC5H6p Nipah F donor plasmid for the production of recombinant ALVAC canarypox virus expressing Nipah F. Name of plasmidIs pSL-6851-29, pC5H 6pNipah F. The plasmid backbone was pCXL-148-2, pC5H6p containing the H6 vaccinia promoter, and the left and right arms of the C5 locus of the insert.

The internal T5NT sequence in Nipah F was removed by site-directed mutagenesis. Fragments encoding the 3 'end of the H6 promoter and the 5' end of the Nipah F gene were PCR amplified using primers 11457.SL and 11458. SL. In the amplified fragment, the T5NT sequence was removed and an ApaI site was introduced for cloning purposes (fig. 5A). The fragment was cloned into pCR2.1, resulting in pSL-6797-3-1 (pCR2.1H 6p 5' -Nipah F, without T5NT), which was confirmed by sequence analysis (FIG. 5B). The 3' -Nipah F fragment was PCR amplified using primers 11456.SL and 11459. SL. In the amplified fragment, the T5NT sequence was removed and an ApaI site was introduced for cloning purposes (fig. 5A). The fragment was cloned into pCR2.1, resulting in pSL-6797-4-1 (pCR2.13' -Nipah F, without T5NT), which was confirmed by sequence analysis (FIG. 5B). The-0.7 kb Nru I-BamH I H6p5 '-Nipah F fragment from pSL-6797-3-1 was inserted into pSL-6427-2-1(pF 8H 6p) to yield pSL-6830-1(pF 8H6p 5' -Nipah F). A1.0 kb Apa I-BamHI 3' -Nipah F fragment from pSL-6797-4-1 was inserted between Apa I and Bam H I of pSL-6830-1 to yield pSL-6839-1(pF 8H6p Nipah F), which was confirmed by sequence analysis. A1.7 kb Nru I-Xma I H6p Nipah F fragment from pSL-6839-1 was inserted into pCXL-148-2(pC 5H 6p) to yield pSL-6851-29(pC 5H6p Nipah F), which was confirmed by sequence analysis (FIGS. 5B and 5C).

Construction of recombinant fowlpox Virus (fowlpox) vFP2207 expressing Nipah F. The gene is Nipah F. The donor plasmid was pSL-6839-1. The insertion site is the F8 locus of fowlpox virus (fowlpox). The promoter is the vaccinia virus H6 promoter. Cells used for in vitro recombination were primary chicken embryo fibroblasts (1 ° CEF) grown in 10% FBS and DMEM.

In vitro recombination was performed by transfecting 1 ° CEF cells with Not I-linearized donor plasmid pSL-6839-1(20 ug). Transfected cells were subsequently infected with fowlpox virus (fowlpox) as a rescue virus at a MOI of 10. After 48 hours, the transfected-infected cells were harvested, sonicated and used for recombinant virus screening. Recombinant plaques were screened based on a plaque transfer hybridization method using a Nipah F specific probe labeled with horseradish peroxidase. After 4 consecutive rounds of plaque purification, recombinants designated vCP2207 were generated and confirmed by hybridization 100% positive for the insert and 100% negative for the F8 ORF.

Construction of recombinant canarypox virus vCP2199 for expressing Nipah G. The gene is NipahG. The donor plasmid was pSL-6802-1-4. The insertion site is C5. The promoter is the H6 promoter.

Cells used for in vitro recombination were primary chicken embryo fibroblasts (1 ° CEF) grown in 10% FBS and DMEM.

In vitro recombination was performed by transfecting 1 ° CEF cells with Not I-linearized donor plasmid pSL-6802-1-4(15 ug). Transfected cells were subsequently infected with ALVAC as a rescue virus at MOI 10. After 24 hours, the transfected-infected cells were harvested, sonicated and used for recombinant virus screening. Recombinant plaques were screened based on a plaque transfer hybridization method using a Nipah G specific probe labeled with horseradish peroxidase. After 5 consecutive rounds of plaque purification, recombinants designated vCP2199 were generated and confirmed by hybridization 100% positive for the Nipah G insert and 100% negative for the C5 ORF.

Construction of recombinant avian jaundice Virus (fowlpox) vCP2200 expressing Nipah G. The gene is Nipah G. The donor plasmid was pSL-6802-2-5. The insertion site is F8. The promoter is the H6 promoter. Cells used for in vitro recombination were primary chicken embryo fibroblasts (1 ° CEF) grown in 10% FBS and DMEM.

In vitro recombination was performed by transfecting 1 ° CEF cells with Not I-linearized donor plasmid pSL-6802-2-5(15 ug). Transfected cells were subsequently infected with fowlpox virus (fowlpox) as a rescue virus at a MOI of 8. After 48 hours, the transfected-infected cells were harvested, sonicated and used for recombinant virus screening. Recombinant plaques were screened based on a plaque transfer hybridization method using a Nipah G specific probe labeled with horseradish peroxidase. After 4 consecutive rounds of plaque purification, recombinants designated vFP2200 were generated. They were confirmed by hybridization 100% positive for the Nipah G insert and 100% negative for the F8 ORF.

Construction of recombinant canarypox virus vCP2208 expressing Nipah F. The gene is NipahF. The donor plasmid was pSL6851.29(pC 5H6p Nipah F). The insertion site is C5. The promoter was the vaccinia H6 promoter. Cells used for in vitro recombination were primary chicken embryo fibroblasts (1 ° CEF) grown in 10% FBS and DMEM.

In vitro recombination was performed by transfecting 1 ° CEF cells with Not I-linearized donor plasmid psl6851.29(10 ug). Transfected cells were subsequently infected with ALVAC as a rescue virus at MOI 10. After 24 hours, the transfected-infected cells were harvested, sonicated and used for recombinant virus screening. Recombinant plaques were screened based on a plaque transfer hybridization method using a Nipah F-specific probe labeled with horseradish peroxidase. After 4 consecutive rounds of plaque purification, recombinants designated vCP2208 were generated and confirmed by hybridization 100% positive for the Nipah F insert and 100% negative for the C5 ORF.

Example 2: expression of

Western blot of fowlpox Virus (fowlpox) Nipah G vFP2200(FIG. 6). Primary CEF cells were infected with vCP2199(ALVAC C5H6p Nipah G) and vFP2200 (fowlpox virus (fowlpox) F8H6p Nipah G) at an MOI of 10 and incubated for 24 hours at 37 ℃. Cells and culture supernatant were then harvested. Sample proteins were separated on a 10% SDS-PAGE gel and transferred to Immobilon nylon membranes. Guinea pig antisera and a chemiluminescent system were used. Nipah G was expressed in cell pellets of vCP2199 and vFP 2200. It does not appear in the supernatant.

Western blot of ALVAC Nipah F vCP2208(drawing)7A and 7B). Primary CEF cells were infected with vCP2208(ALVAC C5H6p Nipah F) at an MOI of 10 and incubated for 24 hours. The supernatant was harvested and clarified. Cells were harvested and suspended in water for lysis. Lysates and supernatants were separated by 10% SDS-PAGE. Proteins were transferred to nylon membranes and blocked with Western blocking buffer. Expression of F protein from vCP2208(ALVAC C5H6p Nipah F) was shown using a guinea pig antiserum and chemiluminescent chromogenic system. The use of pig antiserum and horseradish peroxidase system also showed expression of F protein from vCP2208, but at a lower intensity.

Western blot of ALVAC Nipah G vCP2199(FIG. 6). Primary CEF cells were infected with vCP2199(ALVAC C5H6p Nipah G) and vFP2200 (fowlpox virus (fowlpox) F8H6p Nipah G) at an MOI of 10 and incubated for 24 hours at 37 ℃. Cells and culture supernatant were then harvested. Sample proteins were separated on a 10% SDS-PAGE gel and transferred to Immobilon nylon membranes. Guinea pig antisera and a chemiluminescent system were used. Nipah G was expressed in cell pellets of vCP2199 and vFP 2200. It does not appear in the supernatant.

Western blot of fowlpox Virus (fowlpox) Nipah F vFP2207(FIGS. 7A and 7B). Primary CEF cells were infected with vFP2207 (fowlpox virus (fowlpox) F8H6p NipahF) at an MOI of 10 and incubated for 24 hours. The supernatant was harvested and clarified. Cells were harvested and suspended in water for lysis. Lysates and supernatants were separated by 10% SDS-PAGE. Proteins were transferred to nylon membranes and blocked with Western blocking buffer. Expression of the F protein from vFP2207 (fowlpox virus (fowlpox) F8H 6pNipah F) was shown using a guinea pig antiserum and chemiluminescent chromogenic system. The use of pig antiserum and horseradish peroxidase system also showed expression of F protein from vFP2207, but at a lower intensity.

Example 3: serology and protection

The 16 pigs were randomly divided into 4 groups. Nipah virus F protein expression for animals in group F10 of a mass⁸pfu/dose of VCP 2208. Nipah virus G protein-expressing 10 for group G animals⁸pfu/dose of VCP 2199. Group G + F animals contain 10 expressing the G and F proteins of the Nipah viruses, respectively⁸pfu/dose of VCP2199 and 10⁸pfu/dose of VCP2208 mixture. Animals in the challenge group were unvaccinated control animals.

Pigs were injected intramuscularly on days 0 and 14. Pigs were vaccinated intranasally at day 28 by 2.5X 10⁵pfu of Nipah virus. 7 days after challenge, the presence of virus was identified by RT-PCR or virus isolation in various organs and nasal swabs. Blood samples were collected at day 0, day 7, day 14, day 21, day 28, day 29, day 30, day 31, day 32, day 34, and day 35 after the first injection, and antibody titers were measured by IgG indirect ELISA or serum neutralization assay. Neutralizing antibodies were determined in a microtiter plaque reduction neutralization assay (mPRNT) as previously described (H.Weingartl et al, Can.J.Vet.Rrs.2003, 67, 128-one 132) using Vero V-76 cells and 1% carboxymethylcellulose coverage. Wells with 90% reduction in plaque were considered positive for Nipah virus neutralizing antibodies. Data for ELISA and Neutralization Titers (NTs) are presented in table 1. Combined Nipah F/G induced the highest neutralizing titer prior to challenge, followed by the G vaccine. The F vaccine induced lower neutralizing antibodies.

Viral plaque assay: the viral plaque assay was performed in 12-well plates (Costar, Corning, NY) using a confluent monolayer of Vero 76 or PT-K75. Virus inoculum (400. mu.l/well) was plated on cells at 33 ℃ with 5% CO₂Incubate for 1 hour, then replace with 2ml of 2% carboxymethylcellulose, sodium salt, media viscosity/DMEM (Sigma Chemical, St. Louis, Mo.)/2% FBS cover, and at 33 ℃, 5% CO₂And (4) incubating. After 5 days the cells were fixed with 4% formaldehyde and stained with 0.5% crystal violet/80% methanol/PBS. According to V.Guillame et al J.Virol. method.2004, 120, 229-onal) real-time RT-PCR was performed only on serum/plasma and PMBC samples. Forward primer GCA CTT GAT GTG ATT AGA (SEQ ID NO: 29) and reverse primer GGC AGT GTC GGG AGC TGT AA (SEQ ID NO: 12) were located within the N gene, producing a 395bp amplicon. Real-time RT-PCR was normalized with sensitivity of 300 copies/reaction in 100. mu.l samples using the Nipah virus N gene cloned into pSHAME2a plasmid. Samples that became positive at 35 cycles were considered negative.

Nipah virus was isolated at very low titers in the trigeminal ganglia of pigs #33(1 plaque), #35(1 plaque) and # 36. In control animals, Nipah virus could be isolated from many tissues at up to 10e3 pfu/ml: pig #39 was positive in nasal concha, trachea, olfactory bulb, trigeminal ganglia, bronchiolar lymph node and submandibular Lymph Node (LN); pig #40 was positive in the turbinate, trachea, olfactory bulb, meninges, trigeminal ganglia, bronchiolar LN, submandibular LN, and brain. The RT-PCR results are provided in tables 2 and 3. The number is the threshold number of cycles. No RNA was detected in the plasma, serum or PBMC of immunized pigs immunized with the F/G vaccine.

These results show clear protection using recombinants expressing Nipah virus F or G proteins, as well as complete protection using Nipah virus F + G protein combinations.

Example 4: cross neutralization

The 18 pigs were randomly divided into 4 groups. 10 animals of group F expressing Nipah Virus F protein⁸pfu/dose of vCP 2208. 10 animals of group G expressing Nipah Virus G protein⁸pfu/dose of vCP 2199. Group G + F4 animals were treated with a composition containing 10 proteins expressing the Nipah viruses G and F, respectively⁸pfu/dose of vCP2199 and 10⁸pfu/dose of vCP2208 mixture. As a control group without vaccination, 6 animals were naturally infected with Nipah virus and carried up to 28 days post infection (dpi). This group was named "long-term infection".

F、Pigs in groups G and G + F were injected by intramuscular route on day 0 and 14. Blood samples were collected at day 27 post vaccination (dpv or days post vaccination) and antibody titers were measured by serum neutralization assay. Neutralizing antibodies were assayed in the microtiter plaque reduction neutralization assay (mPRNT) as previously described (H.Weingartl et al, Can.J.Vet.Rrs.2003, 67, 128-one 132) using Vero V-76 cells at 1.2X 10⁵Cells/cm²(40,000 cells/well) were seeded in 96-well plates and grown in DMEM medium supplemented with 10% FBS at 5% CO₂Incubate at 37 ℃. 100 μ L of serial 2-fold dilutions of serum (1/10-1/1280) in combination with 100 μ L of Hendra or Nipah virus in 5% CO₂After incubation for 1 hour at 37 ℃ the virus was adjusted to contain 1000 PFU/100. mu.L. All dilutions were performed in DMEM.

After incubation, 100. mu.L of the above mixture was transferred to a monolayer of V76 cells. Plates containing inoculum at 5% CO₂Incubate at 37 ℃ for 1 hour.

The inoculum was removed after 1 hour and replaced with 100 μ L of 2% carboxymethyl cellulose solution in DMEM supplemented with 2% FBS. Plates at 5% CO₂Incubate at 37 ℃ for 72 hours.

Back titration for Nipah virus yielded results with a working solution dilution of 500 PFU/well, and for Hendra virus: 625 PFU/well.

Note that: serum from pigs vaccinated with the F protein was 2-fold diluted from 1/50 to 1/2400.

Wells with 90% reduction in plaque were considered positive for the presence of Nipah virus neutralizing antibody or for the presence of Hendra virus neutralizing antibody.

Data for neutralization titers are presented in table 4. The combined Nipah F/G induced a synergistic effect for the production of antibodies against Nipah virus, which did not occur during natural infection with Nipah virus (see results for long-term infection group). The G vaccine or F vaccine alone did not induce neutralizing antibodies against Nipah virus, or induced less neutralizing antibodies than the F + G vaccine. There was no correlation between the antibody titer levels against Nipah virus and those against Nipah virus.

[0174]

[0176]

[0180] The invention is further described by the following numbered paragraphs:

1. an avipoxvirus expression vector comprising a polynucleotide encoding a Nipah virus glycoprotein.

2. The fowlpox expression vector of paragraph 1 wherein the Nipah virus glycoprotein is an attachment (G) protein.

3. The fowlpox expression vector of paragraph 1 wherein the Nipah virus glycoprotein is a fusion (F) protein.

4. The fowlpox expression vector of paragraph 1 wherein said Nipah virus glycoprotein is an attachment (G) protein and a fusion (F) protein.

5. The fowlpox expression vector of paragraph 2, wherein said polynucleotide comprises the sequence of SEQ id no: 1, nucleotide base sequence from nucleotide 8943 to nucleotide 10751.

6. The fowlpox expression vector of paragraph 3, wherein said polynucleotide comprises the sequence of SEQ id no: nucleotide base sequence of nucleotide 6654-nucleotide 8294 of 1.

7. The fowlpox expression vector of paragraph 4, wherein said polynucleotide encodes the amino acid sequence of SEQ id no: 8.

8. The fowlpox expression vector of paragraph 2, wherein said polynucleotide encodes the amino acid sequence of SEQ id no: 7.

9. The fowlpox expression vector of paragraph 3 wherein said polynucleotide encodes the amino acid sequence of SEQ id no: 7 and SEQ ID NO: 8.

10. The fowlpox expression vector of paragraph 5, wherein said polynucleotide comprises the sequence of SEQ id no: 1 from nucleotide 6654 to nucleotide base sequence of nucleotide 10751.

11. The avian jaundice virus expression vector of paragraphs 1-10, wherein the avipox virus expression vector is an attenuated avipox virus expression vector.

12. The fowlpox expression vector of paragraphs 1-11, wherein said fowlpox expression vector is a canarypox vector.

13. The canary pox viral vector of paragraph 12, wherein the canary pox viral vector is ALVAC.

14. The fowlpox expression vector of paragraphs 1-11, wherein said fowlpox expression vector is a fowlpox (fowlpox) vector.

15. The fowlpox virus (fowlpox) vector of paragraph 14, wherein said fowlpox virus (fowlpox) vector is a TROVAC.

16. An expression vector, wherein said expression vector is vCP 2199.

17. An expression vector, wherein said expression vector is vCP 2208.

18. An expression vector, wherein said expression vector is vFP 2200.

19. An expression vector, wherein the expression vector is vVP 2207.

20. A formulation for delivery and expression of a Nipah virus glycoprotein, wherein the formulation comprises the vector of any of paragraphs 1-19 and a pharmaceutically or veterinarily acceptable carrier, vehicle or excipient.

21. The formulation of paragraph 20, wherein the carrier, vehicle or excipient facilitates transfection of the vector and/or improves preservation thereof.

22. A method of delivering Nipah virus glycoprotein to an animal comprising administering the formulation of paragraph 21 or 22 to the animal.

23. The method of paragraph 22, wherein the animal is a pig.

24. A method of eliciting an immune response in an animal comprising administering a composition comprising the vector of any one of paragraphs 1-19 in an effective amount for eliciting an immune response.

25. A method of eliciting an immune response in an animal comprising administering a composition comprising cells in an effective amount for eliciting an immune response, wherein the cells comprise the vector of any one of paragraphs 1-19.

26. A method of inducing an immune or protective response in an animal comprising administering a composition comprising the vector of any of paragraphs 1-19 in an effective amount for inducing an immune response.

27. A method of inducing an immune or protective response in an animal comprising administering a composition comprising cells in an effective amount for inducing an immune response, wherein the cells comprise the vector of any one of paragraphs 1-19.

28. The method of any of paragraphs 24-27, wherein the animal is a pig.

29. A method for preventing transmission of Nipah virus between a first animal and a second animal comprising the method of any one of paragraphs 24-27, wherein the animal of any one of paragraphs 24-27 is the first animal.

30. The method of paragraph 29 wherein the first animal is a pig.

31. The method of paragraph 29 or 30, wherein the second animal is a human.

32. The method of paragraph 29 or 30 wherein the second animal is a cat or dog.

33. A kit for performing the method of any of paragraphs 22-32 comprising the vector of any of paragraphs 1-19 or the formulation of any of paragraphs 20 or 21 and instructions for performing the method.

***

In view of the advantageous embodiments of the present invention thus described in detail, it is to be understood that the invention defined by the above paragraphs is not limited to particular details set forth in the above description, as many apparent variations thereof may be made without departing from the spirit or scope thereof.

Claims (15)

1. An avipox expression vector comprising a polynucleotide encoding a Nipah virus glycoprotein, wherein the Nipah virus glycoprotein is an attachment (G) protein, wherein the vector is administered to an animal selected from the group consisting of a pig, a cat, a dog, and a horse.

2. An avipox expression vector comprising a polynucleotide encoding a Nipah virus glycoprotein, wherein the Nipah virus glycoprotein is the fusion (F) protein, wherein the vector is administered to an animal selected from the group consisting of a pig, a cat, a dog, and a horse.

3. The fowlpox expression vector of claim 1 or 2, wherein said fowlpox expression vector is an attenuated fowlpox expression vector.

4. The fowlpox expression vector of claim 1 or 2, wherein said fowlpox expression vector is a canarypox vector.

5. The avipox expression vector of claim 4, wherein the canarypox vector comprises a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 7, or a fragment thereof.

6. The avipox expression vector of claim 4, wherein the canarypox vector comprises a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 8, or a polynucleotide of the G protein of the sequence shown in figure 8.

7. The fowlpox expression vector of claim 1 or 2, wherein said fowlpox expression vector is a fowlpox vector.

8. The fowlpox expression vector of claim 7, wherein the fowlpox vector comprises a nucleic acid sequence encoding a polypeptide having the sequence of SEQ ID NO: 7, or a fragment thereof.

9. The fowlpox expression vector of claim 7, wherein the fowlpox vector comprises a nucleic acid sequence encoding a polypeptide having the sequence of SEQ ID NO: 8, or a polynucleotide of the G protein of the sequence shown in figure 8.

10. The avipox expression vector of claim 6, wherein the canarypox vector comprises a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

11. The avipox expression vector of claim 5, wherein the canarypox vector comprises a nucleic acid sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 28, or a polynucleotide having the sequence shown in figure 28.

12. The fowlpox expression vector of claim 9, wherein the fowlpox vector comprises a nucleic acid sequence encoding a polypeptide having the sequence of SEQ ID NO: 18 or SBQ ID NO: 19.

13. The fowlpox expression vector of claim 8, wherein the fowlpox vector comprises a nucleic acid sequence encoding a polypeptide having the sequence of SEQ ID NO: 24 or SEQ ID NO: 25, or a variant thereof.

14. A formulation for delivery and expression of Nipah virus glycoprotein, wherein the formulation comprises the avipox expression vector of claim 1 or 2 and a pharmaceutically or veterinarily acceptable carrier, vehicle or excipient.

15. The formulation of claim 14, wherein the carrier, vehicle or excipient facilitates transfection of the vector and/or improves preservation thereof.